Phosphate Solubilizing Microbes for Crop Improvement [1 ed.] 9781617285615, 9781608761128

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Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved. Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved. Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

AGRICULTURE ISSUES AND POLICIES SERIES

PHOSPHATE SOLUBILIZING MICROBES FOR CROP IMPROVEMENT

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Agriculture Issues and Policies Series Agriculture Issues & Policies, Volume I Alexander Berk (Editor) 2001. ISBN 1-56072-947-3 Agricultural Conservation Anthony G. Hargis (Editor) 2009. ISBN 978-1-60692-273-6 Hired Farmworkers: Profile and Labor Issues Rea S. Berube (Editor) 2009. ISBN 978-1-60741-232-8 Environmental Services and Agriculture Karl T. Poston (Editor) 2009 ISBN: 978-1-60741-053-9 Weeds: Management, Economic Impacts and Biology Rudolph V. Kingely (Editor) 2009 ISBN 978-1-60741-010-2

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Effects of Liberalizing World Agricultural Trade Henrik J. Ehrstrom (Editor) 2009 ISBN: 978-1-60741-198-7 Economic Impacts of Foreign-Source Animal Disease Jace R. Corder (Editor) 2009 ISBN: 978-1-60741-601-2 Agricultural Wastes Geoffrey S. Ashworth and Pablo Azevedo (Editors) 2009 ISBN: 978-1-60741-305-9 Soybean and Wheat Crops: Growth, Fertilization, and Yield Samuel Davies and George Evans 2009 ISBN: 978-1-60741-173-4 Phosphate Solubilizing Microbes for Crop Improvement Mohammad Saghir Khan and Almas Zaidi (Editors) 2009 ISBN: 978-1-60876-112-8

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

AGRICULTURE ISSUES AND POLICIES SERIES

PHOSPHATE SOLUBILIZING MICROBES FOR CROP IMPROVEMENT

MOHAMMAD SAGHIR KHAN AND Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

ALMAS ZAIDI EDITORS

Nova Science Publishers, Inc. New York

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Khan, Mohammad Saghir. Phosphate solubilising microbes for crop improvement / authors: Mohammad Saghir Khan and Almas Zaidi. p. cm. Includes index. ISBN 978-1-61728-561-5 (E-Book) 1. Soil microbiology. 2. Phosphates--Solubility. 3. Crop improvement. I. Zaidi, Almas. II. Title. QR111.K46 2009 579'.1757--dc22 2009029203

Published by Nova Science Publishers, Inc.  New York

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

CONTENTS Preface Chapter 1

Chapter 2

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Chapter 3

Chapter 4

Chapter 5

vii Biological Importance of Phosphorus and Phosphate Solubilizing Microbes -An Overview Munees Ahemad, Almas Zaidi, Mohd. Saghir Khan and Mohammad Oves Novel Approaches for Analysis of Biodiversity of PhosphateSolubilizing Bacteria Martha-Helena Ramírez-Bahena, Alvaro Peix, Eustoquio Martínez-Molina and Encarna Velázquez Effects of Phosphate Solubilizing Microorganism on Soil Phosphorus Fractions Metin Turan Role of Plant Growth Promoting Microorganisms for Sustainable Crop Production B. Hameeda, G. Harini, B. Keerthi Kiran, O. P. Rupela and Gopal Reddy Genetic and Functional Diversity of Phosphate Solubilizing Fluorescent Pseudomonads and Their Simultaneous Role in Promotion of Plant Growth and Soil Health M. Jaharamma, K. Badri Narayanan and N. Sakthivel

Chapter 6

Practical Use of Phosphate Solubilizing Soil Microorganisms Olga Mikanova and Jaromir Kubat

Chapter 7

Phosphate-Solubilization by Psychrophilic and Psychrotolerant Microorganisms: An Asset for Sustainable Agriculture at Low Temperatures Harshita Negi, Kuheli Das, Anil Kapri and Reeta Goel

Chapter 8

Beneficial Microbes in Sustainable Tropical Crop Production Zulkifli Haji Shamsuddin, O. Radziah and Halimi Mohd. Saud

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43

63

111 129

145 161

vi Chapter 9

Chapter 10

Chapter 11

Chapter 12

Chapter 13

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Chapter 14

Contents Molecular Genetics of Phosphate Solubilization in Rhizosphere Bacteria and Its Role in Plant Growth Promotion S. S. Sindhu, M. K. Verma and Suman Mor

199

Strategies for Development of Microphos and Mechanisms of Phosphate-Solubilization Almas Zaidi, Md. Saghir Khan, Mohd. Oves and Munees Ahemad

229

Variation in Plant Growth Promoting Activities of PhosphateSolubilizing Microbes and Factors Affecting Their Colonization and Solubilizing Efficiency in Different Agro- Ecosystems Mohammad Oves, Almas Zaidi, Mohd. Saghir Khan and Munees Ahemad

247

Management of Plant Diseases Using Phosphate-Solubilizing Microbes Adel Ahmed El-Mehalawy

265

Phosphate Solubilizing Microbes: Potentials and Success in Greenhouse and Field Applications Diriba Muleta

281

Genetic and Phenotypic Characterization of Phosphate-Solubilizing Bacteria and Their Effects on Growth and Symbiotic Properties of Alfalfa Plants Lorena B. Guiñazú, Javier A. Andrés, Nicolás A. Pastor, Marisa Rovera and Susana B. Rosas

309

Chapter 15

Microbial Facilitation of Phosphorus Nutrition in Sugarcane B. Sundara

Chapter 16

Phosphate Solubilizing Microorganisms for Augmenting Crop Nutrition Parvaze Ahmad Wani and Geeta Singh

337

Phosphate Solubilizing Microorganisms: Prospects, Promises and Problems Aftab Afzal and Asghari Bano

357

Genetic Manipulations of Metal Accumulation and Heavy Metal Tolerance: Improving Plants for Environmental Remediation M. Nedkovska and N. Gorinova

377

Chapter 17

Chapter 18

Chapter 19

Biological Control of Plant Nematodes with PhosphateSolubilizing Microorganisms Mujeebur Rahman Khan, Shahana Altaf, Fayaz A. Mohidin, Uzma Khan and Arshad Anwer

Index

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PREFACE Globally, most agronomic soils contain large reserves of phosphorus, but the fixation and precipitation, cause a major deficiency of P in soil, and in turn, impede the growth of crops seriously. Phosphorus replenishment, especially in sustainable production systems, remains a major challenge as it is mainly fertilizer-dependent. Though, the use of chemical phosphatic fertilizers is obviously the best means to circumvent P deficiency in different agroecosystems, their use is always restricted due to its spiralling cost. Interest has recently been generated to find an alternative yet inexpensive technology that could provide sufficient P to plants while reducing the dependence on expensive chemical P fertilizers. Thus, poor availability of P in soil and consequent P-deficiency represents a major constraint to crop production. How to improve phosphorus fertilizer efficiency and utilize the potential nutrient in soil has therefore, long been a major concern for field practitioners. This has led to a search for environment-friendly and economically attractive alternative strategies for improving crop production in low or even P deficient soils. In this context, natural soil inhabitants endowed with P-solubilizing [PS] activity, often termed phosphate-solubilizing microorganisms [PSM], provides a viable substitute to chemical P fertilizers. Inoculation of phosphatesolubilizing microorganisms, like, bacteria, fungi and actinomycetes in soil has been shown to profoundly affect the growth of plants by making not only P available to plants but also facilitate the growth of plants by other mechanisms leading to higher crop yields. Besides providing phosphorus, these microbes also substantially enhance the growth by providing N to the plants through N2 fixation, increasing the availability of nutrients and phyto-hormones, inducing increases in root surface area, enhancing other beneficial symbioses of the host, reducing or preventing the deleterious effects of phyto-pathogenic organisms, synthesizing enzymes, mitigating metal toxicity etc. Accordingly, these microbes augment the growth and yields of a wide range of crops under both conventional and stressed soil environment. Tremendous amount of research work has been done to highlight the role of phosphatesolubilizing microbes in the improvement of crops but very little attempt is made to organize such findings that could help students/teachers/scientists and to progressive farmers in a big way. Phosphate Solubilizing Microbes for Crop Improvement provides a comprehensive source of information on strategies and concepts of microbial technology especially phosphate- solubilizing microbes for the improvement of crops in different agro-ecosystems. The book presents the biological importance of phosphorus and strategies adopted for isolation and screening of PSM (s), mechanisms of P solubilization, mechanisms of plant growth promotion, and method for the development of microphos. Furthermore, some novel

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viii

Mohammad Saghir Khan and Almas Zaidi

approaches including molecular tools used to identify the potential phosphate-solubilizing microbes are presented. The recent advances in understanding the genetics and molecular biology of phosphate-solubilizing bacteria and the genetic engineering of bacterial strains with enhanced phosphate-solubilizing activity are discussed that is likely to lead to improve the efficiency of microphos inoculants and crop productivity. The problems, prospect and potentials of phosphate-solubilizing microbes and their impact on agronomically important crops grown in conventional soils are discussed separately. Special attention is paid to highlight the functional variations within phosphate-solubilizing microbes and to understand the impact of various factors on the phosphate-solubilizing efficiency and colonization of such naturally occurring organisms. The synergism between phosphate-solubilizing microbes and other plant growth promoting rhizobacteria/arbuscular mycorrhizal fungi and their interactive effect on crop productivity is highlighted separately. The book also presents a broad and updated view of the management of plant diseases using phosphate-solubilizing microbes. The book further describes as to how the growth promoting rhizobacteria facilitate plant growth and how advanced information strategies can be used to manipulate and modify the soil environment. The impact of phosphate-solubilizing microorganisms on the growth and development of sugarcane is discussed. A separate chapter on phenotypic and genotypic characterization including 16S rDNA of phosphate-solubilizing bacteria and their consequent inoculation effects on alfalfa is included. The major aim of Phosphate Solubilizing Microbes for Crop Improvement is to highlight and assess the significance of the most important findings as they occur in this area so as to provide insightful and authoritative information to the readers. The book provides a more detailed, structured coverage of research in the area, exclusively, the phosphate-solubilizing microbes-plant interactions and how these interactions could be exploited for maintaining the fertility of soils and consequently promoting the growth of various agronomic crops in different agro-ecosystem across the globe, reported in different scientific media. This book is likely to be of special interest to the postgraduate students, research scholars, teachers, scientists and professionals working in the field of microbiology, soil microbiology, biotechnology, agronomy, plant sciences, plant physiology, mycology and plant protection sciences. The book addresses the problems of agrarian communities who are interested in exploiting the inherent potentials of phosphate-solubilizing microbes for the sustainability of crops and restoration of soil fertility. Besides fulfilling the needs of the academicians/professionals, Phosphate Solubilizing Microbes for Crop Improvement also provides information to the policy makers, inoculant making industries and the practicing scientists of agriculture, soil science and microbial biotechnology across the world. The book highlights both the basic concepts and the most exciting and fascinating aspect of phosphatesolubilizing microbes. Each chapter is contributed by highly experienced academicians/professionals from across the globe and efforts are made to bring in the most recent advances made in the field of phosphate-solubilizing microbes and their utility in the sustainable agriculture. We are extremely thankful to our scientific colleagues here and abroad who very strongly and generously cooperated in this strive and willingly provided us their most recent and detailed scientific information and supported in the development of Phosphate Solubilizing Microbes for Crop Improvement without whom it would have been extremely difficult to complete this book. Also we express our gratitude to scientific colleagues who spared their valuable time to respond to our queries regarding the preparation of this book and who have

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Preface

ix

informally made suggestions to us to improve the overall presentation of the book. We are also indebted to our research scholars for the care and attention exercised in typing and designing portion of the manuscript and their supportive services in finalizing this book. We also gratefully thank the publisher of this book. We are extremely grateful to our family members Dr. Meena Zaidi, Dr. S.A.H. Zaidi, Ms Zainab Zaidi, Ms Butool Zaidi, Dr. M. Shakil Khan and Ms. Saman Khan for their constant support, patience, encouragement and sacrifices during the compilation of this book. Finally, we believe this book may have some conceptual or printing mistakes arising unintentionally during preparation for which we feel sorry in anticipation and if pointed out to us, shall surely correct them in subsequent print/edition. We also invite suggestions and healthy criticism from the readers of this book so that the information on the concerned subject could be improved in future.

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Mohammad Saghir Khan Almas Zaidi

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ABOUT EDITORS Mohammad Saghir Khan, Ph.D. is an Associate Professor at the Department of Agricultural Microbiology, Aligarh Muslim University, Aligarh, India. Dr. Khan received his Ph.D. (Microbiology) degree from Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, India. He has been teaching Microbiology to post-graduate students for the last 13 years and has research experience of 17 years. In addition to his teaching, Dr. Khan is engaged in guiding students for their doctoral degree in Microbiology. He has published over 50 scientific papers including, original research articles, review articles and book chapters in various national and international publication media. Dr. Khan has also edited a book published by one of the leading publishers. His area of research includes rhizobiology, microbiology, environmental microbiology especially heavy metals-microbeslegume interaction, bioremediation, pesticide-PGPR-plant interaction and biofertilizers technology. Almas Zaidi, received her Ph.D. (Agricultural Microbiology) degree from Aligarh Muslim University, Aligarh, India and currently serving as Guest faculty/Assistant Professor at the Department of Agricultural Microbiology, Aligarh Muslim University, Aligarh, India. She has been teaching Microbiology at post graduate level for the last five years and has research experience of 13 years. She has published about 40 research papers and reviews in journals of national and international repute. She has also contributed chapters to different books. Dr. Zaidi has also edited a book published by one of the leading publishers. Her main focus of research is to address problems related with rhizo-microbiology, microbiology, environmental microbiology, and biofertilizer technology.

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CONTRIBUTORS Adel Ahmed El-Mehalawy Microbiology Department, Ain Shams University, Faculty of Science, Egypt Aftab Afzal Department of Plant Sciences, Quaid-I-Azam University Islamabad, Pakistan Almas Zaidi Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim, University, Aligarh-202002; U.P., India

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Alvaro Peix Instituto de Recursos Naturales y Agrobiología.IRNASA-CSIC. C/ Cordel de Merinas, 40-52. 37008 Salamanca, Spain Anil Kapri Department of Microbiology, College of Basic Sciences & Humanities, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar-263145, India Arshad Anwer Department of Plant Protection, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh 202 002, India Asghari Bano Department of Plant Sciences, Quaid-I-Azam University Islamabad, Pakistan Babupoojary Sundara Agronomy Section, Division of Crop Production, Sugarcane Breeding Institute, Coimbatore-641007, Tamil Nadu, India

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Contributors

Diriba Muleta College of Agriculture and Veterinary Medicine, P.O.Box 307; Jimma University, Ethiopia Encarna Velázquez Departamento de Microbiología y Genética, Universidad de Salamanca, Spain Eustoquio Martínez-Molina Departamento de Microbiología y Genética, Universidad de Salamanca, Spain. Fayaz A Mohidin Department of Botany, University of Kashmir, Srinagar, India Geeta Singh Division of Microbiology, Indian Agricultural Research Institute, New Delhi-12; India Gopal Reddy International Crops Research Institute for the Semi-Arid Tropics [ICRISAT], Patancheru, India 502 324

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Gorinova, N AgroBioInstitute, Department of Phytoremediation, Dragan Tzankov Blvd. N8, Sofia 1164, Bulgaria Halimi Mohd. Saud Department of Agriculture Technology, Faculty of Agriculture, Universiti Putra Malaysia Serdang, Selangor, Malaysia Hameeda, B Department of Microbiology, Osmania University, Hyderabad 500 007 Harini, G International Crops Research Institute for the Semi-Arid Tropics [ICRISAT], Patancheru, India 502 324 Harshita Negi Department of Microbiology, College of Basic Sciences & Humanities, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar-263145, India Jaharamma, M Department of Biotechnology, Pondicherry University, Kalapet, Puducherry 605014, India

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Jaromir Kubat Research Institute of Crop Production, Drnovska 507, 161 06 Prague, Czech Republic Javier A. Andrés Microbiología Agrícola, Facultad de Agronomía y Veterinaria, Universidad Nacional de Río Cuarto. Campus Universitario. X5804BYA, Río Cuarto, Córdoba, Argentina. Kiran, BK

International Crops Research Institute for the Semi-Arid Tropics [ICRISAT], Patancheru, India 502 324 Kuheli Das Department of Microbiology, College of Basic Sciences & Humanities, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar-263145, India Lorena B. Guiñazú Laboratorio de Interacción Microorganismo - Planta, Facultad de Ciencias Exactas, FísicoQuímicas y Naturales, Universidad Nacional de Río Cuarto. Campus Universitario. X5804BYA, Río Cuarto, Córdoba, Argentina

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Martha-Helena Ramírez-Bahena Departamento de Microbiología y Genética, Universidad de Salamanca, Spain Metin Turan Atatürk University, Faculty of Agriculture, Department of Soil Science, 25240, ErzurumTurkey

Marisa Rovera Laboratorio de Interacción Microorganismo - Planta, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto. Campus Universitario. X5804BYA, Río Cuarto, Córdoba, Argentina Mohammad Saghir Khan Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim, University, Aligarh-202002; U.P., India Mohammad Oves Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh-202002; U.P., India

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Contributors

Mujeebur Rahman Khan Department of Plant Protection, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh 202002, India Munees Ahemad Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh 202002; U.P., India Narayanan, KB Department of Biotechnology, Pondicherry University, Kalapet, Puducherry 605014, India Nedkovska AgroBioInstitute, Department of Phytoremediation, Dragan Tzankov Blvd. N8, Sofia 1164, Bulgaria Nicolás A. Pastor Laboratorio de Interacción Microorganismo - Planta, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto. Campus Universitario. X5804BYA, Río Cuarto, Córdoba, Argentina

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Olga Mikanova Research Institute of Crop Production, Drnovska 507, 161 06 Prague, Czech Republic Parvaze Ahmad Wani Division of Microbiology, Indian Agricultural Research Institute, New Delhi-12; India Radziah, O Department of Land Management, Faculty of Agriculture, Universiti Putra Malaysia Serdang, Selangor, Malaysia Reeta Goel Department of Microbiology, College of Basic Sciences & Humanities, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar-263145, India Rupela, O.P.

International Crops Research Institute for the Semi-Arid Tropics [ICRISAT], Patancheru, India 502 324

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Sakthivel, N Department of Biotechnology, Pondicherry University, Kalapet, Puducherry 605014, India Shahana Altaf Cancer Research Institute, University of California, San Francisco, USA Sindhu, S.S. Department of Microbiology, CCS Haryana Agricultural University, Hisar 125004, Haryana, India Suman Mor Department of Microbiology, CCS Haryana Agricultural University, Hisar 125004, Haryana, India Susana B. ROSAS Laboratorio de Interacción Microorganismo - Planta, Facultad de Ciencias Exactas, Físico Químicas y Naturales, Universidad Nacional de Río Cuarto Campus Universitario. X5804BYA, Río Cuarto, Córdoba, Argentina

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Uzma Khan Department of Plant Protection, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh 202 002, India Verma, M.K. Department of Microbiology, CCS Haryana Agricultural University, Hisar 125004, Haryana, India Zulkifli Haji Shamsuddin Department of Land Management, Faculty of Agriculture, Universiti Putra Malaysia Serdang, Selangor, Malaysia

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DEDICATED TO THE FOND MEMORIES OF OUR FATHERS LATE SYED ZAKIUL HASAN ZAIDI AND LATE MOHAMMAD SALIM KHAN

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In: Phosphate Solubilising Microbes for Crop Improvement ISBN 978-1-60876-112-8 Editors: M. S. Khan and A. Zaidi © 2009 Nova Science Publishers, Inc.

Chapter 1

BIOLOGICAL IMPORTANCE OF PHOSPHORUS AND PHOSPHATE SOLUBILIZING MICROBES - AN OVERVIEW Munees Ahemad, Almas Zaidi, Mohd. Saghir Khan∗ and Mohammad Oves Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh-202002; U.P., India

ABSTRACT Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Phosphorus [P] is a major element and performs vital functions for sustenance, growth and development of plants. It is involved in several key plant functions, including energy transfer, photosynthesis, transformation of sugars and starches, nutrient movement within the plant and transfer of genetic characteristics from one generation to the next. Deficiency of P in soil severely affects the plant metabolism and in turns their yields. In soil, P occurs in organic as well as inorganic forms which exist in different phases and in equilibrium. Due to many factors, it remains unavailable to plants despite its abundance in soil. One of the eco-friendly approaches to provide the soils with optimum concentration of P to overcome the constraints which hinder the uptake of P by plants is the introduction of phosphate-solubilizing microorganisms in agricultural fields. Such phosphate-solubilizing microorganisms convert the unavailable forms of P into one that is easily assimilated by plants and promote the growth of the plants leading to more yield. However, the response of different phosphate-solubilizing microorganisms like, bacteria, fungi and actinomycetes varies from crop to crop and therefore, there is need of exploring the more potent phosphate- solubilizers that may work in diverse agroecological niches.

∗

Correspondence to: [email protected]

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Munees Ahemad, Almas Zaidi, Mohd. Saghir Khan et al.

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1.1. INTRODUCTION Phosphorus [fos'furus = light-bearing], a nonmetallic chemical element, is a component of key molecules such as, nucleic acids, phospholipids, and ATP, and, consequently, plants cannot grow without a reliable supply of this nutrient. Phosphorus is also involved in controlling important enzyme reactions and in the regulation of metabolic pathways [Theodorou and Plaxton, 1993]. Phosphorus is classified as a major nutrient, because it is frequently deficient for crop production and is required by plants in relatively large amounts. The total P concentration in agricultural crops generally varies from 0.1 to 0.5 percent. It can only be assimilated as soluble phosphate species [Illmer and Schinner, 1992] but when deficient, exhibit a significant adverse impact on overall performance of crops and biodiversity [George and Richardson, 2008]. Sub-optimal levels of P, leads to a 5-15% loss in the yield of crops [Hinsinger, 2001]. Therefore, in high input agricultural practices, a larger amount of phosphatic fertilizer is applied to circumvent the P deficiency and consequently to enhance the yield of various crops. Most of the soluble P however, is rapidly immobilized or form a complex with other elements of soils after application and becomes inaccessible to plants. For instance, in the United States, an average 29% of P added in fertilizer and manure is removed by harvesting crops [Sharpley, 2006]. Thus, poor availability of P in soil and consequent P-deficiency represents a major constraint to crop production globally. Therefore, how to improve phosphorus fertilizer efficiency and utilize the potential phosphorus in soil has long been a major concern for field practitioners. This has led to a search for environment-friendly and economically feasible alternative strategies for improving crop production in low or P deficient soils. In this context, organisms endowed with P-solubilizing [PS] activity, often termed phosphate-solubilizing microorganisms [PSM], may provide a viable substitute to chemical P fertilizers. Inoculation of phosphate-solubilizing microorganisms, like, bacteria [Zaidi et al. 2003; Zaidi and Khan, 2007; Wani et al. 2007a; Naik et al. 2008], fungi [Mittal et al. 2008] and actinomycetes [Mba, 1994; Hanane et al. 2008; Barreto et al. 2008] in soil has been shown to improve solubilization of insoluble phosphates resulting in a higher crop yields [Khan et al. 2007; Linu et al. 2009; Hameeda et al. 2008]. In addition to fertilization, mineralization and enzymatic decomposition of organic compounds, solubilization of P by microbes also increase plant available P [Illmer and Schinner, 1992]. The organic acid secreted by such microbes is considered as principal mechanisms of P solubilization. The synthesis of organic acid and their accumulation acidify microbial cells and their surroundings causing the release of P ions from the P mineral by H+ substitution for Ca2+ [Goldstein, 1994]. The acidification by H+ excretion theory was introduced by Illmer and Schinner in 1995 to explain Ca-P solubilization accompanied by a decrease in pH. Recently, Yanmei et al. [2008] suggested that exopolysaccharide [EPS] which has the ability of phosphorus-holding may be a novel factor in the microbial dissolution of tri-calcium phosphates. Attempt by plant nutrition scientists are also directed towards improving the ability of plants to utilize maximum phosphorus and to breed new varieties with high-P efficiency [Raghothama, 2000; Wang et al. 1998]. Soil scientists on the contrary, are searching for a solution to delay the formation of unavailable P or to accelerate the activation of insoluble P. In follow up studies, success has been achieved where addition of organic acid, organic

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Biological Importance of Phosphorus and Phosphate Solubilizing Microbes

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manure and NH4-zeoli obviously accelerated activation and release of accumulated phosphorus in soil [Zhang et al. 1998a; Zhang et al. 1998b; Shao and Zhao, 2002].

1.2. BIOLOGICAL FUNCTIONS OF PHOSPHORUS

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Phosphorus, a mobile element enters the plant through root hairs, root tips, and the outermost layers of root cells. Phosphorus is picked up by plants mostly as the primary orthophosphate ion, but it is also absorbed as secondary orthophosphate. After they enter into plant root, may be stored in the root or translocated to the above ground part of the plants [Schachtman et al. 1998]. The accumulated phosphorus in plants are then incorporated into organic compounds, including nucleic acids [DNA and RNA], phosphoproteins, phospholipids, sugar phosphates, enzymes, and energy-rich phosphate compounds [e.g. adenosine triphosphate [ATP]]. Such organic forms of P or the inorganic phosphate ion then move throughout the plants and affect virtually every plant process [e.g. nutrient transport, photosynthesis etc.] that involves energy transfer. One of the most vital chemical reactions in nature is photosynthesis which utilizes light energy in the presence of chlorophyll to combine CO2 and H2O2 leading to the formation of sugars with subsequent release of the energy [ATP]. This ATP is later on used to drive many other reactions that occur within the plant, while sugars serve as building blocks to generate other cell structural and storage components. Phosphorus is also a vital component of genes and chromosomes, and thus help in transfer of genetic information from one generation to the next, providing the “blueprint” for all aspects of plant growth and development. Moreover, P is also required for seed formation and development; an inadequate supply of P can reduce seed size, seed number, and viability.

1.2.1. Phosphorus Deficiency When P concentration in plants is sufficient, it allows the plants to operate at optimum rates and growth and development of the plant to proceed at a normal pace. However, when P is inadequate or deficient, it reduces [i] leaf expansion [ii] leaf surface area [iii] number of leaves [iv] root and shoot growth [v] premature senescence of leaves [vi] delay blooming and maturity [vii] delay flowering [viii] reduce quality of forage, fruit, vegetable, and grain crops and [ix] decrease disease resistance. The deficiency of P in plants in general, is manifested in terms of stunted growth, reduced yield and delayed maturity [Sawyer and Creswell, 2000]. Furthermore, the deficiency of P slows the processes of carbohydrate utilization, while carbohydrate production through photosynthesis continues. This in turn, results in a build-up of carbohydrates and the development of a dark green leaf color. In some plants [e.g., tomatoes and corn, canola] P-deficient leaves develop a purple color or leaves may become a dark bluish green [Figure 1]

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Figure 1. Phosphorus deficiency symptoms on sugarcane [Plate A], alfalfa [Plate B], potato [Plate C] and grapes [Plate D]. Adapted from LSU Agricultural Centre Louisiana Cooperative Extension Service [Better Crops/Vol. 83: 1999, No. 1].

1.3. PHOSPHORUS IN THE SOIL SYSTEM AND ITS AVAILABILITY TO PLANTS Phosphorus occurs in soil both in organic and inorganic forms and each form has a variety of P compounds which exist in different phases and in equilibrium with each other. Principally, on the basis of the availability of P to plants, the different forms of P can be categorized as soluble P and insoluble P. The soluble form is easily available for uptake by plants. In contrast, insoluble form is very stable in soil and persists in unavailable form. There is a dynamic and complex relationship among the different forms of P involving soils, plants and variety of microorganisms [Figure 2]. Organic P compounds are found in humus and other organic materials including decayed plant, animal and microbial tissues. Organic P is also the major form of P in manure. Generally, organic P is combined with oxygen to form ester compounds [Thompson and Troch, 1978]. These esters make up about 50 to 70% of identified organic P. In the Chernozemic top soils in western Canada, organic P was estimated at 25 to 55%, which could be available for plant growth after mineralization [Stewart et al. 1980].

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Figure 2. Phosphorus cycle presenting different forms of P and their interrelationships.

Phosphorus in labile organic compounds can be slowly mineralized [broken down and released] as available inorganic phosphate or it can be immobilized [incorporated into more stable organic materials] as part of the soil organic matter [Tate, 1984; Mckenzie and Roberts, 1990]. The process of mineralization or immobilization is carried out by microorganisms and is highly influenced by soil moisture and temperature. Mineralization and immobilization are most rapid in warm, well-drained soils [Busman et al. 2002]. Approximately 70 to 80% of P found in cultivated soils is inorganic [Foth, 1990]. Phosphorus fertilizers are the main input of inorganic P in agriculture soils. Despite its wide application, after N, P is the major nutrient limiting plant growth [Fernandez, et al., 2007]. Worldwide, 5.7 billion hectares contain too little available P for sustaining optimal crop production [Hinsinger, 2001]. Phosphorus ion concentration in most soils ranges from 0.1 to 10 μM; P required for optimal growth ranges from 1 to 5 μM for grasses and 5 to 60 μM for high demanding crops such as, tomato [Lycopersicon lycopersicum] and pea [Pisum sativum] [Raghothama, 1999; Hinsinger, 2001]. Plant available P in 29 southern Alberta soils was approximately 1% of total soil P [Kucey, 1983] [Figure 3]. Phosphorus in fertilizers is converted to water-soluble Pi as orthophosphate ions H2PO4and HPO42- in soil within a few hours after application [Schulte and Kelling, 1996]. As the fertilizer enters the soil, moisture from the soil begins to dissolve the fertilizer particles. The concentration of Pi in solution increases around the dissolved fertilizer particles and diffuses at short distance from the fertilizer particles [Busman et al., 2002]. In most soils, orthophosphate ions H2PO4- and HPO42- dominate at pH below 7 and above 7.2, respectively [Hinsinger, 2001]. These negatively charged P ions attach strongly to the surfaces of minerals containing positively charged ions such as, iron [Fe3+] and aluminum [Al3+] in acidic soils via sorption/desorption processes. The Fe3+ and Al3+ acts as the sorption sites for the negatively charged P [Sato and Comerford, 2005]. These P anions also precipitate with the calcium [Ca2+] in calcium carbonate minerals in calcareous soils forming relatively insoluble compounds. Both processes result in P being fixed or bound, thus removed from the soil solution and unavailable for plants [Foth, 1990; Schulte and Kelling, 1996]. The conversion from stable P to labile P is a slow process and does not occur over the course of one growing

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season [Guo and Yost, 1998]. However, the conversion from labile P to plant available P is a rapid process [Tate and Salcedo, 1988].

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Figure 3. Plant acquisition of soil P [adapted from Schachtman et al. 1998].

In most soils, maximum P availability occurs between pH 5.5 to 7. Within this pH range, P is fixed by hydrous oxides of Fe, Al, and Mn. Between pH 6 to 8 and pH 6.5 to 8.5, P is fixed by silicate minerals and Ca, respectively. As a result, the most efficient use of P in neutral and calcareous soils occurs between pH 6 to 7 [Sharpley, 2006]. With high levels of exchangeable Ca, available P ions react with solid phase CaCO3 and precipitate on the surface of these particles to form Ca-P minerals: Ca[H2PO4]2 [monocalcium P], CaHPO4.2H2O [dicalcium phosphate ehydrate, DCPD, brushite], CaHPO4 [dicalcium phosphate, DCP, monetite], Ca3[PO4]2 [tri-calcium phosphate, TCP], Ca4H[PO4]32.5H2O [octacalcium P, OCP], Ca5[PO4]3.OH [hydroxyapatite] and least soluble apatites. The finer is the size of solid phase CaCO3 the higher the fixation of P. The solubility of Ca-P minerals is in the order: DCPD > DCP > TCP > hydroxyapatite. In alkaline soils, the initial products of reaction of fertilizer triple superphosphate are mainly DCPD and DCP [Russell, 1980; Whitelaw et al. 1999]. Different phases of Ca-P compounds are transferable and, at a given pH, can be dissolved from unstable phases to become precipitated as stable phases. For example, a relatively soluble brushite when applied as fertilizer to calcareous soils was transformed to monetite and slowly to octacalcium P, which in turn, became stable for years if fertilizer was applied continually leading to the formation of hydroxyapatite [Sposito, 1989]. Soil solution Pi concentration increases when water soluble P fertilizer applied to soil is readily dissolved. Over time, the soil fixes P by processes such as precipitation, thereby reducing its concentration in the soil solution. As a result, Pi in the soil solution is general low. The Pi content is usually greater at surface horizons than in sub-soils due to its immobility. The Pi accumulation in topsoil can be a problem especially in a reduced tillage system because of minimal or no mechanical incorporation when fertilizer is applied [Sharpley, 2006].

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1.4. EFFECTS OF COMPOST AND FERTILIZER ON SOIL PHOSPHORUS The application of compost, chemical fertilizers, or combination of both over a period of time builds up total and available phosphorus in soil. Available P level in soil was related to total P and P input. The high level of total P in soil also increased fixed forms of P with Fe, Al and Ca. Continuous application of compost and chemical fertilizers increased inorganic P in fractions more than organic P in the soil. Excessive application of P to soil could increase P fixation, largely in form of Fe-P in paddy soil. The proportion of organic P in total P however, declines with the application of compost and chemical fertilizers. For example, long-term annual use of compost and chemical fertilizers has shown accumulation of P in soil. Although the P remaining in soil can be available to succeeding crops, yet the long-term effects of P surplus would lead to a gradual saturation of the P-sorption capacity and might cause the P loss arising from such soil and in turn contaminate water with P [Park et al. 2004]. Therefore, P management is important to protect the natural environments. In this regard, phosphate-solubilizing microorganisms may be an answer for maintaining the supply of plant available P as these organisms bring about the conversion from labile P to plant available P.

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1.5. PLANT AVAILABLE PHOSPHORUS IN THE SOIL The term available-P, also referred to as labile P is often used to express the amount of soil P in solution which can be absorbed or assimilated and utilized by plants for growth and development during its life cycle. It is required continuously by plants, and hence, its concentration in soil decreases. This is further compounded by the slow replenishment of the extracted P from the soil solution by the labile pool which is dictated by the soil P equilibria [Holford, 1997]. This is however, supplied by phosphatic fertilizers or manure. The concentration of available-P pool is greatly influenced by the soil conditions and the capabibility of the plants to take up P from the soil solution [Raven and Hossner, 1993; Holford, 1997]. Phosphorus is released at a faster rate from the labile pool into the soil solution at lower buffering capacity. In a study, Holford [1997] reported three important soil components controlling the supply of P from the labile pool to replenish crop extraction. These include the amount or concentration of P in the soil solution; the amount of P in the replenishment source that enters into equilibrium with the soil solution phase and P buffering capacity of the soil.

1.6. BIOLOGICAL FACTORS AFFECTING AVAILABILITY OF PHOSPHORUS Soil contains low molecular weight organic acids, such as, citrate, oxalate, acetate, malate, isocitrate and tartrate, which have one or more carboxylic groups. Such organic acids are released by plant root exudates and microorganisms; they can also be products of the degradation of complex organic molecules [Jones, 1998; Geelhoed et al. 1999; Jones et al.

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2003; Yadav and Tarafdar, 2003]. The adsorption of organic acids onto soil surface produces charge, allowing metal cation complexation in solution and anion displacement from the soil matrix [Jones, 1998]. Organic acids have been found to be involved in nutrient mobilization, mainly utilization of insoluble P [Bolan et al. 1994; Ström et al. 2002] with P adsorption decreasing due to competition for active sites for the adsorption and dissolution of light insoluble P compounds [Geelhoed et al. 1999; Jones et al. 2003]. However, released P depends on factors such as solution pH, organic acid characteristics and soil mineralogy [Bolan et al., 1994; Ström et al. 2002]. Other factors affecting the availability of P in soil include enzymes which catalyze numerous biological reactions and are of great agronomic and ecological value. They are released into soil by microorganisms and plant root exudates, participate in the formation and degradation of molecules and contribute to nutrient cycling. Of the various enzymes, phosphatase [includes phytases, acid and alkaline phosphatases, phospholipase and pyrophosphatase], an extra-cellular enzyme catalyzes the hydrolysis of both phosphate ester bonds and anhydride bonds allowing orthophosphate to be released from organic and inorganic compounds, thereby increasing the bioavailable P [Thien and Myers, 1992; Deng and Tabatabai, 1997; Rao et al. 2000]. Acid phosphatase and alkaline phosphatase are orthophosphoric monoester phosphohydrolases [Deng and Tabatabai, 1997; Pant and Warman, 2000] that hydrolyze orthophosphate monoesters while phospholipase and nuclease are orthophosphoric diester phosphohydrolases [Deng and Tabatabai, 1997; Pant and Warman, 2000] that hydrolyze compound type orthophosphate diesters. Inorganic pyrophosphatases act on acid anhydride bonds in phosphoryl-containing anhydrides [Deng and Tabatabai, 1997]. Many microorganisms like members of basidiomycetes [Goud et al. 2008] and bacteria [Bursík and Němec, 1999] have been reported to produce phosphatases.

1.7. AGRICULTURAL IMPORTANCE OF PHOSPHATE SOLUBILIZING BACTERIA The inoculation of phosphate-solubilizing microorganisms has revealed that such microbes not only improve the availability of soluble P to plants but also facilitate the biomass and yield of different agronomically important crops by other mechanisms [Wani et al. 2007a, Wani et al. 2007b]. For instance, In a study, Poonguzhali et al. [2008] reported that phosphate-solubilizing strains of Pseudomonas capable of synthesizing indole-3-acetic acid [IAA], 1-aminocyclopropane-1-carboxylate [ACC] deaminase, and siderophores, substantially enhanced the root length and biomass of Chinese cabbage [Brassica rapa] without affecting P uptake. This study suggested that plant growth promotion by phosphatesolubilizing bacteria could be due to phytohormones or mechanisms other than Psolubilization. In a follow up studies, cold-tolerant phosphate-solubilizing Serratia marcescens with inherent plant growth facilitating activities, such as, IAA, HCN and siderophore, substantially increased dry matter accumulation and nutrient uptake of wheat [Triticum aestivum] seedlings grown in cold temperatures [Selvakumar et al., 2008]. Likewise, Chen et al. [2008] demonstrated that phosphate-solubilizing bacterium strain 9320SD had a significant effect not only on plant biomass but also on total P in winter wheat under both pot and field conditions, although no effect on plant height was found compared to the control. Furthermore, to consolidate the hypothesis that P-solubilizing- microbes improve

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the growth of plants by mechanisms other than phosphate solubilization, a series of experiments was conducted recently. For example, phosphate-solubilizing and hexavalent chromium resistant strains of Pseudomonas sp. PsA4 and Bacillus sp. Ba32 [recovered from heavy metal contaminated soils] were tested to assess their impact on the performance of Indian mustard [Brassica juncea] grown in soil treated with different concentrations of toxic hexavalent chromium [Rajkumar et al. 2006]. Inoculation of both strains promoted the growth of plants even at 95.3 and 198.3 µg of Cr6+ g-1 soil suggesting that the strains PsA4 and Ba32 protected the plants not only against the inhibitory effects of carcinogenic and mutagenic chromium but also synthesized IAA and siderophores in addition to their inherent Psolubilizing activities. All these factors together might have accounted for the better performance of canola when grown in chromium contaminated soils. In a similar study, cadmium-resistant phosphate-solubilizing Pseudomonas aeruginosa has shown to reduce cadmium toxicity and subsequently improved the productivity of chickpea [Cicer arietinum] plants grown in soil treated with different concentration of CdCl2 [Ganesan 2008]. However, the effect of phosphate-solubilizing microbes as inoculant is variable, and depends upon different factors like, soil type, physico-chemical properties of soils, plant genotypes, type and concentrations of root exudates and inherent P-solubilizing efficiency of microbes [Khan et al. 2007]. For instance, Linu et al. [2009] reported that seed inoculation of cowpea [Vigna sinensis] by phosphate solubilizers Gluconactobacter sp. and Burkholderia sp. improved nodulation, root and shoot biomass, straw and grain yield and P and N uptake of the crop. The dehydrogenase, phosphatase and the available P contents of the soil were stimulated by the inoculation with the phosphate- solubilizing bacteria. Among the bacterial strains, the best effect on yield was obtained with Burkholderia sp. Recently, an increase of 99% [under glasshouse] and 66% [under field conditions] following application of two strains of Serratia marcescens and Pseudomonas sp. in biomass and 85% in grain yield of maize [Zea mays] is reported [Hameeda et al. 2008]. The agronomic impact of phosphatesolubilizers, like, Burkholderia sp., Enterobacter sp., and Bradyrhizobium sp., on biological and chemical properties of soybean [Glycine max] under greenhouse conditions was suggested to be not due to the improvement in P nutrition in soybean but by unknown mechanisms [Fernándezet et al. 2007]. However, they concluded that the selection of efficient phosphate-solubilizing strains as possible inoculation tools for P-deficient soils should focus on the integral interpretation of soil plate assays, greenhouse experiments, and field trials. The phosphate-solubilizing microorganisms can also be applied simultaneously in order to improve the yield efficiency of various crops. For example, when two phosphatesolubilizing stains Pseudomonas fluorescens and P. fluorescens were used together along with different rates of N, P, and K [at 0%, 25%, 50%, 75%, and 100% of recommended doses] enhanced the growth and yield of wheat both under pot and field trials. This finding concluded that Pseudomonads could be applied in combination with appropriate doses of fertilizers for better plant growth and savings of fertilizers [Shaharoona et al. 2008]. Like the co-culturing of phosphate-solubilizing bacteria, phosphate-solubilizing fungi can also be used either alone or in combination for improvement of crops under various agro-ecosystems. For example, Penicillium albidum when used alone increased the growth of the red clover [Trifolium pratense L.] by inducing root development and enhancing phosphate mobilization from the soil into the plant [Morales et al. [2007]. Similarly the composite inoculation of Tilemsi phosphate rock solubilizing fungi Aspergillus awamori and Penicillium chrysogenum increased the root dry matter yields of wheat by 60% [Babana and Antoun [2005] as also

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observed for dual inoculation of phosphate-solubilizing fungi Aspergillus awamori, and Penicillium citrinum on the growth and seed production of chickpea plants [Mittal et al. 2008]. Following such success, Wakelin et al. [2007] assessed the impact of triple combinations of phosphate-solubilizing fungi namely, Penicillium radicum, Penicillium bilaiae [strain RS7B-SD1], and an unidentified Penicillium sp. [strain KC6-W2] on the growth and P nutrition of wheat and medic lentil [Lens culinaris] grown at neutral to alkaline soil pH. Following inoculation, a dramatic increase in above ground growth, dry mass, concentration of P in shoots and yields of wheat and lentil was recorded. These and other studies, suggest that consortia of either phosphate-solubilizing bacteria alone, or phosphatesolubilizing fungi alone, or phosphate-solubilizing bacteria with phosphate-solubilizing fungi or phosphate-solubilizing microbes with other plant growth promoting rhizobacteria [PGPR] or arbuscular mycorhhizal fungi [Zaidi et al. 2003; Zaidi and Khan 2007] can be very effective in promoting the biological and chemical properties of crops under different agroecological niches.

CONCLUSION

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Despite various forms of P in soil, P uptake by plants is not carried out completely due to many biological, physical and chemical factors of soils. Phosphate-solubilizing microorganisms might play an important and feasible role in increasing the availability of labile P without affecting the soil fertility and its biochemical composition. There has been a tremendous research on plant mineral nutrition applying different techniques to find out the mechanisms of nutrient acquisition, transportation and utilization. The challenge now is to develop different strategies to explore phosphate-solubilizing bacteria that may work in agronomically different soils and climate efficiently and therefore, paving way to economically and ecologically sound and sustainable agriculture.

REFERENCES Babana, A; Antoun, H. Biological system for improving the availability of Tilemsi phosphate rock for wheat [Triticum aestivum L.] cultivated in Mali. Nutrient Cycling in Agro ecosystems, [2005], 72, 147-157. Barreto, TR; da Silva, ACM; Soares, ACF; de Souza, JT. Population densities and genetic diversity of actinomycetes associated to the rhizosphere of Theobroma cacao. Braz J Microbiol, [2008], 39, 464-470. Bolan, NS; Naidu, R; Mahimairaja, S; Baskaran, S. Influence of low-molecular-weight organic acid on the solubilization of phosphates. Biol. Fertil. Soils., [1994], 18, 311-319. Bursík, M; Němec, M. Alkaline phosphatase production during sporulation of Bacillus cereus. Folia Microbiologica, [1999], 44, 90-92. Busman, L; Lamb, J; Randall G; Rehm, G; Schmitt, M. The nature of phosphorus in soils. [2002]. University of Minnesota Extension Service, Available at http://www.extension.umn.edu/distributution/cropsystems/DC6795.html.

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Chen, Z; Ma, S; Liu, LL. Studies on phosphorus solubilizing activity of a strain of phosphobacteria isolated from chestnut type soil in China. Bioresour Technol. [2008], 99, 6702-6707. Deng, SP; Tabatabai, MA. Effect of tillage and residue management on enzyme activities in soils:III.Phosphatases and arylsulfatase. Biol. Fertil Soils., [1997], 24, 141-146. Fernández, LA; Zalba, P; Gómez, MA; Sagardoy, MA. Phosphate-solubilization activity of bacterial strains in soil and their effect on soybean growth under greenhouse conditions. Biol. Fertil. Soils, [2007], 43, 805-809. Foth, HD. Fundamentals of Soil Science. 8th edition, New York: John Wiley and Sons NY; [1990]. Ganesan, V. Rhizoremediation of cadmium soil using a cadmium-resistant plant growthpromoting rhizopseudomonad. Curr. Microbiol. [2008], 56, 403-407. Geelhoed, JS; Van Riemsdijk, WH; Findenegg, GR. Simulation of the effect of citrate exudation from roots on the plant availability of phosphate adsorbed on goethite. Eur. J. Soil Sci., [1999], 50, 379-390. George, TS; Richardson, AE. Potential and limitations to improving crops for enhanced phosphorus utilization. In: George TS, Richardson AE, editors. The Ecophysiology of Plant-Phosphorus Interactions. Netherlands: Springer; [2008], 247-270. Goldstein, AH. Involvement of the quinoprotein glucose dehydrogenase in the solubilization of exogenous phosphates by gram-negative bacteria. In: Torriani-Gorini A, Yagil E, Silver S, editors. Phosphate in microorganisms: cellular and molecular biology. Washington, DC: ASM Press; [1994], 197-203. Goud, MJP; Goud, JVS; Charya MAS. Replica plate screening method for detecting phosphatase activity in basidiomycetes using 1-napthyl phosphate as a chromogenic substrate. Sci. World J., [2008], 3, 13-15. Guo, F; Yost, RS. Partitioning soil phosphorus into three discrete pools of differing availability. Soil Sci., [1998], 163, 822-833. Hameeda, B; Harini, G; Rupela, OP; Wani, SP; Reddy, G. Growth promotion of maize by phosphate-solubilizing bacteria isolated from composts and macrofauna. Microbiol Res., [2008], 163, 234-242. Hanane, H; Brahim, B; Mohamed, H; Ahmed , L; Marie JV; Yedir, O. Screening for rock phosphate solubilizing Actinomycetes from Moroccan phosphate mines. Agric Ecosys Environ, [2008], 38, 12-19. Hinsinger, P. Bioavailability of soil inorganic P in the rhizosphere as affected by root-induced chemical changes: a review. Plant Soil, 2001, 237, 173-195. Holford, JCR. Soil phosphorus: its measurement, and its uptake by plants. Aust. J. Soil Res., [1997], 35, 227-239. Illmer, P; Schinner, F. Solubilisation of inorganic phosphates by microorganisms isolated from forest soils. Soil Biol. Biochem., [1992], 24, 389-395. Jones, DL. Organic acid in the rhizosphere- a critical review. Plant Soil, [1998, 205, 25-44. Jones, DL; Dennis, PG; Owen, AG; Van Hees, PAW. Organic acid behavior in soilsmisconceptions and knowledge gaps. Plant and Soil, [2003], 248, 31-41. Kucey, RMN. Phosphate-solubilizing bacteria and fungi in various cultivated and virgin Alberta soils. Can. J. Soil Sci., [1983], 63, 671-678. Lindsay, WL. Chemical Equilibrium in Soils. New York: Wiley-Interscience, NY; [1979].

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Linu, MS; Stephen, J; Jisha, MS. Phosphate solubilizing Gluconacetobacter sp., Burkholderia sp. and their potential interaction with cowpea [Vigna unguiculata [L.] Walp.]. Int. J. Agric. Sci., [2009], 4, 79-87. Mba, CC. Field studies on two rock phosphate solubilizing actinomycete isolates as biofertilizer sources. Environ. Mgmt., [1994], 18, 263-269. McKenzie, RH; Roberts TL. Soil and fertilizers phosphorus update. In: Proc. Alberta Soil Science Workshop Proceedings, Edmonton, Alberta. Feb. 20-22, [1990], 84-104. Mittal, V; Singh, O; Nayyar, H; Kaur, J; Tewari, R. Stimulatory effect of phosphatesolubilizing fungal strains [Aspergillus awamori and Penicillium citrinum] on the yield of chickpea [Cicer arietinum L. cv. GPF2]. Soil Biol. Biochem., [2008], 40, 718-727. Morales, A; Alvear, M; Valenzuela, E; Rubio, R; Borie, F. Effect of inoculation with Penicillium albidum, a phosphate-solubilizing fungus, on the growth of Trifolium pratense cropped in a volcanic soil. J Basic Microbiol., [2007], 47, 275-280. Naik, PR; Raman,G; Narayanan,KB; Sakthivel N.Assessment of genetic and functional diversity of phosphate solubilizing fluorescent pseudomonads isolated from rhizospheric soil. BMC Microbiol. [2008], 8, 230. Pant, HK; Warman, PR. Enzymatic hydrolysis of soil organic phosphorus by immobilized phosphatases. Boil. Fertil Soils., [2000], 30, 306-311. Park, M; Singvilay, O; Shin, W; Kim, E; Chung, J; Sa, T. Effects of long-term compost and fertilizer application on soil phosphorus status under paddy cropping system. Comm. Soil Sci. Pl. Anal., [2004], 35, 1635-1644. Poonguzhali, S; Madhaiyan, M; Sa, T. Isolation and identification of phosphate solubilizing bacteria from chinese cabbage and their effect on growth and phosphorus utilization of plants. J Microbiol Biotechnol., [2008], 18, 773-777. Raghothama, KG. Phosphate acquisition. Ann. Rev. Plant Physiol. Mol. Biol., [1999], 50, 665-693. Raghothama, KG. Phosphorus acquisition: plant in the driver’s seat. Trends Plant Sci., [2000], 5, 412–413. Rajkumar, M; Nagendran, R; Lee, K J; Lee, WH; Kim, SZ. Influence of plant growth promoting bacteria and Cr6+ on the growth of Indian mustard. Chemosphere, [2006], 62, 741-748. Rao, MA; Violante, A; Gianfreda, L. Interaction of acid phosphatase with clays, organic molecules and organo-mineral complexes: kinetic and stability. Soil Biol. Biochem., [2000], 32, 1007-1014. Raven, KP; Hossner, LR. Phosphate desorption quantity-intensity relationships in soil. Soil Sci. Soc. Am. J., [1993], 57, 1505-1508. Russell, EW. Soil conditions and plant growth. 10th edition. London: Longman; [1980]. Sato, S; Comerford,NB. Influence of soil pH on inorganic phosphorus sorption and desorption in a humid Brazilian Ultisol. Rev. Bras. Cienc. Solo., [2005], 29. at http://www.scielo.br/ Sawyer, J; Creswell, J. Integrated crop management. In Phosphorus basics. Iowa: Iowa State University, Ames; [2000]; 182-183. Schachtman, DP; Reid RJ; Ayling, SM. Phosphorus uptake by plants: From Soil to Cell. Plant Physiol., [1998], 116, 447-453.

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Schulte, EE; Kelling, KA. Soil and applied phosphorus: Undersatand plant nutrient. Wisconsin: University of Wisconsin Extension, University of Wisconsin, Madison; [1996]. Selvakumar, G; Mohan, M; Kundu, S; Gupta, AD; Joshi, P; Nazim, S; Gupta, HS. Cold tolerance and plant growth promotion potential of Serratia marcescens strain SRM [MTCC 8708] isolated from flowers of summer squash [Cucurbita pepo]. Lett. Appl. Microbiol., [2008], 46, 171-175. Shaharoona, B; Naveed, M; Arshad, M; Zahir, ZA. Fertilizer-dependent efficiency of Pseudomonads for improving growth, yield, and nutrient use efficiency of wheat [Triticum aestivum L.] Appl. Microbiol. Biotechnol, [2008], 79, 147-155. Sharpley, A. Agricultural phosphorus management: Protecting production and water quality. Agricultural Phosphate Management: Protecting Production and Water Quality Lesson 34. USDA-Agricultural Research Service, MidWest Plant Service. Iowa State Iowa. University, Ames, [2006]. at http:/www.lpes.org/Lessons/Lessons34/34-PhosphorusManagement.html Sposito, G. The chemistry of soils. New York: Oxford University Press, NY; [1989]. Stevenson, FJ. Cycles of soil, carbon, nitrogen, phosphorus, sulfur, micronutrients. New York: John Wiley; [1986]. Stewart, JBW; Hedley, MJ; Chauhan, BS. The immobilization, mineralization and distribution of phosphorous in soils. In Western Canada phosphate symposium-Proc. Alberta Soil Science Workshop, Edmonton, AB, [1980], 276-306. Ström, L; Owen, AG; Godbold, DL; Jones, DL. Organic acid mediated P mobilization in the rhizosphere and uptake by maize roots. Soil Biol. Biochem., [2002], 34, 703-710. Taalab, AS; Badr, MA. Phosphorus availability from compacted rock phosphate with nitrogen to sorghum Inoculated with phospho-bacterium. J. Appl. Sci. Res., [2007], 3, 195-201. Tate, KR. The biological transformation of P in the soil. Plant Soil, [1984], 76, 245-256. Tate, KR; Salcedo, I. Phosphorus control of soil organic matter accumulation and cycling. Biogeochem., [1988], 5, 99-107. Theodorou, ME; Plaxton, WC. Metabolic adaptations of plant respiration to nutritional phosphate deprivation. Plant Physiol, [1993], 101: 339-344. Thien, SJ; Myers, R. Determination of bioavailable phosphorus in soil. Soil. Sci. Soc. Am. J., [1992], 56, 814- 818. Thompson, LM; Troch FR. Soils and soil fertility. 4th edition. New York: McGraw-Hill Inc., NY; [1978]. Wakelin, SA; Gupta, VVSR.; Harvey, PR.; Ryder, MH. The effect of Penicillium fungi on plant growth and phosphorus mobilization in neutral to alkaline soils from southern Australia. Canadian J. Microbiol., [2007], 53, 106-115. Wang, QR; Li, JY; Li, ZS. Dynamics and prospect on study of high acquisition of soil unavailable phosphorus by plants. Plant Nutr. Fertil. Sci. [in Chinese], [1998], 4, 107116. Wani, PA; Khan, MS; Zaidi, A. Chromium reduction, plant growth promoting potentials and metal solubilization by Bacillus sp. isolated from alluvial soil. Current Microbiol., [2007b], 54, 237–243.

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Wani, PA; Khan, MS; Zaidi, A. Synergistic effects of the inoculation with nitrogen fixing and phosphate-solubilizing rhizobacteria on the performance of field grown chickpea. J. Plant Nutr. Soil Sci., [2007a], 170, 283-287. Whitelaw, MA; Harden, TJ; Helyar, KR. Phosphate solubilization in solution culture by the soil fungus Penicillium radicum. Soil Biol. Biochem., [1999], 31, 655-665. Yadav, RS; Tarafdar, JC. Phytase and phosphatase producing fungi in arid and semi-arid soils and their efficiency in hydrolyzing different organic P compounds. Soil Biol. Biochem., [2003], 35, 1-7. Zaidi, A; Khan, MS. Stimulatory effects of dual inoculation with phosphate solubilizing microorganisms and arbuscular mycorrhizal fungus on chickpea. Aus. J. Expt. Agri., [2007], 47, 1016-1022. Zaidi, A; Khan, MS; Amil, M. Interactive effect of rhizotrophic microorganisms on yield and nutrient uptake of chickpea [Cicer arietinum L.]. Eur. J. Agron., [2003], 19, 15-21. Zhang, YS; Lin, XY; Luo, AC. Studies on activation of phosphorus by organic manure in soils and its mechanisms, I. Organic acids from decomposition of organic manure [matter] and their effect on activation to different artificial phosphate. Plant Nutr. Fertil. Sci. [in Chinese], [1998a], 4, 151-155. Zhang, YS; Lin, XY; Luo, AC; Su, L. Effect of organic manure [matter] on activation to different phosphate in soils. Plant Nutr. Fertil. Sci. [in Chinese], [1998b], 4, 145-150.

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Chapter 2

NOVEL APPROACHES FOR ANALYSIS OF BIODIVERSITY OF PHOSPHATE-SOLUBILIZING BACTERIA Martha-Helena Ramírez-Bahena1, Alvaro Peix∗2, Eustoquio Martínez-Molina1 and Encarna Velázquez1 1

Departamento de Microbiología y Genética, Universidad de Salamanca, Spain 2 Instituto de Recursos Naturales y Agrobiología. IRNASA-CSIC. C/ Cordel de Merinas, 40-52. 37008 Salamanca, Spain

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Phosphorus is, after nitrogen, the second most important limiting nutrient factor for plant growth and biological development. Only 1-5% of the total phosphorus present in soil is available for plants, and thus processes involved in P cycling and transformations in soil and environment deserve interest and scientific attention. Microorganisms are known to play a central role in P cycling, and some of them are able to solubilize inorganic phosphate or mobilize organic P, for which they have been studied as PGPR to be used in agriculture or water bioremediation. For many years the studies on diversity of phosphate-solubilizing bacteria [PSB] have been mainly performed through isolation and analysis of culturable inhabitants of soil and water. During the last 20 years, an enormous advancement in the study of biodiversity and ecology of microbial communities has been achieved thanks to the development of new techniques, such as culture-independent molecular approaches involving fingerprinting of DNA directly extracted from soil, water or plant tissues [metagenomics], the analyses of RNA transcripts [transcriptomics], proteins [proteomics] or metabolites [metabolomics]. Also, methods for studying microoganisms “in vivo”, such as, fluorescent labelling, GFP, FISH, laser scanning confocal microscopy, etc. have led to the discovery that microbial diversity is much higher than ever thought, and surely a great diversity of phosphate-solubilizing bacteria remains still unknown. This chapter aims to give an overview of novel approaches

∗

Corresponding author: [email protected]

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Martha-Helena Ramírez-Bahena, Alvaro Peix, Eustoquio Martínez-Molina et al. currently developed which are expected to provide useful information on PSB diversity in different agro-ecological niches.

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2.1. INTRODUCTION Microbial biodiversity provides the foundation for biotechnology, being the base for new product discovery, such as, new drugs for disease treatment, purification of water, bioremediation, development of new industrial processes, etc. The advances achieved in molecular biology have allowed unravelling previously unforeseen applications of biotechnology and hence, has given more value to the microbial genetic diversity. A basic requisite of biotechnology is the genetic characterization of biodiversity of species. The total number of existing bacterial species on earth is unknown, we only know less than 9,000 species of bacteria [including cyanobacteria], but attempts to estimate bacterial diversity have ranged from 107 to 109 total species and even these diverse estimates may be off by many orders of magnitude. Most bacteria have not been characterized yet because only a few species from the total populations have been successfully cultured in the laboratory which represents a small fraction of the prokaryotic biota. It is important to emphasize that bacteria are essential in recycling nutrients, and many important steps in nutrient cycles such as, fixation of nitrogen from the atmosphere and mobilization of nutrients, depend on these organisms. Within the great diversity of bacterial groups involved in these proccesses, the phosphate-solubilizing bacteria [PSB] have a great practical value to agriculture since they play an important role in plant P-uptake and growth promotion [Chabot et al., 1996; Peix et al., 2001; Vassilev et al., 2006; Ehteshami et al., 2007; Khan et al., 2007; Hameeda et al., 2008; Poonguzhali et al., 2008; Chan and Yang, 2009; Gulati et al., 2009; Peix et al., 2009] and bioremediation [Kim et al., 2005; Jiang et al., 2008; Rajkumar et al., 2008]. Within prokaryotes, pseudomonads, bacilli and rhizobia have been considered as the main groups of PSB [Rodríguez and Fraga, 1999], however, it is necessary to take into account that with the reclassification of some species of genus Pseudomonas into the genus Burkholderia [Yabuuchii et al., 1992] some phosphatesolubilizing species such as, B. cepacia [formerly Pseudomonas cepacia] currently belong to this genus [Lin et al., 2006]. Moreover, phosphate-solubilizing ability has recently been reported among bacteria belonging to the genera Rhodococcus, Arthrobacter, Serratia, Chryseobacterium, Delftia, Gordonia, Swaminathania, Phyllobacterium, Pantoea, Enterobacter, Streptomyces and Microbacterium [Kim et al., 2003; Loganathan and Nair, 2004; Rivas et al., 2004; Kämpfer, 2007; Sahu et al., 2007]. In addition, Acinetobacter strains have been found to show ability to remove phosphate from water and wastewater [Mino, 2000; Blackall et al., 2002] and soil strains have been reported as phosphate solubilizers [Kang et al., 2009; Gulati et al., 2009; Peix et al., in press]. However, we have just started to explore its diversity, and we begin to understand their ecological features. The intrinsic complexity of PSB groups, populations and communities and the habitats where they are present make necessary to have reliable methodology to study their diversity, aiming to analyze microbial population,s structure and dynamics. This chapter provides an overview of some of the novel approaches and methodologies that are currently being applied to assess the biodiversity of microbial communities in the environment, which have been applied or can be applicable to the study of phosphate-solubilizing bacterial populations. In addition, attention

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will be paid to address those approaches which are based only on nucleic acid studies, but there are many more culture dependent and independent approaches for this purpose within chemotaxonomy, proteomics, metabolomics, etc. which have been widely reviewed elsewhere.

2.2. MOLECULAR METHODS FOR ANALYSIS OF MICROORGANISMS: THE GENOMICS ERA

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Microbial communities can be characterized based on fundamental properties encompassing diversity, identity and quantity. Although most of the molecular techniques currently used for microbial community analyses reveal a considerable view on the community, none of them presents an integral view. To assess bacterial diversity, the biochemical characteristics have been the primary tool for a long time, but these techniques reflect only a very small subset of those characters that allow bacteria to use different resources in the environment, and only show a little fraction of the true existent diversity in this superkingdom of life. More recently, molecular methods, particularly DNA-DNA hybridization [Wayne et al. 1987, Stackebrandt, et al., 2002] and several PCR and sequencing-based methods have helped to study its diversity. rRNA sequence surveys, particularly those based on 16S rRNA gene allow the classification of bacteria [Woese et al., 1984], revealing the extraordinary variety of microbial life, much of it uncultured [Tyson and Banfield, 2005]. Beyond this, taxa too similar to be distinguished and circumscribed by rRNA sequences have revealed further diversity through multilocus sequence analysis [MLSA] [Cladera et al., 2006, Hanage, et al., 2006] and metagenomic studies [Tyson and Banfield, 2005].

2.2.1. Gene Sequencing From the 90’s ownward, the routine sequencing of 16S rRNA gene of all the known bacteria started, partial at the beginning and later complete sequences were and are currently being deposited in public databases as Genbank. The sequencing of the 16S rRNA gene together with the development of mathematical models for construction of trees representing the similarity of the sequences allowed the phylogenetic classification of prokaryotes. The sequencing of 16S rRNA gene is nowadays routinely used for classification and identification of phosphate-solubilizing prokaryotes [Kuklinsky-Sobral et al., 2004; Pandey et al., 2006; Gulati et al., 2008; Naik et al., 2008]. Although the 16S rRNA gene is the basis of the current prokaryotic classification, at present it is known that very closely related species cannot be differentiated based on this gene [Lechner et al., 1998; Valverde et al., 2006]. Therefore, in the last decade other gene sequences have been used as phylogenetic molecular markers in taxonomic studies such as the “housekeeping” genes recA, atpD, carA, gyrB, rpoB, etc., whose usefulness for differentiation of phosphate-solubilizing species of Pseudomonas has been demonstrated [Hilario et al., 2004; Peix et al., 2007]. Several housekeeping genes have been also sequenced in some Burkholderia species [Baldwin et al., 2005]. For example, recA has been proposed as a tool for differentiation of the entire Burkholderia genus [Payne et al.,

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2005]. In other bacteria like rhizobia, several housekeeping genes, such as, recA and atpD, glnII, dnaK or dnaJ, are commonly used in taxonomic studies, phylogeny and diversity [Gaunt et al., 2001; Alexandre et al., 2008; Ramírez-Bahena et al., 2008; Santillana et al., 2008; Rivas et al., 2009]. Several housekeeping genes have been also used in diversity assessment of clinical and environmental Acinetobacter strains [Yamamoto et al., 1999; Peix et al., in press] allowing the differentiation of strains within A. calcoaceticus-A. baumanii complex to which most of phosphate removing strains belong [Srivastava et al., 2006]. In the case of bacilli, the housekeeping genes have been analysed mainly in clinical isolates from B. cereus group [Sorokin et al., 2006; Cardazzo et al., 2008], nevertheless as more species are analysed, its usefulness to identify environmental isolates will increase. The housekeeping genes are appropriate for diversity analysis because their evolutionary rate is higher than those of 16rRNA gene which is not considered very useful for analysis of bacterial diversity. For this reason, several housekeeping loci have recently been analysed together in several phosphate-solubilizing bacterial groups by Multilocus sequence analysis [MLSA] or Multilocus sequence typing [MLST] schemes [Kahn et al., 2008; Rivas et al., 2009]. In the last decade the improvement of sequencing technologies such as, pyrosequencing has led to the sequencing of the complete genome of an increasing number of strains, which is expected to revolutionize our view on bacterial functional and genetic diversity in the next few years.

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2.2.2. Methods Based on DNA Polymorphisms DNA profiling obtained with restriction endonucleases leading to restriction fragment length polymorphisms [RFLP] has been used for many years in microbiology. At the beginning, this technique was performed with genomic DNA, which displayed very complex profiles difficult to analyse. After discovery of PCR [Mullis and Faloona, 1987], RFLP profiling has been applied to specific genes, mainly 16S rRNA with the aim to group different isolates of a collection before sequencing the complete gene for identification. 16S-RFLP pattern analysis has been applied to population analysis of the different groups of PSB, for example phosphate-solubilizing sinorhizobia from the legume Mucuna [Kumar et al., 2006]. However, RFLP has been extended to different housekeeping or functional genes, allowing the study of numerous PSB populations within pseudomonads [Cho and Tiedje, 2000] or bacilli [Beneduzi et al., 2008]. Also, since PCR publication, DNA profiling through amplification of diverse genome regions has been reported. These techniques are based on direct electrophoresis of PCR amplified fragments by using one or two primers providing random amplifications within the bacterial genome [Welsh and McClelland, 1990; Williams et al., 1990; Versalovic et al., 1994]. Several techniques are based on the use of a single primer to obtain random amplifications in the genome [Koeuth et al., 1995; Williams et al., 1990]. Within them the RAPD patterns are the most commonly used to analyze diversity of main groups of phosphate-solubilizing bacteria such as, Pseudomonas and Burkholderia [Chiarini et al., 2000; Rangarajan et al., 2001] and rhizobia [Moschetti et al., 2005; Rivas et al., 2006; Valverde et al., 2006, Iglesias et al., 2007]. Recently RAPD-PCR schemes have been developed to analyse biodiversity in Bacillus [Kwon et al., 2009] and Acinetobacter [Ercolini et al., 2009]. The BOX-PCR is based on the amplification of regions that are repeated through the genome using a single primer and is designed from repetive bacterial genome sequences named BOX elements [Koeuth et al., 1995]. This technique has been

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applied to analyze phosphate-solubilizing Pseudomonas [Freitas et al., 2008; Naik et al., 2008], Burkholderia [Cottyn et al., 2001], rhizobia [Seguin et al., 2001] and Acinetobacter [Freitas et al., 2008]. Other techniques involve the use of two primers and allow amplification of repetitive genome regions [Versalovic et al., 1994]. For instance, REP-PCR [repetitive extragenic palindromic] provide amplification of palindromic reiterative extragenic elements [Versalovic et al., 1994; de Bruijn, 1992]. The ERIC-PCR [enterobacterial repetitive intergenic consensus] also includes two primers designed from enterobacterial repetitive intergenic consensus elements [Hulton et al., 1991]. These fingerprinting techniques have been applied to the population analysis of rhizobia [de Bruijn et al., 1992], Pseudomonas [Beaz-Hidalgo et al., 2008], Burkholderia [Liu et al., 1995] and Acinetobacter [Vila et al., 1996]. All these techniques are strain-specific and allow the differentiation of strains within the same species or subspecies. TP-RAPD fingerprinting is another technique that permit the grouping of bacteria at taxonomically accepted levels [Rivas et al., 2001] which display patterns through PCR amplifications using two primers targeting 16S rDNA conserved regions and relatively high annealing temperatures [50ºC]. It has been applied to different bacterial populations such as, rhizobia [Rodríguez-Navarro et al., 2004; Zurdo-Piñeiro et al., 2004], Pseudomonas and Acinetobacter [Velázquez et al., 2008].

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2.2.3. Low Molecular Weight RNA [LMW RNA] Profiles Most identification methods in bacteria are based on DNA molecules, nevertheless in 1988 Höfle proposed a new approach to identify bacteria basing a direct electrophoresis of low molecular weight RNA in polyacrylamide gels and later the same author proposed the tRNA profiles as bacterial fingerprints [Höfle, 1990]. The low resolution of band patterns was solved with the development of a new electrophoretic technique, Staircase electrophoresis [SCE], which allowed the separation of very close bands specially in the zone of tRNA’s [Cruz Sánchez et al., 1997]. The application of SCE to LMW RNA profiling led to the study of very diverse microbial groups, and to the conclusion that LMW RNA profiles acts as molecular signatures for organisms, both prokaryotes and eukaryotes [Velázquez et al., 2001]. Given the difficulties to handle the gels for their size [40 cm gels were required], a new approach was recently developed adapting the technique to non-sequencing gels in order to facilitate the use of LMW RNA profiling in all laboratories [Velázquez et al., 2006]. This technique has been applied to the identification of species from PSB genera such as, Pseudomonas [Velázquez et al., 2001], Burkholderia [Peix et al., 2001; Velázquez et al., 2006], Bacillus [Velázquez et al., 2001], Acinetobacter [Velázquez et al., 2001] and rhizobia [Velázquez et al., 1998, Rivas et al., 2006; del Villar et al., 2008; Zurdo-Piñeiro et al., 2009]. Although some authors have used LMW RNA profiles to study bacterial populations without isolation [Höfle, 1992], the complexity of LMW RNA profiles in isolated bacteria is too high for a reliable analysis of complex populations.

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2.2.4. Inmunofluorescence Methods: Fluorescent Labeling of Gene Products Fluorescence has long been used to visualize cell biology at many levels, from molecules to complete organisms. Originally, fluorescence involved small organic dyes attached by means of antibodies to the protein of interest. Later, fluorophores could directly recognize organelles, nucleic acids, and certain important ions in living cells. In the past decade, fluorescent proteins have enabled non-invasive imaging in living cells and organisms of reporter gene expression, protein trafficking, and many dynamic biochemical signals. There exist two principal techniques of fluorescent labeling of molecules, conventional fluorescent staining and molecular tagging. Since we are now able to know the complete genome of any organism, the function of each gene product in the cell has attracted interest. The development of techniques of fluorescent protein [FP] labeling and computer-controlled systems for fluorescence and laserconfocal microscopes has also raised the interest and has open the possibility to see directly the behavior of particular proteins in living cells. Depending on such requirements to visualize directly into the molecules in living cells, a specific technique for fluorescent labeling of molecules has been developed. Live-cell imaging techniques make it possible to detect the molecular basis of a variety of functions in the cell. The fluorescence microscopy itself has been a powerful technique to visualize the location of particular molecules in the cell [Watt, et al., 2007]. The combination of multiple techniques with advanced fluorescence microscopy has provided us with new tools to analyze the dynamic behavior of cellular molecules [Giepmans, et al., 2006] and have been widely used in the study of soil communities [Lenaerts, et al., 2007] and other complex communities [Díaz, et al. 2006]. Fluorescent protein such as, GFP have made possible to observe not only the localization per se but also the dynamics of the labeled molecules in the cell, allowing the development of new ways to analyze the functional behavior of proteins of interest in living cells. Labeling with FPs is carried out by genetic engineering including recombinant DNA technology. The behavior of FP-labeled molecules thus is sometimes different from that of endogenous ones. Moreover, tagging with monomeric FPs cannot completely exclude the possibility of adverse effects on the behavior and/or localization of fusion proteins [Watt, et al., 2007, RakosyTican and Aurori, 2007]. Some of GFP-based techniques have been used for assessing the genetic diversity of Pseudomonas and for its surveillance in poplar trees or wheat [Chen, et al. 1997; van Bruggen et al., 2008; Taghavi et al., 2009]. The GFP labelling has been used for rhizobia demonstrating its ability to invade tissues of non-legumes [Chi et al., 2005], in Burkholderia to display its competitiveness in Mimosa nodulation [Elliot et al., 2008] and in Bacillus to examine its ability to colonize plant tissues [Ji et al., 2008]. This technology is also being applied to genus Acinetobacter [Simpson et al., 2007].

2.2.5. Microarrays DNA array technology was originally designed for gene expression or single-nucleotide polymorphism profiling, being nowadays the most suitable technique that makes possible to evaluate thousands of genes in one experiment, giving a more comprehensive view of the genes involved in a specific cellular event [Dharmadi and González, 2004, Rogers and Cambrosio, 2007]. The microarray process can be divided into two main parts. First is the

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printing of known gene sequences onto glass slides or other solid support followed by hibridization of fluorescent labelled cDNA containing the unknown sequences to be screened to the known genes immobilized on the glass slide. After hibridization, arrays are scanned using a fluorescent microarray scanner. Analyzing the relative fluorescent intensity of different genes provides a mesure of the differences in gene expression [Lucchini et al., 2001, Binder, 2006]. This strategy has proved to be successful for microbial identification, even when species can only be discriminated by a single-nucleotide polymorphism. DNA arrays have the capacity to overcome some limitations associated with the restricted resolution of many fingerprinting methods, and the requirement of large sample numbers and associated costs for sequencing clones. In addition, since hybridization signals are proportional to the quantity of target DNA, this technique may also provide quantitative information [Lucchini et al., 2001]. DNA arrays can be divided into different categories based on the genes targeted by the array such as phylogenetic arrays, functional arrays and metagenomic arrays [Lucchini et al., 2001]. The phylogenetic arrays are based on a diagnostic marker such as the 16S rRNA gene and are used for microbial identification. A major advantage of phylogenetic or functional gene arrays is the unlimited expanding capacity to detect numerous microorganisms or genes of special interest. However, a need of specific target organisms or genes are some its limitation. As a result, DNA arrays fail to identify taxa for which no oligonucleotides are developed yet. Furthermore, it is not always possible to have perfect specificity for all detector oligonucleotides since they all need to be hybridized under the same conditions. Nevertheless, this problem can be circumvented by spotting multiple oligonucleotides for the same target [Lucchini et al., 2001; Wu et al., 2001]. Functional gene arrays, designed for the detection of key functional genes in a specific environment, has been widely applied in the study of some members of phosphate-solubilizing bacterial groups as, Bacillus spp. and Pseudomonas spp. [van der Werf et al., 2006; Bavykin et al., 2008]. Metagenomic arrays unlike the other arrays, contain DNA fragments produced directly from environmental DNA and can be applied with no prior sequence knowledge [Sergeev et al., 2006]. Undoubtedly, this is an expanding methodology in the study of microbial communities diversity and biogeography at large scale.

2.3. CULTURE-INDEPENDENT MOLECULAR METHODS FOR ANALYSIS OF MICROBIAL DIVERSITY: THE METAGENOMICS ERA At population or community levels, different molecular fingerprinting techniques are being used since the 90’s which involves directly extracted DNA from environmental samples. Such techniques are very effective in the study of dynamics and structure of microbial communities thus allowing comparative analysis among different geographical locations, monitoring of microbial communities after soil manipulations, etc. The assessment of microbial communities has limitations associated with traditional culture based methods but at the same time such impediments have prompted to develop culture-independent techniques. In this context, the new molecular methods highlight the important role of the non-culturable bacteria that are involved in the complex biological cycles.

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There exist several molecular techniques that have been widely applied for genetic analysis of microbial communities because they can provide a description of a specific community. Such techniques include denaturing and temperature gradient gel electrophoresis [DGGE/TGGE] [Muyzer et al., 1993], single-strand conformation polymorphism [SSCP] [Schwieger and Tebbe, 1998], terminal restriction fragment length polymorphism [T-RFLP] [Liu et al., 1997], amplified ribosomal DNA restriction analysis [ARDRA] and [automated] ribosomal intergenic spacer analysis [[A] RISA] [Ranjard et al., 2001]. These techniques are suitable to compare microbial community composition between different treatments, environments or situations and are based on detection of differences in sequence composition, size or conformation of PCR-amplified genes. They however, differ primarily in the targeted amplified genes and in the method for separating amplicons, so the resolution power of the method depends on this combination and hence the convenience of choosing one or another. The targeted genes most commonly used are structural conserved genes such as ribosomal genes, which are used in intermediate resolution techniques as DGGE, TGGE, SSCP, TRFLP, not having resolution at infrageneric taxonomic levels. On the contrary, the high resolution techniques such as RISA or ARISA target non-codifying and codifying elements as repetitive sequences or the ribosomal intergenic spacers [ITS], having resolution at species level or even at intraspecific level. Besides, other techniques based on fluorescence such as fluorescent in situ hybridization [FISH] and quantitative real time PCR has the potential to overcome culture-based methods.

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2.3.1. Fluorescence “In Situ” Hybridization [FISH] FISH is based on the antisense single-stranded DNAs for the particular genes or sequences are labeled with proper fluorochromes and used as the molecular probes. The fluorescent-labeled probes are hybridized with the complementary RNA or DNA molecules in the cells and are used to detect the particular gene expression in the cells or the number of copies and placement of particular genes or sequences in chromosomes. The most commonly used oligonucleotid probes for bacterial diversity studies are those targeting the 16S or 23S rRNA, and depending on the degree of conserved sequence of the probe, it is possible to detect microorganisms at different taxonomic levels, from species to wider phylogenetic groups as genus, phylum, etc [Amann et al., 1995]. This technique is applied for bacterial identification and combines the simplicity of microscopy observation and the specificity of DNA hybridization [DeLong et al., 1989] and, in general, the whole procedure can be completed in a few hours [Amann et al., 1995]. Although, FISH could detect single cell but practically, the detection level is often 103 cells/ml, rendering this technique in general less sensitive than PCR-based techniques. Another limitation is the insufficient automation for high sample throughput. Furthermore, an extensive knowledge of the community is required since the probes need to be designed beforehand and after all, only a limited number of probes can be applied in one hybridization run. This drawback limits the application of FISH for community analysis on a high level of phylogenetic resolution. However, as the understanding of the ecology of complex microbial communities is enhanced by analyses in minimally disturbed samples, FISH is widely used in environmental microbiology [Maszenan et al., 2000] and reveals the morphology of the target organisms, their abundance in a given ecosystenm and their interactions with other microbiota. FISH has been used for several

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applications including detection and identification of soil bacteria [Watt et al., 2006; Lenaerts et al., 2007; Martín et al., 2008] and therefore this technology could also be used to detect phosphate-solubilizing bacteria by the designing of specific probes. Moreover, FISH has been used for the monitoring of phosphate-accumulating bacteria [Barat et al., 2008]

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2.3.2. Specific PCR Based Methods The exhaustive knowledge of many genes sequences gained over the years has led to the use of single DNA bands as microbial markers. These specific bands can be visualized by direct electrophoresis allowing the rapid identification of microorganisms in complex environment after the designing of primers targeting specific zones of different genes. For example, nested PCR based on different ribosomal and housekeeping genes and intergenic spacer regions [ITS] is widely used in diagnosis of animal, human and plant pathogenic bacteria but also in identification of bacteria as, Pseudomonas [Evans et al., 2004], Acinetobacter [Vanbroekhoven et al., 2004], Burkholderia [Payne et al., 2006] and Rhizobium [Tan et al., 2001; Pulawska and Sobiczewski, 2005] in the environmental samples. The amplification and detection of sequence characterized amplified regions [SCARs] is a recently developed technique applied to bacterial diagnostic which has not yet been widely applied for identification of bacteria in environmental samples. Latest studies have however, suggested its role in the identification of bacterial strains, Pseudomonas [Holmberg et al., 2009] and Burkholderia [Gobbin et al., 2007], in environmental samples demonstrating the suitability of this technique for the fore mentioned purpose. There are many approaches based on PCR-amplification and sequencing of specific genes using as template DNA directly extracted from samples, allowing the identification of culturable and non-culturable bacteria. This methodology has been applied to identification of Bacillus [Felici et al., 2008], Pseudomonas [Pesaro and Widmer, 2006], Burkholderia [Pirone et al., 2005], Acinetobacter [Abd-El-Haleem et al., 2002] and Rhizobium [Muresu et al., 2005]. A variant of this methodology named Multiplex PCR involves the use of different primer pairs and DNA templates in the same reaction and allows the detection and identification of bacteria from complex samples such as, soil and water [Felske et al., 2003; Malik et al., 2008].

2.3.3. Denaturing Gradient Gel Electrophoresis [DGGE] and Temperature Gradient Gel Electrophoresis [TGGE] Both DGGE and TGGE help to examine microbial diversity based upon electrophoresis of small PCR-amplified DNA fragments [200–700 bp] on an acrylamide gel having a low to high denaturant gradient. For DGGE, denaturing chemicals such as, formamide and urea are used while a temperature gradient is applied in TGGE. As describe earlier, these intermediate resolution techniques usually target the 16SrRNA gene, amplifying partial sequences of this gene. Both DGGE and TGGE are useful for comparisons among microbial communities and spatial or temporal changes, but the organisms are identified only at genus level. Moreover, specialized equipment are required to perform such analysis. DNA fragments of similar length but with different sequences can be separated according to their melting properties [Muyzer et al., 1993, 2004]. Generally, the melting behavior depends on the length of the

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product, its GC-content and the nucleotide sequence. Initially the melting process is partial, with discrete domains becoming single stranded, decreasing the mobility of the DNA fragments through the gel. Eventually, strand separation stretches over the entire length of the product with the exception of a GC-rich clamp [40–45 base GC-rich sequence], which is usually attached to the 5’-end of the forward primer [Sheffield et al., 1989] to increase the percentage of sequence variants that can be detected in DNA framents [Myers et al., 1985; Muyzer et al., 2004]. This clamp is highly stable and holds the strands partially together leading to a molecule whose migration is extremely retarded. Although the gradients might have to be adjusted to a specific sample for optimal resolution [Ogier et al., 2002], these methods have the theoretical potential to detect differences as little as a single or a few base pairs. Major advantages of both DGGE and TGGE are their affordability for molecular laboratories and the relatively easy interpretation of results. Furthermore, individual bands can be excised from the gel and identified by sequencing. However, reliable identification by sequencing may be hampered by the small fragment sizes of the PCR products, which might not contain enough information for precise taxonomic classification. In addition, it should be noted that different sequences may have identical electrophoretic mobility, resulting in comigration of different fragments [Sekiguchi et al., 2001]. A major drawback often associated with DGGE/TGGE is lack of reproducibility while handling big gels; primer– dimer formation and variable gel staining all affect reproducibility. However, reproducibility can be enhanced by the inclusion of an internal standard, facilitating normalization of samples within and between gels. The DGGE has been used for the study of some solubilizing bacterial genera as Bacillus spp. [Miambi et al., 2003], Pseudomonas spp. [Hunter, et al., 2006], and specific rhizobial communities in soil [de Oliveira et al., 2006], and TGGE has recently been successfully used for isolation and identification of P mobilizing bacteria, specifically phosphonoacetate-degrading bacteria [Panas et al., 2007].

2.3.4. Single-Strand Conformation Polymorphisms Single strand conformation polymorphism [SSCP] is a PCR-based fingerprinting technique initially designed for mutation detection and is now widely used for this purpose. Later on it was adapted for the fingerprinting of cultivated soil microorganisms and noncultivated rhizosphere bacterial communities [Lee et al., 1996; Schwieger and Tebbe, 1998]. This method is based on the different electrophoretic migration of the amplicons by means of the sequence-dependent spatial conformation of single stranded DNA. It has been usually applied to resolve amplicons of 16S rRNA variable regions up to 400 bp, whose reproducibility and differentiation power depends on the fragment size. However, other targets such as 16S-23S rDNA ITS have also been used [Mora et al., 2003]. It is simple to perform than DGGE/TGGE since no special equipment is required and no GC-clamp is needed to be added to the primers. It also permits excision and sequencing of bands for phylogenetic affiliation of microbial community members. As drawbacks, the rate of reannealing of DNA, the existence of more than one band from a double stranded product after electrophoresis and the several possible conformations for the same product coexisting in the same gel must be taken into account, for which modifications of the methodology have been developed to make it suitable for complex microbial populations studies [Schwieger and

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Tebbe, 1998]. SSCP approach have been applied for ribotyping of potential PSB, as, rhizobia [Clapp et al., 2001], pseudomonads and bacilli [Mittal and Johri, 2008]. The SSCP profiles have been also analysed by capillary electrophoresis through fluorescence detection [King et al., 2005]

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2.3.5. Ribosomal Intergenic Spacer Analysis and Automatized Ribosomal Intergenic Spacer Analysis Ribosomal intergenic spacer analysis [RISA] [Borneman and Triplett, 1997] involves PCR amplification of total bacterial community DNA of the intergenic region between the 16S and 23S ribosomal genes. The intergenic spacer [ITS] region displays significantly more heterogeneity in length and nucleotide sequence than the flanking 16S and 23S ribosomal genes. In RISA, size differences of the spacers are exploited for subtyping of bacterial strains or in cases where fingerprinting of ribosomal sequences does not provide sufficient resolution. In general, intergenic spacer lengths may vary between approximately 400 and 1200 bp, and community profiles are affected by rRNA operon copy number. After gel electrophoresis of the PCR product, a complex community-specific banding pattern is generated, with each band corresponding to at least one organism in the original community. RISA is a high resolution technique with discrimination power at species or intraspecies level, since ITS regions are extremely variable in size and sequence. Moreover, no special equipment is required, and could be adapted even to agarose gels in horizontal staircase electrophoresis [Peix et al., 2005]. This technique is-[i] simple to handle [ii] easy to analyse qualitative composition of microbial communities and [iii] allows the simultaneous phylogenetic analysis of different microbial groups. However, even when ITS sequences deposited in databases are increasing consistently, currently their number is very far from that of 16S rRNA sequences. The lack of sensitivity associated with this gel-based method however, led to the development of automated RISA [Ranjard et al., 2001], in which the original steps of DNA extraction and PCR amplifications are identical, except that a fluorescently labeled forward primer is used in the PCR. The electrophoretic step is subsequently performed on an automated system, with laser detection of fluorescent DNA fragments. Potential problems associated with [A] RISA are the preferential amplification of shorter templates [Fisher and Triplett, 1999] and the fact that, because of ITS length variation within a single genome, a single organism can contribute to more than one signal. In order to increase reproducibility and standardization, Cardinale et al. [2004] evaluated different primer sets with respect to universality, sensitivity and reproducibility and selected the most suitable primers for ARISA for environmental bacterial communities. As for DGGE/TGGE and T-RFLP, ARISA has been regularly applied in microbial ecology [Ranjard et al., 2001], and the main groups containing phosphate solubilizers have been also targeted by RISA and ARISA [Ikeda et al., 2008].

2.3.6. Terminal Restriction Fragment Length Polymorphism Terminal restriction fragment length polymorphism [T-RFLP] analysis is a PCR-based community profiling method that is commonly used for comparative microbial community

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analysis [Liu et al., 1997]. For this technique, marker genes are amplified with fluorescently labeled primer [s], followed by restriction digestion [typically using 4-base cutters] and separation and detection on an automated sequencer. There are more than 20 tetra-base cutter restriction enzymes commercially available and almost the 16 palindromic combinations of the four bases are recognized by one or more of these restriction endonucleases, allowing the search for numerous polymorphic sites [Nesme and Normand, 2004]. Only labeled terminal restriction fragments [TRFs] are detected and their length heterogeneity indicates the complexity of the community visualized by an electropherogram. An internal size standard labeled with different fluorescent dye, allows precise length assignment with single-base pair resolution. With the 16S rRNA gene as target, obtained TRFs can be compared with the rapidly expanding sequence database of the Ribosomal Database Project, allowing predictions of the organisms present in the analyzed sample [Marsh et al., 2000]. The use of the multiple restriction enzymes available increases the specificity and the reliability of the assay. The choice of the endonucleases is based on the desired degree of discriminative power, or to track a specific population. Several free access online tools such as TAP-TRFLP [http://rdp.cme.msu.edu], torast [http://www.torast.com], and MiCA [http://mica.ibest. uidaho.edu/] allow the in silico prediction of TRFs, enabling selection of endonucleases [Marsh et al., 2000; Ricke et al., 2005]. T-RFLP allows a sensitive detection and because of its high throughput capacity, it performs well in surveys with large sample numbers for example to ascertain spatial or temporal changes in a community structure. However, this method also has some limitations in accurately predicting microbial community structures. Incomplete or non-specific restriction leads to overestimation of the diversity since the number of fragments increases. However, restriction efficiency can be checked by inclusion of an internal standard in the restriction step. Overestimation of diversity can also be generated by pseudoterminal restriction fragments as reported by Egert and Friedrich [2003]. These fragments can be produced upon intramolecular folding of single-stranded products, creating transient structures that are accessible for digestion. However, this problem can be overcome by treatments with single-strand-specific nucleases [Egert and Friedrich, 2003]. One of the major restrictions for identification is variation between the in silico predicted and the experimentally obtained TRF lengths. Despite these limitations, T-RFLP has become a valuable molecular tool for microbial community analysis, especially when high throughput and high sensitivity are required without the need for direct sequence information. Increasingly, T-RFLP is successfully used to describe microbial populations, and the main phosphate-solubilizing groups have been studied with this technique such as rhizobia [Sarita et al., 2005], Pseudomonas [Bankhead et al., 2004] or Bacillus [Graff and Conrad, 2005]

2.3.7. Amplified rDNA Restriction Analysis Amplified ribosomal DNA restriction analysis [ARDRA] is a relatively simple PCRbased fingerprinting technique based on the digestion of amplified ribosomal community DNA followed by gel electrophoresis that can be used for microbial identification [Laguerre et al., 1994] or comparison of microbial communities and dynamics [Moyer et al., 1994; Martínez-Murcia et al., 1995]. In contrast to T-RFLP, all digested fragments are detected, increasing the level of resolution. One single restriction enzyme generally does not provide sufficient resolution and hence, multiple restriction enzymes are used either separately or in

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combination to obtain the desired resolution. Another drawback of this method is the limited staining sensitivity in gels resulting in the suppression of bands from less abundant community members or in a loss of phylogenetic information [Tiedje et al., 1999]. As a consequence, this technique is only preferred when the community is dominated by a few members, but is of limited use for specific phylogenetic groups or for complex communities. Nevertheless, this is a high resolution technique with discrimination power at species level and has been applied to study microbial diversity including rhizobia [Laguerre et al., 1994] and phosphate- solubilizing Pseudomonas fluorescens populations [Frey-Klett et al., 2005].

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2.3.8. Real-Time PCR Besides analyzing the identity of the main members of a community, it is desirable to know the size of the respective populations. For example, whether the magnitude is expected to be pernicious in the case of pathogens, or beneficial in the case of PSB. Therefore, in addition to the microbial community structure and identity determinations, quantitative assay of the microbial populations is becoming more and more significant. In order to quantify the presence of certain microorganisms by DNA-based techniques, the amount of genomic DNA should be correlated to the amount of biomass. However, the non-linear nature of PCR-amplification makes it challenging to relate the final amount of sequences to the initial amount of genomic DNA in the sample. As a consequence, PCRbased fingerprinting techniques such as DGGE and T-RFLP or DNA arrays are often considered to be semi-quantitative. In contrast, accurate quantification of DNA can be performed using real-time [RT]-PCR. This procedure is a reproducible, high-throughput, highly precise and accurate for quantification of specific gene/bacterial populations in environmental samples. Moreover, it is possible to target different genes in one step using probes labelled with different reporter dyes and multiplex PCR. It has been used targeting either 16S rRNA, 16S-23S ITS or functional genes for several bacterial groups, some of them containing important representatives of PSB [Saikaly et al., 2007]. This method enables an online detection of the PCR product, avoiding the need for postPCR processing. In addition, RT-PCR allows accurate template quantification over a wide dynamic range [4107-fold]. Typically, DNA amplification is continuously monitored based on the emission of fluorescence. In general, the initial concentration of target DNA is linked to a precise threshold cycle, defined as the cycle number at which fluorescence increases above the background level. Ultimately, the target DNA is quantified using a calibration curve that relates threshold cycles to exact concentrations of template DNA. Accumulating amplicons can be detected with several methods that make use of either fluorescent DNA-intercalating dyes or sequence-specific fluorescent probes [Heid et al., 1996; Mackay, 2004]. Concentrations of reporter dye labelled probes and primers can be optimized empirically to minimize the cycle threshold values, standard curves and limits of detection have been assayed in different studies [Hermansson and Lindgren, 2001].

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2.3.9. Complete Genomes and Future Perspectives

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Genotypic diversity of phosphate-solubilizing bacteria such as, Pseudomonas strains is being currently studied in detail after the advent and improvement of sequencing technologies such as pyrosequencing [Iacono et al., 2008]. Such techniques have allowed to sequence 25 million bases in just one 4h run with 99.9% accuracy, which in turn has led to the sequencing of the complete genome of an increasing number of strains. Furtheromore, using substractive hybridization, genome comparisons have become possible to detect strain-specific regulatory genes and genomic islands [Battle et al., 2009]. Since the first Pseudomonas complete genome sequence was obtained, which was the genome of the important opportunistic pathogen strain P. aeruginosa PA01 published in 2000 [Stover et al., 2000], a total of 16 complete Pseudomonas genomes have been sequenced and deposited in databases up to date according to the Genomes Online Database [http://www.genomesonline.org/gold.cgi]. Also, 12 rhizobial strains, 7 Acinetobacter and 17 Bacillus strains have been completely sequenced and deposited. Interestingly, there is also a strain of Thiobacillus and another of Acidithiobacillus completely sequenced, these genera are being studied for their ability to solubilize insoluble phosphates [rock phosphate] by releasing inorganic acids into soils [Somani, 1990, Stamford et al., 2007]. The comparative studies on some of these genomes have provided very useful and intresting informations demonstrating that genome sizes are extremely variable, and different genes involved in pathogenicity and other phytobeneficial traits have been detected to be transferred horizontally among strains. In the future, sequencing of more and more complete genomes of phosphate- solubilizing bacteria are expected which is likely to help to understand the genetic and molecular bases of microbial phosphate solubilization and the genetic diversity of this complex group of bacteria, as well as to clarifying the phylogenetic relationships and taxonomy of the different taxa.

CONCLUSION In conclusion, a lot of different molecular approaches are being increasingly developed for the study of microbial communities with specific ecological traits such as phosphate solubilisation. Assessment of biodiversity of complex bacterial groups is important due to the overwhelming diversity existent in the different ecosystems. However, lack of proper methodologies to identify the structural and functional variation within heterogenous microbial communities in different ecosystems warrants new methods for similar assessment. As reviwed and discussed in this chapter, though different approaches have both advantages and limitation, yet a combination of them can provide a nice picture of the genotypic diversity, structure, dynamics, spatial scaling and biogeography of PSB communities. In any case, microbiologists must be encouraged enough to continue doing research in this fascinating field so that more informations are generated to unravel bacterial diversity and hence to preserve such natural treasures. In this context, As E. O. Wilson has very rightly stated that “biological diversity must be treated more seriously as a global resource, to be indexed, used, and above all, preserved.

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REFERENCES Abd-El-Haleem, D., Moawad, H., Zaki, EA and Zaki, S. [2002]. Molecular characterization of phenol-degrading bacteria isolated from different Egyptian ecosystems. Microb. Ecol, 43, 217-224. Alexandre, A., Laranjo, M., Young, JP and Oliveira, S. [2008]. DNAJ is a useful phylogenetic marker for alphaproteobacteria. Internatl J. Syst Evol. Microbiol., 58, 28392849. Amann, RI., Ludwig, W and Schleifer, KH. [1995]. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev., 59, 143-169. Baldwin, A., Mahenthiralingam, E., Thickett, KM., Honeybourne, D., Maiden, MC., Govan, JR., Speert, DP., Lipuma, JJ., Vandamme, P and Dowson, CG. [2005]. Multilocus sequence typing scheme that provides both species and strain differentiation for the Burkholderia cepacia complex. Journal of Clinical Microbiol, 43, 4665-4673. Bankhead, SB., Landa, BB., Lutton, E., Weller, DM and Gardener, BBM. [2004]. Minimal changes in rhizobacterial population structure following root colonization by wild type and transgenic biocontrol strains. FEMS Microbiol Ecol, 49, 307-318. Barat, R., Montoya, T., Borrás, L., Ferrer, Jand Seco, A. [2008]. Interactions between calcium precipitation and the polyphosphate-accumulating bacteria metabolism. Water Res, 42, 3415-3424. Battle, SE., Rello, J and Hauser, AR. [2009]. Genomic islands of Pseudomonas aeruginosa. FEMS Microbiol Lett, 290, 70-78. Bavykin, SG., Mikhailovich, VM., Zakharyev, VM., Lysov, YP., Kelly, JJ., Alferov, OS., Gavin, IM., Kukhtin, AV., Jackman, J., Stahl, DA., Chandler, D and Mirzabekov, AD. [2008]. Discrimination of Bacillus anthracis and closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microarray. Chemico Biological Interactions, 171, 212-235. Beaz-Hidalgo, R., López-Romalde, S., Toranzo, AE and Romalde, JL. [2008]. Polymerase chain reaction amplification of repetitive intergenic consensus and repetitive extragenic palindromic sequences for molecular typing of Pseudomonas anguilliseptica and Aeromonas salmonicida. J. Aquatic Animal Health, 20, 75-85. Beneduzi, A., Peres, D., da Costa, PB., Zanettini, MHB; Passaglia, LMP. [2008]. Genetic and phenotypic diversity of plant-growth-promoting bacilli isolated from wheat fields in southern Brazil. Research in Microbiology, 159, 244-250. Binder, H. [2006]. Thermodynamics of competitive surface adsorption on DNA microarrays. Journal of Physics: Condensed Matter, 18, S491-S523. Blackall, LL., Crocetti, GR., Saunders, AM and Bond, PL. [2002]. A review and update of the microbiology of enhanced biological phosphorus removal in wastewater treatment plants. Antonie van Leeuwenhoek, 81, 681-691. Borneman, J and Triplett, EW. [1997]. Molecular microbial diversity in soils from eastern Amazonia: evidence for unusual microorganisms and microbial population shifts associated with deforestation. Appl. Environ. Microbiol, 63, 2647-2653. Cardinale, M., Brusetti, L., Quatrini, P., Borin, S., Puglia AM., Rizzi, AE., Zanardini, C. Sorlini, Corselli, C and Daffonchio, D. [2004]. Comparison of different primer sets for

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30

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use in automated ribosomal intergenic spacer analysis of complex bacterial communities. Appl. Environ. Microbiol., 70, 6147-6156. Cardazzo, B., Negrisolo, E., Carraro, L., Alberghini, L., Patarnello, T and Giaccone, V. [2008]. Multiple-locus sequence typing and analysis of toxin genes in Bacillus cereus food-borne isolates. Appl. Environ. Microbiol., 74, 850-860. Chabot, R., Antoun, H and Cescas, MP. [1996]. Growth promotion of maize and lettuce by phosphate solubilizing Rhizobium leguminosarum biovar phaseoli. Plant Soil, 184, 311321. Chang, CH and Yang, SS. [2009]. Thermo-tolerant phosphate-solubilizing microbes for multi-functional biofertilizer preparation. Biores Technol., 100,1648-1658. Chen, F., González, JM., Dustman, WA., Moran, MA and Hodson, RE. [1997]. In situ reverse transcription, an approach to characterize genetic diversity and activities of Prokaryotes. Appl. Environ. Microbiol., 63, 4907-4913. Chi, F., Shen, SH., Cheng, HP., Jing, YX., Yanni, YG and Dazzo, FB. [2005]. Ascending migration of endophytic rhizobia, from roots to leaves, inside rice plants and assessment of benefits to rice growth physiology. Appl. Environ. Microbiol., 71, 7271-7278. Chiarini, L., Giovannelli, V., Bevivino, A., Dalmastri, C and Tabacchioni, S. [2000]. Different portions of the maize root system host Burkholderia cepacia populations with different degrees of genetic polymorphism. Environ Microbiol, 2, 111-118. Cho, JC and Tiedje, JM. [2000]. Biogeography and degree of endemicity of fluorescent Pseudomonas strains in soil. Appl. Environ. Microbiol., 66, 5448-5456. Cladera, AM., Sepúlveda-Torres, L., Valens-Vadell, M., Meyer, JM., Lalucat, J; GarcíaValdés, E. [2006]. A detailed phenotypic and genotypic description of Pseudomonas strain OX1. Syst Appl Microbiol, 29, 422-430. Clapp, JP., Mansur, I., Dodd, JC and Jeffries, P. [2001]. Ribotyping of rhizobia nodulating Acacia mangium and Paraserianthes falcataria from different geographical areas in Indonesia using PCR-RFLP-SSCP [PRS] and sequencing. Environ Microbiol, 3, 273280. Cottyn, B., Regalado, E., Lanoot, B., De Cleene, M., Mew, TW and Swings, J. [2001]. Bacterial populations associated with rice seed in the tropical environment. Phytopathol, 91[3], 282-292. Cruz-Sánchez, JM., Velázquez, E., Mateos, P., Velázquez, E and Martínez-Molina, E. [1997]. Enhancement of resolution of low molecular weight RNA profiles by staircase electrophoresis. Electrophoresis, 18, 1909–1911. de Bruijn, FJ. [1992]. Use of repetitive [repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus] sequences and the polymerase chain reaction to fingerprint the genomes of Rhizobium meliloti isolates and other soil bacteria. Appl. Environ. Microbiol., 58, 2180-2187. DeLong, EF., Wickham, GS and Pace NR. [1989]. Phylogenetic stains: ribosomal RNAbased probes for the identification of single cells. Science, 243, 1360-1363. de Oliveira, VM., Manfio, GP., da Costa Coutinho, HL., Keijzer-Wolters, AC and van Elsas, JD. [2006]. A ribosomal RNA gene intergenic spacer based PCR and DGGE fingerprinting method for the analysis of specific rhizobial communities in soil. J. Microbiol Methods, 64, 366-379.

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del Villar, M., Rivas, R., Peix, A., Mateos, PF., Martínez-Molina, E., van Berkum, P., Willems, A and Velázquez, E. [2008]. Stable low molecular weight RNA profiling showed variations within Sinorhizobium meliloti and Sinorhizobium medicae nodulating different legumes from the alfalfa cross-inoculation group. FEMS Microbiol. Lett, 282, 273-281. Dharmadi, Y and González, R. [2004]. DNA microarrays: experimental issues, data analysis, and application to bacterial systems. Biotechnology Progress, 20, 1309-1324. Díaz, EE., Stams, AJM., Amils, R and Sanz, JL. [2006]. Phenotypic properties and microbial diversity of methanogenic granules from a full-scale upflow anaerobic sludge bed reactor treating brewery wastewater. Appl. Environ. Microbiol, 72, 4942-4949. Egert, M and Friedrich, MW. [2003]. Formation of pseudo-terminal restriction fragments, a PCR-related bias affecting terminal restriction fragment length polymorphism analysis of microbial community structure. Appl. Environ. Microbiol., 69, 2555–2562. Ehteshami, SM., Aghaalikhani, M., Khavazi, K and Chaichi, MR. [2007]. Effect of phosphate solubilizing microorganisms on quantitative and qualitative characteristics of maize [Zea mays L.] under water deficit stress. Pak J. Biol. Sci., 10, 3585-3591. Elliott, GN., Chou, JH., Chen, WM., Bloemberg, GV., Bontemps, C., Martínez-Romero, E., Velázquez, E., Young, JP., Sprent, JI and James, EK. [2008]. Burkholderia spp. are the most competitive symbionts of Mimosa, particularly under N-limited conditions. Environ Microbiol, DOI: 10.1111/j.1462 2920.2008.01799.x Ercolini, D., Russo, F., Nasi, A., Ferranti, P and Villani, F. [2009]. Mesophilic and psychrotrophic bacteria from meat and their spoilage potential in vitro and in beef. Appl Environ Microbiol, DOI: AEM.02762-08v1. Evans, FF., Seldin, L., Sebastian, GV., Kjelleberg, S., Holmström, C and Rosado, AS. [2004]. Influence of petroleum contamination and biostimulation treatment on the diversity of Pseudomonas spp. in soil microcosms as evaluated by 16S rRNA based-PCR and DGGE. Lett Appl Microbiol, 38, 93-98. Felici, C., Vettori, L., Toffanin, A and Nuti, M. [2008]. Development of a strain-specific genomic marker for monitoring a Bacillus subtilis biocontrol strain in the rhizosphere of tomato. FEMS Microbiol. Ecol., 65, 289-298. Felske, AD., Heyrman, J., Balcaen, A and de Vos, P. [2003]. Multiplex PCR screening of soil isolates for novel Bacillus-related lineages. J. Microbiol Methods, 55, 447-458. Fisher, MM., Triplett, EW. [1999]. Automated approach for ribosomal intergenic spacer analysis of microbial diversity and its application to freshwater bacterial communities. Appl. Environ. Microbiol., 65, 4630-4636. Freitas, DB., Lima-Bittencourt, CI., Reis, MP., Costa, PS; Assis, PS., Chartone-Souza, E and Nascimento, AM. [2008]. Molecular characterization of early colonizer bacteria from wastes in a steel plant. Lett. Appl. Microbiol, 47, 241-249. Frey-Klett, P., Chavatte, M., Clausse, M.L., Courrier, S., Le Roux, C., Raaijmakers, J., Martinotti, MG., Pierrat, JC and Garbaye, J. [2005]. Ectomycorrhizal symbiosis affects functional diversity of rhizosphere fluorescent pseudomonads. New Phytologist, 165, 317-328. Gaunt, MW., Turner, SL., Rigottier-Gois, L., Lloyd-Macgilp, SA and Young, J.P. [2001]. Phylogenies of atpD and recA support the small subunit rRNA-based classification of rhizobia. International Journal of Syst Evol Microbiol, 51, 2037-2048.

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Martha-Helena Ramírez-Bahena, Alvaro Peix, Eustoquio Martínez-Molina et al.

Giepmans, BNG., Adams, SR., Ellisman, MH and Tsien, RY. [2006]. The fluorescent toolbox for assessing protein location and function. Science, 312, 217-223. Gobbin, D., Rezzonico, F and Gessler, C. [2007]. Quantification of the biocontrol agent Pseudomonas fluorescens Pf153 in soil using a quantitative competitive PCR assay. Soil Biol Biochem, 39, 1609-1619. Graff, A and Conrad, R. [2005]. Impact of flooding on soil bacterial communities associated with poplar [Populus sp.] trees. FEMS Microbiol Ecol, 53, 401-415. Gulati, A., Rahi, P and Vyas, P. [2008]. Characterization of phosphate-solubilizing fluorescent pseudomonads from the rhizosphere of seabuckthorn growing in the cold deserts of Himalayas. Curr. Microbiol., 56, 73-79. Gulati, A., Vyas, P., Rahi, P and Kasana, RC. [2009]. Plant growth-promoting and rhizosphere-competent Acinetobacter rhizosphaerae strain BIHB 723 from the cold deserts of the Himalayas. Curr. Microbiol., 58, 371-377. Hameeda, B., Harini, G., Rupela, OP., Wani, SP and Reddy, G. [2008]. Growth promotion of maize by phosphate-solubilizing bacteria isolated from composts and macrofauna. Microbiol. Res., 163, 234-242. Hanage, WP., Fraser, C and Spratt, BG. [2006]. Sequences, sequence clusters and bacterial species. Philosophical Trans Royal Soc B: Biol Sci, 361, 1917-1927. Heid, CA., Stevens, J., Livak, KJ and Williams, PM. [1996]. Real time quantitative PCR. Genome Res, 6, 986-994. Hermansson, A and Lindgren, PE. [2001]. Quantification of Ammonia-Oxidizing Bacteria in Arable Soil by Real-Time PCR. Appl Environ Microbiol, 67, 972-976. Hilario, E., Buckley, T and Young, J. [2004]. Improved resolution on the phylogenetic relationships among Pseudomonas by the combined analysis of atpD, carA, recA and 16S rDNA. Antonie van Leeuwenhoek, 86, 51–64. Höfle, MG. [1988]. Identification of bacteria by low molecular weight RNA profiles: a new chemotaxonomic approach. J Microbiol Methods, 8, 235-248. Höfle, MG. [1990]. Transfer RNAs as genotypic fingerprints of eubacteria. Arch Microbiol, 153, 299-304. Höfle, MG. [1992]. Bakterioplankton community structure and dynamics after large-scale release of nonindigenous bacteria as revealed by low-molecular-weight-RNA analysis. Appl Environ Microbiol, 58, 3387-3394. Holmberg, AIJ., Melin, P., Levenfors, JP and Sundh, I. [2009]. Development and evaluation of SCAR markers for a Pseudomonas brassicacearum strain used in biological control of snow. Biol Control, 4, 181-187. Hulton, CSJ., Higgings, CP. and Sharp, PM. [1991]. ERIC sequences: a novel family of repetitive elements in the genomes of Escherichia coli, Salmonella typhimurium and other enterobacteria. Molecular Microbiol, 5, 825-834. Hunter, PJ., Petch, GM., Calvo-Bado, LA., Pettitt, TR., Parsons, NR., Morgan, JAW and Whipps, JM. [2006]. Differences in microbial activity and microbial populations of peat associated with suppression of damping-off disease caused by Pythium sylvaticum. Appl .Environ. Microbiol., 72, 6452-6460. Iacono, M., Villa, L., Fortini, D., Bordoni, R., Imperi, F., Bonnal, RJ., Sicheritz-Ponten, T., De Bellis, G., Visca, P., Cassone, A and Carattoli, A. [2008]. Whole-genome pyrosequencing of an epidemic multidrug-resistant Acinetobacter baumannii strain

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belonging to the European clone II group. Antimicrobial Agents and Chemotherapy, 52, 2616-2625. Iglesias, O., Rivas, R., García-Fraile, P., Abril, A., Mateos, PF., Martínez-Molina, E and Velázquez, E. [2007]. Genetic characterization of fast-growing rhizobia able to nodulate Prosopis alba in North Spain. FEMS Microbiol. Lett, 277, 210-216. Ikeda, S., Rallos, L.E.E., Okubo, T. , Eda, S., Inaba, S., Mitsui, H and Minamisawa, K. [2008]. Microbial community analysis of field-grown soybeans with different nodulation phenotypes. Appl. Environ. Microbiol., 74, 5704-5709. Ji, X., Lu, G., Gai, Y., Zheng, C and Mu, Z. [2008]. Biological control against bacterial wilt and colonization of mulberry by an endophytic Bacillus subtilis strain. FEMS Microbiol Ecol, 65, 565-573. Jiang, CY., Sheng, XF., Qian, M and Wang, QY. [2008]. Isolation and characterization of a heavy metal-resistant Burkholderia sp. from heavy metal-contaminated paddy field soil and its potential in promoting plant growth and heavy metal accumulation in metalpolluted soil. Chemosphere, 72, 157-164. Kämpfer, P. [2007]. Taxonomy of phosphate solubilizing bacteria. First International Meeting on Microbial Phosphate Solubilization. In: Velázquez, E. and Rodríguez-Barrueco, C. [Eds.]. Developments in plant and soil sciences. [pp. 101-106]. The Netherlands: Springer. Kang, SM., Joo, GJ., Hamayun, M., Na, CI., Shin, DH., Kim, HY., Hong, JK and Lee, IJ. [2009]. Gibberellin production and phosphate solubilization by newly isolated strain of Acinetobacter calcoaceticus and its effect on plant growth. Biotechnol Letters, 31, 277281. Khan, MS., Zaidi, A and Wani, PA. [2007]. Role of phosphate-solubilizing microorganisms in sustainable agriculture - A review. Agron Sustain Dev 27, 29-43. Khan, NH., Ahsan, M., Yoshizawa, S., Hosoya, S., Yokota, A and Kogure, K. [2008]. Multilocus sequence typing and phylogenetic analyses of Pseudomonas aeruginosa isolates from the ocean. Appl. Environ. Microbiol., 74, 6194-6205. Kim, CH., Han, SH., Kim, KY., Cho, BH., Kim, YH., Koo, BS and Kim, YC. [2003]. Cloning and expression of pyrroloquinoline quinone [PQQ] genes from a phosphatesolubilizing bacterium Enterobacter intermedium. Curr. Microbiol., 47, 457-461. Kim, YH., Bae, B and Choung, YK. [2005]. Optimization of biological phosphorus removal from contaminated sediments with phosphate-solubilizing microorganisms. J. Biosci. Bioengg, 99, 23-29. King, S., McCord, BR and Riefler, RG. [2005]. Capillary electrophoresis single-strand conformation polymorphism analysis for monitoring soil bacteria. J. Microbiol. Methods, 60, 83-92. Koeuth, T., Versalovic, J. and Lupski, JR. [1995]. Differential subsequence conservation of interspersed repetitive Streptococcus pneumoniae BOX elements in diverse bacteria. Genome Research, 5, 408-418. Kuklinsky-Sobral, J., Araújo, WL., Mendes, R., Geraldi, IO., Pizzirani-Kleiner, AA. and Azevedo, JL. [2004]. Isolation and characterization of soybean-associated bacteria and their potential for plant growth promotion. Environ. Microbiol., 6, 1244-1251. Kumar, B., Kumar, MS., Annapurna, K. and Maheshwari, DK. [2006]. Genetic diversity of plant growth-promoting rhizobia isolated from a medicinal legume, Mucuna pruriens Linn. Current Science, 91, 1524-1529.

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Martha-Helena Ramírez-Bahena, Alvaro Peix, Eustoquio Martínez-Molina et al.

Kwon, GH., Lee, HA., Park, JY., Kim, JS., Lim, J., Park, CS., Kwon, DY., Kim, YS. and Kim, JH. [2009]. Development of a RAPD-PCR method for identification of Bacillus species isolated from Cheonggukjang. International J Food Microbiol, 28, 282-287. Laguerre, G., Allard, MR., Revoy, F. and Amarger, N., [1994]. Rapid identification of rhizobia by restriction-fragment-length-polymorphism analysis of PCR-amplified 16S ribosomal RNA-genes. Appl. Environ. Microbiol., 60, 56–63. Lechner, S., Mayr, R., Francis, KP., Prüss , BM., Kaplan, T., Wiessner-Gunkel, E., Stewart, GSAB. and Scherer, S. [1998]. Bacillus weihenstephanensis sp. nov. is a new psychrotolerant species of the Bacillus cereus group. International J. Syst Bacteriol, 48, 1373-1382. Lee, D.H., Zo, YG., and. Kim SJ. [1996]. Non-radioactive method to study genetic profiles of bacterial communities by PCR-single-strand-conformation polymorphism. Appl. Environ. Microbiol., 62, 3112-3120. Lenaerts, J., Lappin-Scott, HM., and Porter, J. [2007]. Improved fluorescent in situ hybridization method for detection of bacteria from activated sludge and river water by using DNA molecular beacons and flow cytometry. Appl. Environ. Microbiol., 73, 20202023. Lin, T.F., Huang, HI., Shen, FT. and Young, CC. [2006]. The protons of gluconic acid are the major factor responsible for the dissolution of tricalcium phosphate by Burkholderia cepacia CC-Al74. Biores Technol, 97, 957-960. Liu, PY., Shi, ZY., Lau, YJ., Hu, BS., Shyr, JM., Tsai, WS., Lin, YH. and Tseng, CY. [1995]. Comparison of different PCR approaches for characterization of Burkholderia [Pseudomonas] cepacia isolates. J. Clin. Microbiol., 33, 3304-3307. Liu, WT., Marsh, TL., Cheng, H. and Forney, LJ. [1997]. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl Environ Microbiol, 63, 4516-4522. Loganathan, P. and Nair, S. [2004]. Swaminathania salitolerans gen. nov., sp. nov., a salttolerant, nitrogen-fixing and phosphate-solubilizing bacterium from wild rice [Porteresia coarctata Tateoka]. Intern. J. Syst. Evol. Microbiol, 54, 1185-1190. Lucchini, S., Thompson, A. and Hinton, JCD. [2001]. Microarrays for microbiologists. Microbiology, 147, 1403-1414. Mackay, IM. [2004]. Real-time PCR in the microbiology laboratory. Clin. Microbiol. Infectious Diseases 10, 190-212. Malik, S., Beer, M., Megharaj, M. and Naidu, R. [2008]. The use of molecular techniques to characterize the microbial communities in contaminated soil and water. Environment International, 34, 265-276. Marsh, TL., Saxman, PR; Cole, JR. and Tiedje, JM. [2000]. Terminal restriction fragment length polymorphism analysis program, a web-based research tool for microbial community analysis. Appl. Environ. Microbiol. 66, 3616-3620. Martín, M., Gibello, A., Lobo, C., Nande, M., Garbi, C., Fajardo, C., Barra-Caracciolo, A., Grenni, P. and Martínez-Iñigo, MJ. [2008]. Application of fluorescence in situ hybridization technique to detect simazine-degrading bacteria in soil samples. Chemosphere, 71, 703-710. Martínez-Murcia, AJ., Acinas, SG. and Rodriguez-Valera F. [1995]. Evaluation of prokaryotic diversity by restrictase digestion of 16S rDNA directly amplified from hypersaline environments. FEMS Microbiol. Ecol, 17, 247-256.

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Novel Approaches for Analysis of Biodiversity of Phosphate-Solubilizing Bacteria

35

Maszenan, A., Seviour, R.J., Patel, BK. and Wanner, J. [2000]. A fluorescently-labelled rRNA targeted oligonucleotide probe for the in situ detection of G-bacteria of the genus Amaricoccus in activated sludge. J. Appl. Microbiol, 88, 826-835. Miambi, E., Guyot, JP. and Ampe, F. [2003]. Identification, isolation and quantification of representative bacteria from fermented cassava dough using an integrated approach of culture-dependent and culture-independent methods. Intern. J. Food Microbiol, 82, 111120. Mino, T. [2000]. Microbial selection of polyphosphate-accumulating bacteria in activated sludge wastewater treatment processes for enhanced biological phosphate removal. Biochemistry [Moscow], 65, 341-348. Mittal, S., and Johri, BN. [2008]. Influence of management practices on the diversity of pseudomonads in rhizosphere soil of wheat cropping system. Biol. Fertil Soils, 44, 823831. Mora, D., Ricci, G., Guglielmetti, S., Daffonchio, D. and Fortina, MG. [2003]. 16S-23S rRNA intergenic spacer region sequence variation in Streptococcus thermophilus and related dairy streptococci and development of a multiplex ITS-SSCP analysis for their identification. Microbiology SGM, 149, 807-813. Moschetti, G., Peluso, A., Protopapa, A., Anastasio, M., Pepe, O. and Defez, R. [2005]. Use of nodulation pattern, stress tolerance, nodC gene amplification, RAPD-PCR and RFLP16S rDNA to discriminate genotypes of Rhizobium leguminosarum biovar viciae. Syst. Appl. Microbiol., 28, 619-631. Moyer, CL., Dobbs, FC. and Karl, DM. [1994]. Estimation of diversity and community structure through restriction fragment length polymorphism distribution analysis of bacterial 16S rRNA genes from a microbial mat at an active, hydrothermal vent system, Loihi seamount, Hawaii. Appl. Environ. Microbiol., 60, 871–879. Mullis, KB. and Faloona, FA. [1987]. Specific synthesis of DNA in vitro via a polymerasecatalyzed chain reaction. Methods Enzymol., 155, 335-350. Muresu, R., Sulas, L., Polone, E. and Squartini, A. [2005]. PCR primers based on different portions of insertion elements can assist genetic relatedness studies, strain fingerprinting and species identification in rhizobia. FEMS Microbiol. Ecol., 54, 445-453. Muyzer G., de Waal EC., Uitterlinden AG. [1993]. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol., 59, 695–700. Muyzer, G., Brinkhoff, T., Nübel, U., Santegoeds, C., Schäfer, H. and Wawer, C. [2004]. Denaturing Gradient Gel Electrophoresis [DGGE] in microbial ecology. In: Kowalchuk GA., De Bruijn, FJ., Head, IM., Akkermans, ADL. and Van Elsas, JD., editors. Molecular Microbial Ecology Manual [pp. 743-770]. The Netherlands: Kluwer Academic Publishers. Myers, RM., Fischer, SG., Lerman, LS. and Maniatis, T. [1985]. Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis. Nucleic Acids Research, 13, 3131-3145. Naik, PR., Raman, G., Narayanan, K.B. and Sakthivel, N. [2008]. Assessment of genetic and functional diversity of phosphate solubilizing fluorescent pseudomonads isolated from rhizospheric soil. BMC Microbiology, 8, 230.

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36

Martha-Helena Ramírez-Bahena, Alvaro Peix, Eustoquio Martínez-Molina et al.

Nesme, X. and Normand, P. [2004]. Easy individual strain and community typing by rDNA ITS1 analysis. In: Kowalchuk G. A., De Bruijn, F. J., Head, I. M., Akkermans, A. D. L. and Van Elsas, J. D., editors. Molecular Microbial Ecology Manual [pp. 671-688]. The Netherlands: Kluwer Academic Publishers. Ogier, JC., Son, O., Gruss, A., Tailliez, P. and Delacroix-Buchet A. [2002]. Identification of the bacterial microflora in dairy products by temporal temperature gradient gel electrophoresis. Appl. Environ. Microbiol., 68, 3691-3701. Panas, P., McMullan, G. and Dooley, JSG. [2007]. RT-TGGE as a guide for the successful isolation of phosphonoacetate degrading bacteria. J. Appl. Microbiol., 103, 237-244. Pandey, A., Trivedi, P., Kumar, B. and Palni, LM. [2006]. Characterization of a phosphate solubilizing and antagonistic strain of Pseudomonas putida [B0] isolated from a subalpine location in the Indian Central Himalaya. Curr. Microbiol., 53, 102-107. Payne, GW., Vandamme, P., Morgan, SH., LiPuma, JJ., Coenye, T., Weightman, AJ., Jones, TH. and Mahenthiralingam, E. [2005]. Development of a recA gene-based identification approach for the entire Burkholderia genus. Appl. Environ. Microbiol., 71, 3917–3920. Payne, GW., Ramette, A., Rose, HL., Weightman, AJ., Jones, TH., Tiedje, JM. and Mahenthiralingam, E. [2006]. Application of a recA gene-based identification approach to the maize rhizosphere reveals novel diversity in Burkholderia species. FEMS Microbiol. Lett, 259, 126-132. Peix, A., Mateos, P.F., Rodriguez-Barrueco, C., Martínez-Molina, E. and Velazquez, E. [2001]. Growth promotion of common bean [Phaseolus vulgaris L.] by a strain of Burkholderia cepacia under growth chamber. Soil Biol. Biochem, 33, 1927-1935. Peix, A., Rivas, R., Velázquez, E., Mateos, P.F., Martínez-Molina, E., Muñoz-Herrera, A., Gómez-Alonso, A. and Velázquez, E. [2005]. Application of horizontal staircase electrophoresis in agarose minigels to the random intergenic spacer analysis of clinical samples. Electrophoresis, 26, 4402-4410. Peix, A., Valverde, A., Rivas, R., Igual, JM., Ramírez-Bahena, MH., Mateos, PF., SantaRegina, I., Rodríguez-Barrueco, C., Martínez-Molina, E. and Velázquez, E., [2007]. Reclassification of Pseudomonas aurantiaca into Pseudomonas chlororaphis and proposal of three subspecies, P. chlororaphis subsp. chlororaphis subsp. nov., P. chlororaphis subsp. aureofaciens subsp. nov. and Pseudomonas chlororaphis subsp. aurantiaca subsp. nov. Intern J. Syst Evol Microbiol, 57, 1286-1290. Peix, A., Lang, E., Verbarg, S., Spröer, C., Rivas, R., Santa-Regina, I., Mateos, PF. MartínezMolina, E., Rodríguez-Barrueco, C. and Velázquez, E. [2009]. Acinetobacter strains IH9 and OCI1, two rhizospheric phosphate solubilizing isolates able to promote plant growth, constitute a new genomovar of Acinetobacter calcoaceticus. Syst. Appl. Microbiol, in press. Pesaro, M. and Widmer, F. [2006]. Identification and specific detection of a novel pseudomonadaceae cluster associated with soils from winter wheat plots of a long-term agricultural field experiment. Applied and Environmental Microbiology, 72, 37-43. Pirone, L., Chiarini, L., Dalmastri, C., Bevivino, A. and Tabacchioni, S. [2005]. Detection of cultured and uncultured Burkholderia cepacia complex bacteria naturally occurring in the maize rhizosphere. Environ. Microbiol., 7, 1734-1742. Poonguzhali, S., Madhaiyan, M. and Sa, T. [2008]. Isolation and identification of phosphate solubilizing bacteria from Chinese cabbage and their effect on growth and phosphorus utilization of plants. Journal of Microbiology and Biotechnology, 18, 773-777.

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37

Pulawska, J. and Sobiczewski, P. [2005]. Development of a semi-nested PCR based method for sensitive detection of tumorigenic Agrobacterium in soil. Journal of Applied Microbiology, 98, 710-721. Rajkumar, M., Ma, Y. and Freitas, H. [2008]. Characterization of metal-resistant plant-growth promoting Bacillus weihenstephanensis isolated from serpentine soil in Portugal. Journal Basic Microbiology, 48, 500-508. Rakosy-Tican, E. and Aurori, CM. [2007]. The usefulness of the gfp reporter gene for monitoring Agrobacterium-mediated transformation of potato dihaploid and tetraploid genotypes. Plant Cell Reports, 26, 661-671. Ramírez-Bahena, MH., García-Fraile, P., Peix, A., Valverde, A., Rivas, R., Igual, JM., Mateos, PF., Martínez-MolinA, E. and Velázquez, E. [2008]. Revision of the taxonomic status of the species Rhizobium leguminosarum [Frank 1879] Frank 1889AL, Rhizobium phaseoli Dangeard 1926AL and Rhizobium trifolii Dangeard 1926AL. R. trifolii is a later synonym of R. leguminosarum. Reclassification of the strain R. leguminosarum DSM 30132 [=NCIMB 11478] as Rhizobium pisi sp. nov. Intern. J. Syst. Evol. Microbiol., 58, 2484-2490. Rangarajan, S., Loganathan, P., Saleena, LM. and Nair, S. [2001]. Diversity of pseudomonads isolated from three different plant rhizospheres. J. Appl. Microbiol., 91, 742-749. Ranjard, L., Poly, F., Lata, J.C., Mougel, C., Thioulouse, J., and Nazaret, S. [2001]. Characterization of bacterial and fungal soil communities by automated ribosomal intergenic spacer analysis fingerprints: biological and methodological variability. Appl. Environ. Microbiol., 67, 4479-4487. Ricke P, Kolb S, Braker G. [2005]. Application of a newly developed ARB softwareintegrated tool for in silico terminal restriction fragment length polymorphism analysis reveals the dominance of a novel pmoA cluster in a forest soil. Appl. Environ. Microbiol. 71, 1671-1673. Rivas, R., Velázquez, E., Valverde, A., Mateos, P.F. and Martínez-Molina, E. [2001]. A two primers random amplified polymorphic DNA procedure to obtain polymerase chain reaction fingerprints of bacterial species. Electrophoresis, 22, 1086-1089. Rivas, R., Trujillo, ME., Sánchez, M., Mateos, P.F., Martínez-Molina, E. and Velázquez, E. [2004]. Microbacterium ulmi sp. nov., a xylanolytic, phosphate-solubilizing bacterium isolated from sawdust of Ulmus nigra. Intern. J. Systematic. Evol. Microbiol, 54, 513517. Rivas, R., Peix, A., Mateos, P.F., Trujillo, ME., Martínez-Molina, E. and Velázquez, E. [2006]. Biodiversity of populations of phosphate solubilizing rhizobia that nodulate chickpea in different Spanish soils. Plant Soil, 287, 23-33. Rivas, R., Martens, M., de Lajudie, P. and Willems, A. [2009]. Multilocus sequence analysis of the genus Bradyrhizobium. Syst. Appl. Microbiol, 32, 101-110. Rodríguez, H. and Fraga, R. [1999]. Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnol. Adv., 17, 319-339. Rodríguez-Navarro, DN., Camacho, M., Leidi, EO., Rivas, R. andVelázquez, E. [2004]. Phenotypic and genotypic characterization of rhizobia from diverse geographical origin that nodulate Pachyrhizus species. Syst. Appl. Microbiol., 27, 737-745.

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38

Martha-Helena Ramírez-Bahena, Alvaro Peix, Eustoquio Martínez-Molina et al.

Rogers, S. and Cambrosio, A. [2007]. Making a New Technology Work: The Standardization and Regulation of Microarrays. The Yale Journal of Biology and Medicine, 80, 165-178. Sahu, MK., Sivakumar, K., Thangaradjou, T. and Kannan, L. [2007]. Phosphate solubilizing actinomycetes in the estuarine environment: an inventory. J. Environ. Biol., 28, 795-798. Saikaly, PE., Barlaz, MA. and de Los Reyes, FL. [2007]. Development of quantitative realtime PCR assays for detection and quantification of surrogate biological warfare agents in building debris and leachate. Appl. Environ. Microbiol., 73, 6557-6565. Santillana, N,. Ramírez-Bahena, MH., García-Fraile, P., Velázquez, E. and Zúñiga, D. [2008]. Phylogenetic diversity based on rrs, atpD, recA genes and 16S-23S intergenic sequence analyses of rhizobial strains isolated from Vicia faba and Pisum sativum in Peru. Arch. Microbiol., 189, 239-247. Sarita, S., Sharma, PK., Priefer, UB. and Prell, J. [2005]. Direct amplification of rhizobial nodC sequences from soil total DNA and comparison to nodC diversity of root nodule isolates, FEMS Microbiol Ecol., 54, 1–11. Schwieger, F. and Tebbe, CC. [1998]. A new approach to utilize PCR-single-strandconformation polymorphism for 16S rRNA gene-based microbial community analysis. Appl. Environ. Microbiol., 64, 4870-4876. Seguin, P., Graham, PH., Sheaffer, CC., Ehlke, NJ. and Russelle, MP. [2001]. Genetic diversity of rhizobia nodulating Trifolium ambiguum in North America. Can. J. Microbiol., 47, 81-85. Sekiguchi, H., Tomioka, N., Nakahara, T. and Uchiyama, H. [2001]. A single band does not always represent single bacterial strains in denaturing gradient gel electrophoresis analysis. Biotechnol. Lett, 23, 1205–1208. Sergeev, N., Distler, M., Vargas, M., Chizhikov, V., Herold, KE. and Rasooly, A. [2006]. Microarray analysis of Bacillus cereus group virulence factors. J. Microbiol. Methods, 65, 488-502. Sheffield, VC., Cox, DR. and Myers, RM. [1989]. Attachment of a 40 bp G+C rich sequence [GC-clamp] to genomic DNA fragments by polymerase chain reaction results in improved detection of single-based changes. Proc. Natl. Acad. Sci. USA, 86, 232-236. Simpson, DJ., Fry, JC., Rogers, HJ. and Day, MJ. [2007]. Transformation of Acinetobacter baylyi in non-sterile soil using recombinant plant nuclear DNA. Environ Biosaf Res, 6, 101-112. Somani, LL., Bhandari, SC., Saxena, SN., and Gulati, IJ. [1990]. Phosphomicroorganisms. In: Somani L.L. [Ed.]. Biofertilizers. Jodhpur, India: Scientific Publishers. Sorokin, A., Candelon, B., Guilloux, K., Galleron, N., Wackerow-Kouzova, N., Ehrlich, SD., Bourguet, D. and Sanchis, V. [2006]. Multiple-locus sequence typing analysis of Bacillus cereus and Bacillus thuringiensis reveals separate clustering and a distinct population structure of psychrotrophic strains. Appl. Environ. Microbiol., 72, 1569-1578. Srivastava, S. and Srivastava, KA. [2006]. Biological phosphate removal by model based continuous cultivation of Acinetobacter calcoaceticus. Process Biochem, 41, 624-630. Stackebrandt, E., Frederiksen, W., Garrity, G.M., Grimont, PAD., Kampfer, P., Maiden, MCJ., Nesme, X., Rossello-Mora, R., Swings, J., Truper, HG., Vauterin, L., Ward, AC. and Whitman, WB. [2002]. Report of the adhoc committee for the re-evaluation of the species definition in bacteriology. Internatl J Syst Evol Microbiol, 52, 1043-1047.

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Stamford, NP., Santos, PR., Santos, CES., Freitas, ADS., Dias, SHL. and Lira, MA. Jr. [2007]. Agronomic efectiveness of biofertilizers with phosphate rock, sulphur and Acidithiobacillus for yam bean grown on a Brazilian tableland acidic soil. Biores Technol, 98, 1311-1318. Stover, CK., Pham, XQ., Erwin, AL., Mizoguchi, SD., Warrener, P., Hickey, MJ., Brinkman, FS., Hufnagle, WO., Kowalik, DJ., Lagrou, M., Garber, RL., Goltry, L., Tolentino, E., Westbrock-Wadman, S., Yuan, Y., Brody, LL., Coulter, SN., Folger, KR., Kas, A., Larbig, K., Lim, R., Smith, K., Spencer, D., Wong, GK., Wu, Z., Paulsen, IT., Reizer, J., Saier, MH., Hancock, RE., Lory, S., Olson, MV. [2000]. Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature, 406, 959-964. Tan, Z., Hurek, T., Vinuesa, P., Müller, P., Ladha, JK. and Reinhold-Hurek, B. [2001]. Specific detection of Bradyrhizobium and Rhizobium strains colonizing rice [Oryza sativa] roots by 16S-23S ribosomal DNA intergenic spacer-targeted PCR. Appl. Environ. Microbiol., 67, 3655-3664. Taghavi, S., Garafola, C., Monchy, S., Newman, L., Hoffman, A., Weyens, N., Barac, T., Vangronsveld, J. and van der Lelie, D. [2009]. Genome survey and characterization of endophytic bacteria exhibiting a beneficial effect on growth and development of poplar trees. Appl. Environ. Microbiol., 75, 748-757. Tiedje, JM., Asuming-Brempong, S., Nüsslein, K., Marsh, TL. and Flynn, SJ. [1999]. Opening the black box of soil microbial diversity. Appl. Soil. Ecol., 13, 109-122. Tyson, GW. and Banfield, JF. [2005]. Cultivating the uncultivated: a community genomics perspective. Trends Microbiol, 13, 411-415. Valverde, A., Igual, JM., Peix, A., Cervantes, E. and Velázquez, E. [2006]. Rhizobium lusitanum sp. nov. a bacterium that nodulates Phaseolus vulgaris. Internatl J. Syst. Evol. Microbiol, 56, 2631-2637. van der Werf, M.J., Pieterse, B., van Luijk, N., Schuren, F.,van der Werff-van der Vat, B., Overkamp, K. and Jellema, RH. [2006]. Multivariate analysis of microarray data by principal component discriminant analysis: prioritizing relevant transcripts linked to the degradation of different carbohydrates in Pseudomonas putida S12. Microbiol, 152, 257272. van Bruggen, AH., Semenov, AM., Zelenev, VV., Semenov, AV., Raaijmakers, JM., Sayler, RJ., de Vos, O. [2008]. Wave-like distribution patterns of gfp-marked Pseudomonas fluorescens along roots of wheat plants grown in two soils. Microb Ecol, 55, 466-475. Vanbroekhoven, K., Ryngaert, A., Wattiau, P., De Mot, R. and Springael, D. [2004]. Acinetobacter diversity in environmental samples assessed by 16S rRNA gene PCRDGGE fingerprinting. FEMS Microbiol. Ecol., 50, 37-50. Vassilev, N., Vassileva, M. and Nikolaeva, I. [2006]. Simultaneous P-solubilizing and biocontrol activity of microorganisms: potentials and future trends. Appl. Microbiol Biotechnol, 71, 137-144. Velázquez, E., Cruz-Sánchez, JM., Mateos, PF. and Martínez-Molina, E. [1998]. Analysis of stable low-molecular-weight RNA profiles of members of the family Rhizobiaceae. Appl. Environ. Microbiol., 64, 1555-1559. Velázquez, E., Trujillo, ME., Peix, A., Palomo, JL., García-Benavides, P., Mateos, PE., Ventosa, A. and Martínez-Molina, E. [2001]. Stable low molecular weight RNA analyzed by staircase electrophoresis, a molecular signature for both prokaryotic and eukaryotic microorganisms. Syst. App. Microbiol., 24, 490-499.

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Velázquez, E., Rivas, R., del Villar, M., Valverde, A., Peix, A., Mateos, PF. and MartínezMolina E. [2006]. A new approach for separating low-molecular-weight RNA molecules by staircase electrophoresis in non-sequencing gels. Electrophoresis, 27, 1732-1738. Velázquez, E., Rojas, M., Lorite, MJ., Rivas, R., Zurdo-Piñeiro, JL., Heydrich, M. and Bedmar, EJ. [2008]. Genetic diversity of endophytic bacteria which could be find in the apoplastic sap of the medullary parenchym of the stem of healthy sugarcane plants. J. Basic Microbiol., 48, 118-124. Versalovic, J., Schneider, M., de Bruijn, FJ., and Lupski, JR. [1994]. Genomic fingerprinting of bacteria using repetitive sequencing-based polymerase chain reaction. Methods Mol Cell Biol, 5, 25-40. Vila, J., Marcos, MA. and Jimenez de Anta, MT. [1996]. A comparative study of different PCR-based DNA fingerprinting techniques for typing of the Acinetobacter calcoaceticusA. baumannii complex. J. Med. Microbiol., 44, 482-489. Watt, M., Hugenholtz, P., White, R. and Vinall K. [2006]. Numbers and locations of native bacteria on field-grown wheat roots quantified by fluorescence in situ hybridization [FISH]. Environ. Microbiol., 8, 871-884. Watt, RM., Wang, J., Leong, M., Kung, H., Cheah, KSE., Liu, D., Danchin, A. and Huang, J. [2007]. Visualizing the proteome of Escherichia coli: an efficiet and versatile method for labeling chromosomal coding DNA sequences [CDSs] with fluorescent protein genes. Nucleic Acids Res., 35, 1-11. Wayne, L., Brenner, GDJ., Colwell, RR., Grimont, PAD., Kandler, O., Krichevsky, MI., Moore, LH., Moore, WEC., Murray, RGE., Stackebrandt, E., Starr, MPL. and Trüper, HG. [1987]. Report of the ad-hoc-committee on reconciliation of approaches to bacterial systematics. Internatl J. Syst. Bacteriol, 37, 463-464. Welsh, J. and McClelland, M. [1990]. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acid Res, 18, 7213-7218. Williams, JGK., Kubelic, AR., Livak, KJ., Rafalski, JA. and Tingey, SV. [1990]. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acid Res., 18, 6531-6535. Wilson, EO. [1988]. The current state of biological diversity. In: Wilson, EO. and Peter, FM. [Eds.] Biodiversity [3-18]. Washington, DC: National Academy Press. Wu, L., Thompson, DK., Li. G., Hurt, RA., Tiedje, JM. and Zhou, J. [2001]. development and evaluation of functional gene arrays for detection of selected genes in the environment. Appl. Environ. Microbiol., 67,5780-5790. Yabuuchi, E., Kosako, Y., Oyaizu, H., Yano, I., Hotta, H., Hashimoto, Y., Ezaki, T. and Arakawa, M. [1992]. Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia [Palleroni and Holmes 1981] comb. nov. Microbiol. Immunol., 36, 1251-1275. Yamamoto, S., Bouvet, PJ. and Harayama, S. [1999]. Phylogenetic structures of the genus Acinetobacter based on gyrB sequences: comparison with the grouping by DNA-DNA hybridization. Internatl J. Syst. and Evol Bacteriol, 49, 87-95. Zurdo-Piñeiro, JL., Velázquez, E., Lorite, MJ., Brelles-Mariño, G., Schröder, EC., Bedmar, EJ., Mateos, PF. and Martínez-Molina, E. [2004]. Identification of fast-growing rhizobia nodulating tropical legumes from Puerto Rico as Rhizobium gallicum and Rhizobium tropici. Syst Appl Microbiol, 27, 469-477.

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Zurdo-Piñeiro, JL., García-Fraile, P., Rivas, R., Peix, A., León-Barrios, M., Willems, A., Mateos, PF., Martínez-Molina, E., Velázquez, E. and van Berkum, P. [2009]. Rhizobia from Lanzarote, the Canary Islands, that nodulate Phaseolus vulgaris have characteristics in common with Sinorhizobium meliloti from mainland Spain. Appl. Environ. Microbiol., DOI: AEM.02811-08v1.

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In: Phosphate Solubilising Microbes for Crop Improvement ISBN 978-1-60876-112-8 Editors: M. S. Khan and A. Zaidi © 2009 Nova Science Publishers, Inc.

Chapter 3

EFFECTS OF PHOSPHATE SOLUBILIZING MICROORGANISM ON SOIL PHOSPHORUS FRACTIONS Metin Turan∗ Atatürk University, Faculty of Agriculture, Department of Soil Science, 25240, Erzurum-Turkey

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ABSTRACT Soil phosphorus [P] fractions can be used as indicators for short- and long-term P availability. The labile organic and inorganic P concentrations are critical sources of P in agro-ecosystems, representing an active reservoir [source and sink] of P. The less available P pools [other than residual P] may be better measures of potentially plant available P, since these pools represent the soil P reservoir that can re-supply labile pools over time. Little information is available on the effect of P-solubilizing microorganism on P pools in soil orders. This chapter focuses on short- and long-term plant availability and efficiency of microorganisms in solubilizing P sources within multiple soil types and to better understand the additional benefits of these microorganisms between soil P pools.

3.1. SOIL P AVAILABILITY AND BEHAVIOR FOR PLANT UPTAKE Phosphorus is an essential part of the energy transfer system in plants. Phosphorus plays a pivotal role in the nutrition of all plants as an essential element participating in a wide array of physiological and biochemical processes occurring in living organisms [Vance et al. 2003]. The element is generally abundant in soil but, due to its highly reactive nature, the amount of phosphorus in form available to plants is a limiting factor; deficiencies lead to slower and often stunted growth and decreased yields. Phosphorus is sequestered mainly through the ∗

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mechanisms of precipitation and adsorption. It is capable of adsorption onto the surface of soil particles and also reacts readily with soil cations, particularly iron, aluminium and calcium, to form insoluble compounds, which plants are unable to utilise [Wild, 1988]. The conventional approach to improve phosphorus nutrition for optimum crop yield is to apply a phosphatic fertilizer which contains phosphorus in soluble form. However, the applied P is also subjected to the same fixation processes, resulting in a large fraction of it unavailable to plants. It has been estimated that the proportion of phosphorus fertilizer used by plants is in the order of only 5–25% [Wild, 1988]. While to maintain high yields, farmers need to continually apply more fertilizers than is used by crops [Goldstein, 1986]. Phosphorus is taken up from the soil solution by plant roots as orthophosphate ions, principally H2PO4and to a lesser extent HPO42-. Several factors can influence both the rate and the amount of P uptaken by the plant and, therefore, can affect the recovery of a single application of P fertilizer. The same factors can also affect the recovery of P reserves accumulated in the soil from past additions of P as fertilizer or manure. The most important factors controlling the availability of P to plant roots are its concentration in the soil solution and the P-buffer capacity of the soil. The latter controls the rate at which P in the soil solution is replenished, i.e. the rate of desorption of P from the solid phase of the soil, which is faster in soils with a high buffer capacity [Syers et al. 2008]. Most soils contain substantial reserves of total P, most of which remains relatively inert, and only less than 10% of soil P enters the plant-animal cycle [Kucey et al. 1989]. Consequently, P deficiency is widespread and P fertilizers are almost universally required to maintain crop production. Although, the P in these fertilizers is initially plant available, it rapidly reacts with the soil constituents and becomes progressively less available for uptake by plant. In acid soils, the reaction products are aluminum and iron phosphates while in the predominantly calcareous soils, the reaction products are calcium phosphates [Sundara et al. 2002]. As a result most of the P applied [often as much as 90%] is rendered unavailable for plant uptake but is retained in insoluble form [Figure 1].

Figure 1. Phosphorus availability in different soil pH degrees [adapted from Brady and Well, 2005].

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Thus, soils commonly have large reserves of “fixed” P that could support long-term crop requirements if it is mobilized through appropriate soil management practices, involving organic matter additions and/or use of P-solubilizing microbes. This view began to be challenged for a number of reasons. Field experiments showed that plant-available P residues could accumulate, at least in some soils, and that these residues increased crop yields. It was also realized that the results from many of the laboratory experiments were unlikely to relate to what happened in field soils because the conditions used for the laboratory experiments were inappropriate. This suggested that new concepts about the behavior of P in soil needed to be formulated.These new concepts, accepted at least among many involved in soil and fertilizer P research, relate to P equilibria in soils which explain reasonably well changes in the extractability of soil P, and the decrease in plant availability of added P with time. These equilibria primarily involve adsorption and absorption reactions that may be largely reversible with time. For P, which in the short- and long-term will be plant available, the current concept is that this P is held by soil components with a continuum of bonding energies. Building on this concept, pools of soil P related to the accessibility and extractability, and thus the plantavailability of the P, can be categorized and conceptualized and expressed diagrammatically [Figure 2]. Phosphorus in the soil solution, the first pool, is immediately available for uptake by plant roots and is present in solution in ionic forms. The second pool represents resin-Pi and Po, NaHCO3 -Pi and Po that is only weakly bonded to the surfaces of soil components [most labile]. This Pi and Po is readily available because it is in equilibrium with Pi in the soil solution and is readily transferred to the soil solution as plant roots take up Pi. Readily available Pi was often described as labile P in papers published in 1950s. The P in the third pool is less readily available NaOH-Pi and Po [less labile] for plant uptake [chemi-adsorbed to amorphıs and crystaline Al and Fe], but it can become available over time.

Figure 2. A simple schematic representation of phosphorus pools in the plant-soil system [adapted from Arau´jo et al. 1993a].

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This P is more strongly bonded to soil components, or is present within the matrices of soil components as absorbed P [i.e. P adsorbed on internal surfaces]. The P in the fourth pool is only very slowly available or H2SO4 –Pi and Po [most resistance], often over periods of many years. It has a low or very low extractability. It is P that is very strongly bonded to soil components, or is P that has been precipitated as slightly-soluble P compounds, or it is part of the soil mineral complex, or it is unavailable due to its position within the soil matrix. Fifth pool is residual [unavailable] for plant uptake [Araujo et al. 2004]. The most important feature of this conceptual model is the reversible transfer of P between the first three pools and it is this that clearly confronts the concept of irreversible fixation of P in soil. However, when a water-soluble P source is added to soil only a small fraction remains in the soil solution, the remaining P becomes distributed between the readily- and less readily-available pools.

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3.2. INGREDIENT OF GROWTH MEDIA INFLUENCE SOIL MICROBIAL ACTIVITY AND P SOLUBILITY Phosphosphate solubilizing microorganisms in general, have been found effective in solubilizing insoluble P in soils [Khan et al. 2007]. The solubilization effect is generally due to the production of organic acids, such as, citric, glutamic, succinic, lactic, oxalic, glyoxalic, maleic, fumaric, tartaric and a-ketobutyric acids; releaded by PSM into the liquid medium [Maliha et al. 2004; Turan et al. 2006]. The action of organic acids has been attributed to their chelation property. Because P-solubilizing microorganisms render more phosphates into solution than is required for their growth and metabolism, the surplus is absorbed by plants [Alexander, 1977]. Plant uptake of nutrients from soil is more marked in the ‘rhizosphere’ surrounding the root than outside this zone [Darrah, 1993]. Root exudation of various chemical molecules into the rhizosphere is largely dependent on the nutritional status of the plant, with some species exuding organic acid anions in response to P and Fe deficiency or phytosiderophores due to Fe and Zn defiency [Jones and Darrah, 1994]. Consequently, the released compounds can cause some nutrient elements to be relatively more available for uptake by plants. The rate of exudation itself is increased by the presence of microbes in the rhizosphere [Gardner et al. 1983] and promoted by the uptake and assimilation of certain nutrient elements. The composition of root exudates can be complex, and often ranges from mucilage, root border cells, extracellular enzymes, simple and complex sugars, phenolics, amino acids, vitamins, organic acids, nitrogenous macromolecules such as purines, and nucleosides to inorganic or gaseous molecules [Uren and Reisenauer, 1988]. Many of these organic substrates excreted into the rhizosphere, particularly amino acids, organic acids, proteins, carbohydrates and vitamins, promote microbial biosynthesis of ethylene [Arshad and Frankenberger, 1990], a powerful plant signal controlling development. On nutrient-rich media, a large number of soil microorganisms can solubilize poorly soluble Ca-phosphates, because high growth rates are often associated with proton release and hence, dissolution of Ca-phosphates. Proton release is not effective in mobilizing P from Fe or Al phosphates or Pi adsorbed onto Fe or Al oxides. For these forms of P, mobilization requires the release of organic acid anions, which release Pi via ligand exchange or chelation

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of Fe or Al [Banik and Dey, 1983]. Soil microorganisms and their activities play an important role in biogeochemical cycles and provides plant with nutrients in available form. Microbial densities and activities are detected largely in rhizosphere of plants than out side of the rhizosphere. So the changes in chemical, physical and biological properties of soil are studied extensively. Several kinds of microorganism in rhizosphere carry out decomposition of organic matter and release several plant nutrients, accelerate the absorption of phosphorus and protect plants from plant pathogen. Under nutrient-poor conditions and hence, lower microbial growth rates, P solubilization are often strongly reduced [Syers et al. 2008]. Therefore, isolates selected as strong P solubilizers in vitro may not be effective in the rhizosphere due to lack of carbon and other nutrients. Additionally, P mobilization may be transient because of the re-formation of poorly soluble P forms [Delvasto et al. 2006] and the uptake of Pi by microorganisms [Hoberg et al. 2005]. Due to the ease of isolating microorganisms with apparently high P solubilization capacity, many studies have been conducted to investigate the effect of inoculation with P solubilizers on plant growth and Pi uptake. In several pot- and field-experiments, inoculation with P-solubilizing microorganisms resulted in increased plant growth and Pi uptake [Kundu and Gaur, 1980; Kumar and Narula, 1999; Wani et al. 2007a and b]. Compared to plants grown in sterile media, Pi uptake by oat [Avena sativa] plants inoculated with a soil microbial community increased by 120% and 320%, when grown with Fe-phosphate and Ca-phosphate [rock-P], respectively [Gerretsen, 1948]. In contrast, there are also reports where inoculation [Rhizobium meliloti] did not increase plant [Medicago sativa L., cv Aragon] growth and Pi uptake [Azcón-Aguilar et al. 1986; Badr el-Din et al. 1986]. The poor effectiveness of inoculated strains to increase the plant available Pi, may be the result of poor growth and survival due to lack of nutrients and/or low competitiveness compared to the indigenous microflora. Successful inoculants must be ‘rhizospherecompetent.’ A number of traits have been shown to be important for rhizosphere competence, including motility, high growth rate, ability to synthesize amino acids and vitamin B1, ability to utilize organic acids, presence of certain cell surface proteins as well as rapid adjustment to changing conditions [Lugtenberg and Dekkers, 1999]. In the rhizosphere, plant and microbial solubilization of nutrient and mineralization processes occur simultaneously. For bulk soil, it has been shown that mineralization/solubilization dominate at low soil C/P ratios, whereas immobilization [uptake of P by the microbial biomass] exceeds mineralization/solubilization at high C/P ratios [He et al. 1997]. Root exudates consist predominantly of sugars and, hence, are C-rich. Therefore, it can be assumed that microbial immobilization of P dominates in the rhizosphere. Thus, plants and microorganisms compete for P. For instance, McLaughlin et al. [1988] investigated the distribution of P in the plant [wheat] -soil [calcixerollic xerochrept] system after addition of isotopically labeled residues or inorganic P fertilizer [32PO4 and 33PO4 derived from mineral fertilizer]. Of the fertilizer P, 18% was taken up by the plants and 29% by the soil microbial biomass. Moreover, a 65% residue P was taken up by the microbial biomass and 16% by plants suggesting that the microbial biomass is more competitive at acquiring P than plants [McLaughlin and Alston, 1986]. In the presence of a readily available carbon source, P is rapidly immobilized in the microbial biomass. However, upon C depletion, microbial growth rates decrease and part of the microbial biomass may die off, releasing P [Philip and Hammond, 2008]. For example, on day two, increasing the amount of glucose-C added to the soil demonstrated a substantial increase in P in the microbial biomass and a decrease in plant available P [resin P]. However,

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microbial biomass P decreased progressively due to depletion of C and, as a result of net P release from the microbial biomass, plant available P increased [Marschner et al. 2006 and 2007]. This clearly showed the importance of C supply in P immobilization in the microbial biomass, the rapid turnover of microbial biomass once C is depleted, and the direct and negative relationship between microbial P and plant available P. Seeling and Zasoski [1993] suggested that P uptake by the microbial biomass could be beneficial for plants, because it would decrease P fixation by maintaining low inorganic P concentrations in the soil solution.

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3.3. SOLUBILIZATION OF MINERAL PHOSPHATES It is generally accepted that the major mechanism of mineral phosphate solubilization [mps] is through the action of organic acids, synthesized by soil microorganisms [Leyval and Berthelin, 1989; Salih et al. 1989, Rodriguez and Fraga, 1999]. Production of organic acids results in acidification of the microbial cell and its surroundings. Consequently, Pi may be released from a mineral phosphate by proton substitution for Ca2+ [Goldstein, 1994]. Among the organic acids, gluconic acid seems to be the most frequent agent of mps and is reported as the principal organic acid produced by phosphate-solubilizing bacteria, such as, Pseudomonas sp. [Illmer and Schinner, 1992], Erwinia herbicola [Liu et al. 1992] and Pseudomonas cepacia [Goldstein et al. 1993]. Other organic acid identified in strains with phosphatesolubilizing ability is 2-ketogluconic acid, which is present in Rhizobium leguminosarum [Halder et al. 1990], Rhizobium meliloti [Halder and Chakrabartty, 1993], Bacillus firmus [Banik and Dey, 1982], and other unidentified soil bacteria [Duff and Webley, 1959]. Strains of Bacillus liqueniformis and Bacillus amyloliquefaciens were found to produce mixtures of lactic, isovaleric, isobutyric, and acetic acids [Rodríguez and Fraga, 1999]. Other organic acids, such as, glycolic, oxalic, malonic, and succinic acid, have also been reported among phosphate solubilizers [Banik and Dey, 1982; Illmer and Schinner, 1992]. Alternative possibilities for mps have been proposed which is based on the lack of a linear correlation between pH and the amount of solubilized P [Asea et al. 1988; Ehrlich, 1990]. In addition, no significant amounts of organic acid production could be detected from a phosphate-solubilizer fungus, Penicillium sp. [Illmer and Schinner, 1992]. Further studies have shown that the release of H+ to the outer surface in exchange for cation uptake or with the help of H+ translocation ATPase could constitute alternative ways for solubilization of mineral phosphates. Other mechanisms have also been considered, such as the production of chelating substances by microorganisms [Duff and Webley, 1959] as well as the production of inorganic acids, such as sulphidric, nitric, and carbonic acid. However, the effectiveness of such processes has been questioned and their contribution to P release in soil appears to be negligible [Vázquez, 1996].

3.4. ORGANIC PHOSPHATE SOLUBILIZATION Soil contains a wide range of organic substrates, which can be a source of P for plant growth. To make this form of P available for plant nutrition, it must be hydrolyzed to inorganic P. Mineralization of most organic phosphorous compounds is carried out by means

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of phosphatase enzymes. The presence of a significant amount of phosphatase activity in soil has been reported [Kremer, 1994; Sarapatka and Kraskova, 1997]. Important levels of microbial phosphatase activity have been detected in different types of soils [Kirchner et al. 1993; Kucharski et al. 1996]. In fact, the major source of phosphatase activity in soil is considered to be of microbial origin [Garcia et al. 1992; Xu and Johnson, 1995]. In particular, phosphatase activity is substantially increased in the rhizosphere [Tarafdar and Junk, 1987]. The pH of most soils ranges from acidic to neutral values. Thus, acid phosphatases should play a major role in this process. Significant acid phosphatase activity was observed in the rhizosphere of slash pine [Pinus ellioti] in two forest Spodosoils [Fox and Comerford, 1992]. Burns [1983] studied the activity of various phosphatases in the rhizosphere of maize [Zea mays], barley [Hordeum vulgare], and wheat [Triticum aestivum] and reported that phosphatase activity was higher in the inner rhizosphere at acidic and neutral soil pH. Soil bacteria expressing a significant level of acid phosphatases included strains from the genera Rhizobium [Abd-Alla, 1994], Enterobacter, Serratia, Citrobacter, Proteus and Klebsiella [Thaller et al. 1995], Pseudomonas [Gügi et al. 1991] and Bacillus [Skrary and Cameron, 1998]. According to Greaves and Webley [1965], approximately 30–48% of culturable soil and rhizosphere microorganisms utilize phytate. On the other hand, Richardson and Hadobas [1997] reported that 63% of culturable soil bacteria were able to grow on this substrate as carbon and P source on agar medium. However, of these, only 39–44% could utilize phytate as a P source in liquid medium, while a very low proportion could use it as a C source. All of these studies provide evidence supporting the role of bacteria in rendering organic P available to plants [Tarafdar and Claassen, 1988]. The degradability of organic phosphorous compounds depends mainly on the physico-chemical and biochemical properties of their molecules. For instance, nucleic acids, phospholipids, and sugar phosphates are degraded easily, while phytic acid, polyphosphates, and phosphonates are decomposed slowly [Ohtake et al. 1996; McGrath et al. 1998]. The mineralization of these compounds is carried out by phosphatases. The dephosphorylating reactions involve hydrolysis of phosphoester or phosphoanhydride bonds. The acid phosphohydrolases, unlike alkaline phosphatases, show optimal catalytic activity at acidic to neutral pH values. Moreover, they can be further classified as specific or nonspecific acid phosphatases, in relation to their substrate specificity. Rossolini et al. [1998] has published a comprehensive review of bacterial nonspecific acid phosphohydrolases. The specific phosphohydrolases with different activities include: 3′-nucleotidases and 5′-nucleotidases [Burns and Beacham, 1986]; hexose phosphatases [Pradel and Boquet, 1988]; and phytases. A specific group of P releasing enzymes is those able to cleave C-P bonds from organophosphonates [Ohtake et al. 1996; McGrath et al. 1998].

3.5. PHOSPHORUS SOLUBILIZING BACTERIA AFFECT DIFFERENTIAL P FRACTION OF SOIL Understanding the short- and long-term dynamics of soil P under different fertilizer treatments is important for the sustainable management of cropping systems. Sequential chemical extraction methods such as, the one developed by Hedley et al. [1982a and b], later modified by Tiessen and Moir [1993], have been widely used to chemically fractionate the

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continium of soil P. Attempts have been made to interpret these fractions in terms of functional inorganic and organic P pools [Cross and Schlesinger, 1995]. It is generally assumed that P availability to plants decreases with increasing strength of the chemicals used in a sequential fractionation procedure. Measurable P fractions such as, resin- or bicarbonateextractable P are thought to be labile-P that contributes more to plant available-P, while hydroxide- and acidextractable P fractions are beleived to be P forms of moderate or low availability [Tiessen et al. 1984; Cross and Schlesinger, 1995]. However, considering the continuous nature of P forms in soils, the functional interpretation of operationally defined P fractions should be carefully monitored. This is even more pronounced in the soils that undergo periodic flooding, which causes significant changes in the proportions of different P fractions extracted with chemical procedures [Huguenin-Elie et al. 2003].

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3.5.1. Total Phosphorus The concentrations of the various P fractions vary with depth, slope position and order of soil [Ozgul et al. 2007]. Total P contents of the aridisol and mollisol were considerably higher than those of the inceptisol and entisol [Table 1]. In general, P fractions were more abundant in the top soil than the deeper soil layers. Phosphorus solubilizing bacteria and fungi can increase soil P availability and may enhance crop yields in P deficient soil. In this context, Turan et al. [2007] conducted a greenhouse study to determine the effect of phosphatesolubilizing bacteria [Bacillus FS-3] on phosphorus contents of tomato [Lycopersicon esculentum L.], growth performance and residual phosphorus in soil. The results have shown that phosphorus availability of soil increased when phosphate-solubilizing bacteria [PSB] was applied. Amount of plant available form of soil phosphorus fraction [resin-Pi + NaHCO3-Pi + NaHCO3-Po + NaOH-Pi + NaOH-Po] increased with PSB application. In all fertilizer types, bacteria application has approximately converted 20% of lesser available phosphorus in to labile forms. In all of the fertilizer, plant shoot and root dry weight and P uptake were generally greater in the PSB applications than without PSB. A statistically significant differences in total P concentrations between treatments with/without phosphate-solubilizing microorganism were recorded [Table 2]. Average soil P contents in treatments without PSM [FS-3] ranged between 385.3 and 410.9 mg kg-1, which was higher than those [380.2 and 404.8 mg kg-1] of the treatments with PSM inoculations suggesting that PSM significantly decreased the total P content of soil, which coul probably be due to microbial solubilization of inorganic and organic phosphorus forms in soil.

3.5.2. Labile P fractions The labile P pool of the surface horizon [sum of resin-Pi, NaHCO3-Pi and NaHCO3-Po] decreased from 21.3 mg kg-1 in the mollisol to 14.6 mg kg-1 in the inceptisol, 10.5 mg kg-1 in the aridisol, and 6.6 mg kg-1 in the entisol [Table 1]. In the same horizon, inorganic labile P [resin Pi + NaHCO3-Pi] accounted for 69%, 67%, 48%, and 5% of the labile P pool in the mollisol, inceptisol, entisol and aridisol, respectively. This fraction accounted for 2.5-0.9% of total P in the horizons of the mollisol, 1.8-0.7% in the inceptisol, 0.7-0.3% in the aridisol and 1.00.7% in the entisol. These proportions are similar to those found in other studies conducted

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Effects of Phosphate Solubilising Microorganism on Soil Phosphorus Fractions

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in the similar climatic region [Arau´jo et al. 1993b; Agbenin and Tiessen, 1994], in semi-arid regions in Brazil [Cross and Schlesinger, 2001] and also in temperate areas in Saskatchewan [Roberts et al. 1985]. For all soils, NaOH-Po was by far the largest fraction of extractable organic P, and the second largest fraction overall. This is not untypical for soils being under native vegetation with no fertilizer application [Hedley et al. 1982a; Tiessen et al. 1992; Crews, 1996]. Areas under cultivation, especially where fertilizer is applied, normally contain a greater fraction of inorganic P [Tiessen et al. 1992; Arau´jo et al. 1993a; Arau´jo et al. 1993b; Ball-Coelho et al. 1993; Arau´jo and Salcedo, 1997]. However, NaOH-Po and NaHCO3-Po concentrations decreased with soil depth and were consistent with organic matter accumulation in the surface layer. Both NaOH-Po and NaHCO3-Po were higher at lower slope position, consistent with higher organic matter levels at lower positions on slope, a trend which may be related to wetter soil water regimes at the bottom of the slopes. The lowinput subsistence agriculture practiced in the area depends on the continuous supply of P from soil reserves along the production cycle. The common crops, like, cereals [wheat and maize], beans and cassava take up a variable amount of P, depending mostly on weather condition and biomass accumulation, but on average P uptake is around 10 kg ha-1yr-1 [Turan and Sezen, 1999]. The resin-Pi and NaHCO3-Pi concentrations in the surface horizons of the mollisol were above the critical levels established for more humid areas [Turan and Sezen 1999], suggesting the potential for crop production without fertilizer application for several years before a P deficiency occur. The inceptisol, aridisol and especially the entisol does not contain enough available P to sustain crop production based on labile P pool sizes. Actual production years could be longer if the labile pool is replenished over time by the more stable fractions [NaOH-Pi, NaOH-Po, and H2SO4-Pi] but continued agricultural productions with low inputs and no or limited fertilizer addition must take into account that in the long run the native supply will be exhausted. As an example, Gunes et al. [2009] conducted a study to determine the effectiveness of Bacillus FS-3 and Aspergillus FS9 in enhancing strawberry [Fragaria x ananasa cultivar Fern] yield and mineral content of leaves and fruits using a P-deficient calcareous aridisol. Phosphorus fertilizer addition increased soil P fractions. In the absence of microbial inoculation, all P pools increased linearly and significantly [R2 > 0.91] with P application. The greatest increases upon P addition [change in P pool upon addition of 200 kg P ha-1 [P0/P200] >10%] occurred in the most plant available inorganic fractions [resin-Pi and NaHCO3-Pi]. Also, the non-occluded organic P fractions associated with soil colloids [NaHCO3-Po] increased [P0/P200 = 6-7%]. In the unamended soil, P application resulted in a 6% increase in P0/P200 of the humic and/or Fe and Al compounds [NaOH-Po] while for all other fractions, independent of micro-organism addition, the addition of 200 kg P ha-1 resulted in less than a 2% increase. However, when Bacillus FS-3 was also used with 50, 100, 150 and 200 kg P ha-1, increased resin-Pi by 44%, 38%, 34%, and 42%, respectively. While application of 50, 100, 150 and 200 kg P ha-1 along with Bacillus FS-3, increased NaHCO3-Pi by 45%, 28%, 28%, 9% and NaHCO3-Po by 42%, 47%, 17% and 8%, respectively, suggesting the effectiveness of the bacteria in increasing these two P pools. These results show that Bacillus FS-3 application mostly enhanced directly available P [sum of resin Pi + NaHCO3-Pi + NaHCO3-Po]. In addition, Aspergillus FS9 addition also followed a trend similar to bacterial inoculation, although the increases were not as great as those observed for bacterial application [Table 3].

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Table 1. Phosphorus fractions for four soil orders under native vegetation in Turkey* Inorganic fractions Soil Order

Horizon

Depth cm

Resin-Pi

NaHCO 3 -Pi NaOH-Pi

Organic fractions H2SO4-Pi

NaHCO 3 -Po

NaOH-Po

Residual-P

Total-P

H2SO4 Pi/tota lP

-1

[mg kg ]

Mollisol

Aridisol

Inceptisol

Entisol

A1

0-40

5.9±1.2a

8.9±2.2a

127.5±7.8b

110.2±6.8a

6.5±1.4a

187.8±12.5a

109.0±14.8ab 582.1±20.2a

0.19a

Bt

40-85

3.8±1.1b

7.6±1.1b

110.7±8.2b

99.3±4.7ab

4.8±1.1b

160.6±10.4b

108.5±10.6ab 506.5±17.6b

0.20a

BC

85-115

1.4±0.4c

3.5±0.8c

192.2±9.1a

84.6±6.7b

3.4±0.8b

108.6±11.3c

128.3±11.5a

533.6±18.2a

0.16b

C

115-+

0.9±.0.3d

2.8±0.9c

78.7±6.7c

62.4±4.3c

1.9±0.7c

98.4±8.9bd

100.8±9.7b

390.4±15.6c

0.16b

Ak

0-22

1.3±0.8a

3.7±0.7a

132.1±10.9a

237.1±5.5a

5.5±1.1a

80.8±7.6a

224.3±14.7b

700.2±19.6a

0.33a

2ABk

22-66

0.6±0.2b

2.1±0.4b

117.5±11.6ab

199.2±8.3b

5.2±0.8a

61.2±6.5b

202.5±10.3c

566.2±15.3b

0.35a

3BCk

66-+

0.4±0.1b

1.4±0.2b

98.7±10.2b

104.9±4.6c

2.5±.0.5b

38.6±3.5c

267.4±9.2a

524.9±14.2c

0.20b

A

0-25

6.6±1.1a

3.2±0.3a

53.1±8.6a

198.6±10.2a 4.8±0.6a

87.5±5.2a

159.0±7.6a

531.4±27.6a

0.37a

Bk

25-70

1.8±0.6b

1.6±0.3b

49.4±7.5a

164.4±11.4b 3.2±0.8ab

68.3±4.8b

138.5±5.6b

423.8±30.5b

0.38a

BCk

70-+

1.5±0.4b

0.9±0.6c

36.4±8.3b

99.6±8.3c

2.1±0.6b

42.4±3.1c

118.3±6.8c

345.2±24.9c

0.29b

A11

0-35

1.2±0.4a

2.1±0.6a

67.5±5.5a

50.2±6.7a

3.3±1.3a

60.4±7.4a

125.0±7.4a

320.1±12.6a

0.16a

A12

35-70

0.8±0.3bc 1.3±0.2b

40.7±3.4b

39.4±4.9b

2.2±0.7ab

52.5±5.6ab

118.2±6.8ab

256.5±11.4b

0.15a

Ab

70-05

0.6±0.1c

1.1±0.1b

20.2±4.2c

14.2±5.2c

1.4±0.5b

37.2±4.8b

98.3±8.9b

183.3±13.5c

0.08b

C

105-+

0.4±0.1d

0.6±0.1c

12.7±1.8d

9.6±4.3c

0.9±0.2c

20.4±2.1c

60.8±7.3b

142.2±14.6d

0.07b

*Within each specific soil order and P fraction, means followed by different letters are significantly different [α < 0.05] Pi; inorganic phosphorus, Po; organic phosphorus [Adapted from Ozgul et al. 2007].

Table 2. Effcets of phosphorus solubilizing micro organism on P availability between P fractions

Resin-Pi

Inorganic fractions NaHCOF-Pi i NaOH-Pi

Organic fractions f i NaOH-Po H2SO4-Pi NaHCO 3 -Po -1 [mg kg ]

T1 No P Fertilizer

0.4±0.1b

0.8±0.2b

72.4±4.4

84.4±5.8

2.6±0.7

T2 NSP

1.7±0.4a

3.9±1.4a

74.3±4.6

85.1±6.3

T3 TSP

1.6±0.3a

3.8±1.2a

73.6±4.2

83.2±6.2

T4 DAP

1.7±0.4a

3.6±1.1a

74.8±4.8

T5 PA

1.8±0.5a

3.7±1.0a

72.2±4.6

T6 RP

0.8±0.1a**

1.6±0.6b

Mean

1.33B*

T7 No P Fertilizer

0.8±0.1b

T8 NSP T9 TSP

Residual-P

Total P

98.2±6.8a

135.7±10.2

385.3±14.9c

2.8±0.9

94.2±6.7ab

136.8±11.4

400.4±15.3b

2.9±1.0

95.5±69ab

135.4±9.10

410.9±16.5ab

84.4±5.7

2.8±0.7

97.4±7.1a

133.2±9.8

415.7±17.2a

79.3±6.4

2.7±0.4

90.2±6.4b

130.6±10.7

406.3±16.3b

75.3±4.5

83.7±6.1

2.7±0.8

94.7±6.8ab

134.8±11.1

410.2±16.7ab

2.9B

73.76A

83.35 A

2.75A

95.03A

134.41 A

404.80A

1.0±0.3b

58.5±4.2

83.2±6.7

0.4±0.1

82.3±8.2a

129.2±10.8

380.2±17.2 b

2.4±0.8a

5.2±1.5a

56.4±3.7

82.4±6.2

0.8±0.1

81.1±7.6ab

130.8±11.7

388.2±14.3 b

2.3±0.7a

4.9±1.4a

55.2±3.5

81.3±6.7

0.6±0.1

80.1±7.5b

131.4±13.2

401.5±17.7 a

T10 DAP

2.2±0.7a

4.8±1.6a

57.6±3.8

82.6±6.4

0.5±0.1

81.6±6.4ab

130.2±10.5

402.3±16.9 a

T11 PA

1.9±0.6a

4.5±1.7a

53.1±3.6

74.6±5.9

0.4±0.1

81.7±5.9ab

128.4±8.4

400.0±15.0 a

T12 RP

1.7±0.5a

4.3±1.2a

55.7±3.7

75.2±5.2

0.5±0.1

80.4±6.2b

131.6±9.8

404.8±14.8 a

Mean

2.88A

4.95A

56.08B

79.88 B

0.53B

81.2B

130.26 B

396.16 B

Without PSM application

Treatment

With PSM application

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Pi; inorganic phosphorus, Po; organic phosphorus, Values are means of ten soil samples in each pot, *Means in columns followed by capital letter with/without PSM application, **Means in columns followed by small letter a different phosphorus fertilizer differ significantly [Adapted fromTuran et al. 2007].

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Table 3. Effect of P-solubilizing bacteria [Bacillus FS-3] or fungi [Aspergillus FS9] on soil P fractions of a calcareous aridisol in eastern Turkey amended with 0 to 200 kg P ha-1 P applied

Treatment

Inorganic P fractions Organic P fractions Residual-P Resin-Pi NaHCO3-Pi NaOH-Pi H2SO4-Pi NaHCO3-Po NaOH-Po kg P ha-1 ------------------------------------------------------------ mg kg-1 ------------------------------------------------------------At the onset of the trial, prior to the pot experiment [no amendments] 1.4 2.8 92.7 215.4 5.9 226.6 338.3 After strawberry harvest 0 Control 1.1 c 2.7 b 91.6 c 212.8 a 5.7 c 223.8 a 328.9 a +Aspergillus FS9 1.8 b 3.2 a 95.4 b 211.4 ab 6.2 b 221.5 b 327.4 c +Bacillus FS-3 2.1 a 3.4 a 99.9 a 210.0 b 7.9 a 219.9 c 327.9 b Control 6.3 c 10.4 b 109.9 c 224.7 a 14.4 b 225.0 a 335.5 a 50 +Aspergillus FS9 7.2 b 10.2 b 117.5 a 224.2 b 8.2 c 223.5 b 332.8 b +Bacillus FS-3 9.1 a 14.9 a 114.8 b 221.7 c 20.5 a 220.5 c 331.1 c Control 9.6 c 15.5 c 134.7 c 236.5 a 17.1 c 256.6 a 341.8 a 100 +Aspergillus FS9 10.0 b 17.3 b 138.6 b 234.7 c 21.1 b 252.8 b 340.0 b +Bacillus FS-3 13.2 a 19.8 a 155.5 a 235.5 b 25.1 a 248.6 c 338.4 c Control 15.1 c 26.1 c 165.6 c 245.3 a 29.4 c 275.1 a 354.6 a 150 +Aspergillus FS9 17.5 b 29.5 b 170.6 b 243.3 b 33.5 b 273.4 b 349.2 b +Bacillus FS-3 20.2 a 33.5 a 186.1 a 241.1 c 34.4 a 273.5 b 346.8 c Control 20.2 c 36.6 c 178.3 c 260.2 a 40.8 b 281.5 a 359.9 a 200 +Aspergillus FS9 23.5 b 37.5 b 182.1 b 258.3 b 39.6 c 278.7 b 358.5 b +Bacillus FS-3 28.6 a 39.8 a 200.6 a 256.0 c 44.2 a 276.9 a 356.1 c

Total-P

927 926 920 916 1108 1106 1078 1127 1185 1096 1234 1196 1175 1343 1320 1281

a b c a b c b a c a b c a b c

Means with different letters within a P treatment and soil P pool are significantly different at P < 0.05; The P fractions were determined with the sequential P fractionation [Adapted from Gunes et al. 2009].

Effects of Phosphate Solubilising Microorganism on Soil Phosphorus Fractions

55

These results are consistent with work by Turan et al. [2006; 2007] who showed that application of Bacillus strain FS-3 and Aspergillus strains FS9 and FS-11, in liquid cultures increased P availability from rock phosphate and three calcium phosphates, as also reported by Sundara et al. [2002] and Wan and Wong [2004] who demonstrated an increase in plantavailable P following Bacillus megatarium inoculation.

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3.5.3. Moderately Labile P Fractions The highest concentrations of moderately labile P [NaOH-Pi, NaOH-Po, and H2SO4-Pi] were found in the aridisol followed by the mollisol, inceptisol and entisol [Table 1]. The NaOH-Pi pool represented 30-50% of the moderately labile P pool in the mollisol, 16-20% in the inceptisol, 29-41% in the aridisol, and 28-38% in the entisol [Table 1]. In the surface horizon, where most of the roots of agricultural crops are found, the mollisol and aridisol had more than twice the amount of NaOH-Pi as compared to the inceptisol and entisol. The NaOH-Po fraction was greatest in the mollisol [187.8 mg kg-1 in the surface horizon] compared to 87.5 mg kg-1 in the inceptisol, 80.8 and 60.4 mg kg-1 in aridisol and entisol, respectively. In the inceptisol and aridisol, H2SO4-Pi accounted for more than 50% of the moderately labile P pool throughout the soil profile except for the BCk [calcic] horizon of the aridisol where H2SO4-Pi accounted for 43% of the moderately labile P pool. High quantities of H2SO4-Pi are thought to be a necessary condition for sustainable and continuous productivity in soils of higher pH [Crews, 1996]. When P mineralized from soil organic matter is not sufficient to sustain crop requirements and no additional P is supplied, P availability is mostly governed by P-Ca dissolution and desorption. This form of P release may be particularly important in the semi-arid regions, where low amounts of organic matter and low input agriculture prevail. In acidic soils in the humid tropics, H2SO4-Pi levels are generally low due to intense weathering and P release from Fe/Al-oxides is often insufficient to support sustainable production [Tiessen et al. 1992]. These results indicate that the H2SO4Pi reserve was the largest in the aridisol and inceptisol followed by the mollisol where the NaOH-Po fraction dominated the moderately labile P pools rather than the inorganic P fractions [Cassagne et al. 2000; Cross and Schlesinger, 2001]. Gunes et al. [2009] evaluated P solubilization study in a calcareous aridisol, and assessed the labile P pool [sum of resin-Pi and NaHCO3-Pi and Po] and moderately labile fraction [NaOH-Pi +NaOH-Po] in soil inoculated with or without PSB [Table 3]. The amount of plant available form of soil phosphorus fraction [resin-Pi + NaHCO3-Pi + NaHCO3-Po + NaOH-Pi+ NaOH-Po] increased with PSB application. The rates changed around 20% for all fertilizer types. For instance, in the NSP type fertilizer, while the amount of labile P fraction [Pi + NaHCO3-Pi + NaHCO3-Po + NaOH-Pi+ NaOH-Po, 1.7+3.9+2.8+74.3+94.2, respectively] was 176.9 mg kg-1, it decreased to 145.9 mg kg-1 with PSB application [Table 3]. The results showed that the labile P pool increased with PSB application and thus enhanced plant phosphorus uptake. Depending upon fertilizer types, PSB application increased plant-P with rates of ferilizers. For instance, effectiveness of PSB application on P uptake was 51% in rock phosphate, but it was only 14% in phosphoric acid, as compared with the treatment without PSB.

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Metin Turan

3.5.4. Rresidual P Fraction Residual-P, the most resistant and insoluble fraction varied from 202.5-267.4 mg kg-1 [3251% of total P] in the aridisol to 118.3-159.9 mg kg-1 [30-34% of total P] in the inceptisol, 100.8-128.3 mg kg-1 [19-26% of total P] in the mollisol, and 60.6-125.0 mg kg-1 [39-54% of total P] in the entisol [Tables 1]. The high proportion of residual P in the aridisol suggests a higher degree of weathering than normal expected for this soil order [Walker and Syers, 1976]. However, the sulphuric acid solution might not extract all Ca- P resulting in a higher residual P fraction [Syers et al. 1967; Syers et al. 1968]. Considering that residual P is not likely to become plant available over time, the aridisol and mollisol have similar amount of labile and moderately labile P but in the mollisol more of these P was in the labile fraction and moderately labile organic P fraction whereas in the aridisol, more P was in the H2SO4-Pi fraction. Furthermore, PSB [Bacillus FS-3] application converted 134.41 mg kg-1 residual-P in to 130.26 mg kg-1 residual-P. However, no significant difference was observed among phosphorus fertilizer forms. High quantities of H2SO4-Pi are beleived to be a necessary condition for sustainable and continuous productivity [Crews, 1996]. Phosphorus availability is mostly governed by P-Ca dissolution rate, when soil organic matter bound P is insufficient to sustain crop requirements. This may be particularly important in the semi-arid, where low amounts of organic matter and low input agriculture prevail. This also contrasts with acid soils from humid tropics, where H2SO4-Pi is generally exhausted due to intense weathering and P release from Fe/Al-oxides is insufficient to support continuous production [Tiessen et al. 1992].

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CONCLUSION For all soil orders, residual P increased over depth while more labile fraction was greater at the surface layers. The mollisol contained the greatest pool of labile P. The aridisol and inceptisol had lower labile P pools but larger P reserves. The entisol had the least amount of total P within the A horizon with 40% unavailable for plant growth [residual P]. These results suggest that in mollisol, the soil P supply is greatest to support crop production. In the inceptisol, more P is directly available than in the aridisol and entisol. But in the aridisol, re-supply from more stable P fractions [NaOH and H2SO4-Pi ] might contribute to P availability over time while in the inceptisol and entisol, re-supply of the labile P fraction from the more stable pools will not be sufficient given small pool sizes. For, these three soil orders, regular P applications will be needed to sustain crop production over time. Intrestingly, PSM application reduced the rate of P fertilizer by 20% and increased plant P content between 12%-50%. As a consequence, the composite application of phosphate- solubilizing bacteria and fungi, could be used to develop microbial inoculants for augmenting the productivity of crops in P-deficient soils.

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REFERENCES Abd-Alla, MH.[1994]. Use of organic phosphorus by Rhizobium leguminosarum biovar viceae phosphatases. Biol. Fertil Soils, 18. 216–8. Agbenin, JO., and Tiessen, H. [1994]. Phosphorus transformations in a toposequence of Lithosols and Cambisols from semi-arid north eastern Brazil. Geoderma, 62. 345-362. Alexander, M. [1977]. Introduction to Soil Microbiology. Wiley New York, Wiley Eastern Ltd, New Delhi, 1985, 467 pp. [Third Reprint] Arau´jo, MSB., Salcedo, IH., and Sampaio, EVSB. [1993a]. Efeito de fertilizacoes fosfatadas anuais em solos cultivados com cana-de-acucar: I. Intensidade e formas de acumulacao. Revista Brasileira de Cieˆncia do Solo, Campinas, SP, Brasil, 17. 389-396. Arau´jo, MSB., Salcedo, IH., and Sampaio, EVSB. [1993b]. Efeito de fertilizacoes fosfatadas anuais em solos cultivados com cana-de-acucar: II. Formas disponı´veis e efeito residual do P acumulado. Revista Brasileira de Cieˆncia do Solo, Campinas, SP, Brasil, 17. 397-403. Arau´jo, MSB., and Salcedo, IH. [1997]. Formas preferenciais de acumulacao de fo´sforo em solos cultivados com cana-de-aciicar na Regia˜o Nordeste. Revista Brasileira de Cieˆncia do Solo, Vic¸osa, MG, Brasil 21. 643-650. Arau´jo, MSB., Schaefer, CER., and Sampaio, VSB., [2004]. Soil Phosphorus toposequences of semi-arid Latosol and Luvisol in northeastern Brazil. Geoderma, 119.309-321. Arshad, M. and Frankenberger, WT. Jr. [1990]. Ethylene accumulation in soil in response to organic amendments. Soil Sci. Soc. Am. J. 54. 1026–1031 Asea, PEA., Kucey, RMN., and Stewart, JWB. [1988]. Inorganic phosphate solubilization by two Penicillium sp. in solution culture and soil. Soil Biol. Biochem. 20. 459–64. Azcón-Aguilar, C., Gianinazzi-Pearson, V., Fardeau, J.C., and Gianinazzi, S. [1986]. Effect of vesicular-arbuscular mycorrhizal fungi and phosphate-solubilizing bacteria on growth and nutrition of soybean in a neutral-calcareus soil amended with 32P-45Ca-tricalcium phosphate. Plant Soil, 96. 3–15. Badr el-Din, SMS., Khalafallah, MA., and Moawad, H. [1986]. Response of soybean to dual inoculation with Rhizobium japonicum and phosphate dissolving bacteria. Z. Pflanz Bodenkunde, 149. 130–135. Ball-Coelho, B., Salcedo, IH., Tiessen, H., and Stewart, JWB. [1993]. Short and long-term phosphorus dynamics in a fertilized ultisol under sugarcane. Soil Sci. Soc. Am. J. 57. 1027-1034. Banik, S., and Dey, BK. [1982]. Available phosphate content of an alluvial soil is influenced by inoculation of some isolated phosphate-solubilizing microorganisms. Plant Soil, 69. 353–364. Banik, S., and Dey, BK. [1983]. Phosphate-solubilizing potentiality of the microorganisms capable of utilizing al phosphate as a sole phosphorus source. Zbl. Mikrobiol. 138. 17–23. Brady, N., and Well, RR. [2005]. The Nature and Properties of Soils. Thirteenth Editon. Published by pearson Education [Singapore] Pte. Ltd., Indian Branch, 482 F.I.E. Patparganj, Delhi, India. Burns, RG. [1983]. Extracellular enzyme-substrate interactions in soil. In: Slater JH, Whittenbury R, Wimpenny JWT, editors. Microbes in their Natural Environment. Cambridge: Cambridge Univ Press. pp. 249–298.

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Burns, DM., and Beacham, IR. [1986]. Nucleotide sequence and transcriptional analysis of the Escherichia coli ushA gene, encoding periplasmic UDP-sugar hydrolase [59nucleotidase]: regulation of the ushA gene, and the signal sequence of its encoded protein product. Nucleic Acids Res. 14. 4325–4342. Cassagne, N., Remaury, M., Gauquelin, T., and Fabre, A. [2000]. Forms and profile distrubition of soil phosphorus in alpine Inceptisols and Spodosols [Pyrenees, France]. Geoderma, 95. 162-172. Crews, TE. [1996]. The supply of phosphorus from native, inorganic phosphorus pools in continuously cultivated Mexican agroecosystems. Agriculture, Ecosyst. Environ. 57. 197208. Cross, AF., and Schlesinger, WH. [1995]. A literature review and evaluation of the Hedley fractionation: Applications to the biogeo chemical cycle of soil phosphorus in natural ecosystems. Geoderma, 64. 197–214. Cross, AF., and Schlesinger, WH. [2001]. Biological and geochemical controls on phosphorus fractions in semiarid soils. Biogeochemistry, 52. 155-172. Delvasto, P., Valverde, A., Ballester, A., Igual, JM., Munoz, JA., Gonzalez, F., Blazques, ML., and Garcia, C. [2006]. Characterization of brushite as a re-crystallization product formed during bacterial solubilization of hydroxyapatite in batch cultures. Soil Biol. Biochem. 38. 2645–2654. Darrah, PR. [1993]. The rhizosphere and plant nutrition: A quantitative approach. Plant Soil, 155. 1–20. Duff, RB., and Webley, DM. [1959]. 2-Ketogluconic acid as a natural chelator produced by soil bacteria. Chem. Ind. 1376–1377. Ehrlich, HL. [1990]. Mikrobiologische und biochemische Verfahrenstechnik. In: Einsele A, Finn RK, Samhaber W, editors. Geomicrobiology, 2nd ed. Weinheim: VCH Verlagsgesellschaft. Fox, TR., and Comerford, NB. [1992]. Rhizosphere phosphatase activity and phosphatase hydrolysable organic phosphorus in two forested spodosols. Soil Biol. Biochem. 24. 579– 583. Garcia, C., Fernandez, T., Costa, F., Cerranti, B., and Masciandaro, G. [1992]. Kinetics of phosphatase activity in organic wastes. Soil Biol. Biochem. 25. 361–5. Gardner, WK., Barber, DA., and Parbery, DG. [1983]. The acquisition of phosphorus by Lupinus albus L. III. The probabale mechanism by which phosphorus movement in the soil/root interface is enhanced. Plant Soil, 70. 107–24. Gerretsen, FC. [1948]. The influence of microorganisms on the phosphate intake by the plant. Plant Soil, 1. 51–81. Goldstein, AH. [1986]. Bacterial solubilization of mineral phosphates: historical perspective and future prospects. Am. J. Altern. Agri. 1. 51–57. Goldstein, AH., Rogers, RD., and Mead, G. [1993]. Mining by microbe. Bio/Technology; 11. 1250–1254. Goldstein, AH. [1994]. Involvement of the quinoprotein glucose dehydrogenase in the solubilization of exogenous phosphates by gram-negative bacteria. In: Torriani-Gorini A, Yagil E, Silver, S, editors. Phosphate in Microorganisms: Cellular and Molecular Biology. Washington, DC: ASM Press, pp. 197–203.

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Effects of Phosphate Solubilising Microorganism on Soil Phosphorus Fractions

59

Greaves, MP., and Webley, DM. [1965]. A study of the breakdown of organic phosphates by microorganisms from the root region of certain pasture grasses. J. Appl. Bact. 28. 454465. Gunes, A., Ataoğlu, N., Turan, M., Esikten, A., and Ketterings, QM. [2009]. Effects of phosphate solubilizing bacteria and fungi on strawberry yield and nutrient content. J. Plant Nut. Soil Sci. Plant Analyses [in press]. Gügi, B., Orange, N., Hellio, F., Burini, JF., Guillou, C., Leriche, F., and Guespin-Michel, JF. [1991]. Effect of growth temperature on several exported enzyme activities in the psychrotropic bacterium Pseudomonas fluorescens. J. Bacteriol, 173. 3814–3820. Halder, AK., Mishra, AK., Bhattacharyya, P., and Chakrabartty, PK. [1990]. Solubilization of rock phosphate by Rhizobium and Bradyrhizobium. J. Gen. Appl. Microbiol. 36. 81-92. Halder, AK., and Chakrabartty, PK. [1993]. Solubilization of inorganic phosphate by Rhizobium. Folia Microbiol. 38. 325–330. He, ZL., O’Donnell, AG., and Syers, JK. [1997]. Seasonal responses in microbial biomass carbon, phosphorus and sulphur in soils under pasture. Biol. Fert. Soils, 24. 421–428. Hedley, MJ., Stewart, JWB., and Chauhan, BS. [1982a]. Changes in inorganic soil phosphorus fractions induced by cultivation practices and by laboratory incubations. Soil Sci. Soc. Am. J. 46. 970-976. Hedley, MJ., White, RE., and Nye, PH. [1982b]. Plant-induced changes in the rhizosphere of rape [Brassica napus var. emerald] seedlings: 3. Changes in L value, soil phosphate fractions and phosphatase activity. New Phytologist, 91. 45-56. Hoberg, E., Marschner, P., and Lieberei, R. [2005]. Organic acid exudation and pH changes by Gordonia sp. and Pseudomonas fluorescens grown with P adsorbed to goethite. Microbiol. Res. 160. 177–187. Huguenin-Elie, O., Kirk, GJD., and Frossard, E. [2003]. Phosphorus uptake by rice from soil that is flooded, drained or flooded then drained. Eur. J. Soil Sci. 54. 77–90. Illmer, P., and Schinner, F. [1992]. Solubilization of inorganic phosphates by microorganisms isolated from forest soil. Soil Biol. Biochem. 24. 389–95. Jones, DL., and Darrah, PR. [1994]. Role of root derived organic acids in the mobilization of nutrients from the rhizosphere. Plant Soil, 166. 247–257. Khan, MS., Zaidi, A., and Wani, PA. [2007]. Role of phosphate-solubilizing microorganisms in sustainable agriculture-A review. Agron. Sustain. Dev. 27. 29-43. Kirchner, MJ., Wollum, AG., and King, LD. [1993]. Soil microbial populations and activities in reduced chemical input agroecosystems. Soil Sci. Soc. Am. J. 57. 1289–1295. Kremer, RJ. [1994]. Determination of soil phosphatase activity using a microplate method. Comun. Soil Sci. Plant Anal. 25. 319–325. Kucey, RMN., Janzen, HH., and Leggett, ME. [1989]. Microbially mediated increases in plant available phosphorus. Adv. Agron. 42. 199-225. Kucharski, J., Ciecko, Z., Niewolak, T., and Niklewska-Larska, T. [1996]. Activity of microrganisms in soils of different agricultural usefulness complexes fertilized with mineral nitrogen. Acta Acad Agric Technicae-Olstenensis, 62. 25–35. Kumar, V., and Narula, N. [1999]. Solubilization of inorganic phosphates and growth emergence of wheat as affected by Azotobacter chroococcum mutants. Biol. Fert. Soils, 28. 301–305.

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Metin Turan

Kundu, BS., and Gaur, AC. [1980]. Establishment of nitrogen-fixing and phosphatesolubilizing bacteria in the rhizosphere and their effect on yield and nurtrient uptake of wheat crop. Plant Soil, 57. 223–230. Leyval, C., and Berthelin, J. [1989]. Interaction between Laccaria laccata, Agrobacterium radiobacter and beech roots: influence on P, K, Mg and Fe movilization from minerals and plant growth. Plant Soil, 117. 103–10. Liu, TS., Lee, LY., Tai, CY., Hung, CH., Chang, YS., Wolfram, JH., Rogers. R., and Goldstein, AH. [1992]. Cloning of an Erwinia herbicola gene necessary for gluconic acid production and enhanced mineral phosphate solubilization in Escherichia coli HB101: Nucleotide sequence and probable involvement in biosynthesis of the coenzyme pyrroloquinoline quinone. J. Bacteriol. 174. 5814–9. Lugtenberg, BJJ., and Dekkers, LC. [1999]. What makes Pseudomonas bacteria rhizosphere competent? Environ. Microbiol. 1. 9–13. Maliha, R., Khalil, S., Ayub, N., Alam, S., and Latif, F. [2004]. Organic acids production and phosphate solubilization by phosphate solubilizng micoorganism Pak J Biol Sci, 7.187186. Marschner, P., Solaiman, Z., and Rengel, Z. [2006]. Rhizosphere properties of Poaceae genotypes under P-limiting conditions. Plant Soil, 283. 11–24. Marschner, P., Solaiman, Z., and Rengel, Z. [2007]. Brassica genotypes differ in growth, phosphorus uptake and rhizosphere properties under P-limiting conditions. Soil Biol Biochem. 39. 87–98. McGrath, JW., Hammerschmidt, F., and Quinn, JP. [1998]. Biodegradation of phosphonomycin by Rhizobium huakuii PMY1. Appl. Environ. Microbiol. 64. 356–58. McLaughlin, MJ., and Alston, AM. [1986]. The relative contribution of plant residues and fertiliser to the phosphorus nutrition of wheat in a pasture/cereal system. Aust. J. Soil Res. 24. 517–526. McLaughlin, MJ., Alston, AM., and Martin, JK. [1988]. Phosphorus cycling in wheat-pasture rotations. II. The role of the microbial biomass in phosphorus cycling. Aust. J. Soil Res. 26. 333–342. Ohtake, H., Wu, H., Imazu, K., Ambe, Y., Kato, J., and Kuroda, A. [1996]. Bacterial phosphonate degradation, phosphite oxidation and polyphosphate accumulation. A Res Conserv and Recycling, 18. 125–134. Ozgul, M., Turan, M., and Kettering, QM. [2007]. Short –and long term phosphorus availaility in four soil orders under indigenous vegetation in Turkey. Acta Agric. Scand. Section B. Soil Plant Sci. 57. 357-364. Philip, JW. and Hammond, JP. [2008]. The Ecophysiology of plant-phosphorus interactions. Springer, p. 289. Pradel, E., and Boquet, PL. [1988]. Acid phosphatases of Escherichia coli: molecular cloning and analysis of agp, the structural gene for a periplasmic acid glucose phosphatase. J. Bacteriol. 170. 4916–4923. Richardson, AE., and Hadobas, PA. [1997]. Soil isolates of Pseudomonas spp. that utilize inositol phosphates. Can J Microbiol. 43. 509–516. Roberts, TL., Stewart, JWB., and Bettany, JR. [1985]. The influence of topography on the distribution of organic and inorganic soil phosphorus across a narrow environmental gradient. Can. J. Soil Sci. 65. 651-665.

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Effects of Phosphate Solubilising Microorganism on Soil Phosphorus Fractions

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Rodríguez, H., and Fraga, R. [1999]. Phosphate solubilizng bacteria and their role in plant growth promotion. Biotech Adv. 17.319–339. Rossolini, GM., Shippa, S., Riccio, ML., Berlutti, F., Macaskie, LE., and Thaller, MC. [1998]. Bacterial nonspecific acid phosphatases: physiology, evolution, and use as tools in microbial biotechnology. Cell Mol Life Sci.54.833–50. Salih, HM., Yahya, AY., Abdul-Rahem, AM., and Munam, BH. [1989]. Availability of phosphorus in a calcareus soil treated with rock phosphate or superphosphate as affected by phosphate dissolving fungi. Plant Soil, 120. 181–185. Sarapatka, B., and Kraskova, M. [1997]. Interactions between phosphatase activity and soil characteristics from some locations in the Czech Republic. Rostlinna-Vyroba-UZPI; 43. 415–419. Seeling, B., and Zasoski, RJ. [1993]. Microbial effects in maintaining organic and inorganic solution phosphorus concentrations in a grassland topsoil. Plant Soil, 148. 277–284. Skrary, FA., and Cameron, DC. [1998]. Purification and characterization of a Bacillus licheniformis phosphatase specific for D-alpha-glycerphosphate. Arch. Biochem. Biophys. 349. 27–35. Sundara, B., Natarajam, V., and Hari, K. [2002]: Influence of phosphorus solubilizing bacteria on the changes in soil available phosphorus and sugarcane and sugar yields. Field Crops Res. 77. 43-49. Syers, JK., Williams, JDH., Campbell, AS., and Walker, TW. [1967]. The significance of apatite inclusions in soil phosphorus studies. Proceedings. Soil Sci. Soc.Am. 31. 752-756. Syers, JK., Williams, JDH., and Walker, TW. [1968]. The determination of total phosphorus in soils and parent materials. New Zeal. J. Agric.Research, 11. 757-762. Syers, JK., Jonston, AE., and Curtion, D. [2008]. Efficiency of soil and Fertilizer phophorus use. FAO. Fertilizer and Plant Nutrition Bulletion 18. Food and Agriculture Organization of the United Nations, Rome. Tarafdar, JC., and Junk, A. [1987]. Phosphatase activity in the rhizosphere and its relation to the depletion of soil organic phosphorus. Biol. Fertil. Soil, 3.199–204. Tarafdar, JC., and Claassen, N. [1988]. Organic phosphorus compounds as a phosphorus source for higher plants through the activity of phosphatases produced by plant roots and microorganisms. Biol. Fertil. Soils, 5. 308–12. Thaller, MC., Berlutti, F., Schippa, S., Iori, P., Passariello, C., and Rossolini, GM. [1995]. Heterogeneous patterns of acid phosphatases containing low-molecular-mass Polipeptides in members of the family Enterobacteriaceae. Int. J. Syst. Bacteriol. 4. 255– 261. Tiessen, H., Stewart, JWB., and Cole, CV. [1984]. Pathways of phosphorus transformation in soils of differing pedogenesis. Soil Sci. Soc. Am. J. 48. 853-858. Tiessen, H., Salcedo, IH. and Sampaio, EVSB. [1992]. Nutrient and soil organic matter dynamics under shifting cultivation in semi-arid north-eastern Brazil. Agr. Ecosyst. Environ. 38. 139-151. Tiessen, H. and Moir, JO. [1993]. Characterization of available P by sequential extraction. In: Carter MR [Ed], Soil Sampling and Methods Analysis. Can. Soc.Soil Sci. Lewis Publishers, pp .75-86. Turan, M. and Sezen, Y. [1999]. Effect of Some Soil Properties on phosphorus availability in various pH Soil. GAP. 1. Agricultural Congres. Şanlıurfa,Turkey, pp.1011-1019.

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Metin Turan

Turan, M., Ataoğlu, N. and Sahin, F. [2006]. Evaluation of the Capacity of Phosphate Solubilizng Bacteria and Fungi on Different Forms of Phosphorus in Liquid Culture. J. Sustainable Agric. 28. 99-108. Turan, M., Ataoğlu, N. and Sahin, F. [2007]. Effcets of Bacillus FS-3 on growth of tomato [Lycopersicon esculentum L.] plants ana vailability of phosphorus in soil. Plant Soil Environ. 53. 58-64. Uren, NC. and Reisenauer, HM. [1988]. The role of root exudates in nutrient acquisition. Adv Plant Nutr. 3. 79–114. Vázquez, P. [1996]. México. Bacterias solubilizadoras de fosfatos inorgánicos asociadas a la rhizosfera de los mangles: Avicennia germinans [L.] L y Laguncularia racemosa [L.] Gerth. Tesis para el título de Biologo Marino. Univ. Autónoma de Baja California Sur. La Paz, B.C.S. Vance, CP., Uhde-Stone, C., and Allan, DL. [2003]. Phosphorus acquisition and use: critical adaptations by plants for securing a nonrenewable resource. New Phytol. 157. 423–447. Walker, TW., and Syers, JK. [1976]. The fate of phosphorus during pedogenesis. Geoderma, 15. 1- 19. Wan, JHC., and Wong, MH. [2004]. Effects of earthworm activity and P-solubilizing bacteria on P availability in soil. J. Plant Nutr. Soil Sci. 167. 209-213. Wani, PA, Khan, MS., and Zaidi, A. [2007a]. Synergistic effects of the inoculation with nitrogen fixing and phosphate-solubilizing rhizobacteria on the performance of field grown chickpea. J Plant Nutr Soil Sci. 170. 283-287. Wani, PA; Khan, MS., and Zaidi, A. [2007b]. Co inoculation of nitrogen fixing and phosphate solubilizing bacteria to promote growth, yield and nutrient uptake in chickpea. Acta Agron Hung. 55. 315-323. Wild, A. [1988]. Plant nutrients in soil: Phosphate. In Soil Conditions and Plant Growth, A. Wild [ed.], Logman Scientific and Technical, Exex. 695-742. Xu, JG. and Johnson, RL. [1995]. Root growth, microbial activity and phosphatase activity in oil-contaminated, remediated and uncontaminated soils planted to barley and field pea. Plant Soil, 173. 3–10.

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Chapter 4

ROLE OF PLANT GROWTH PROMOTING MICROORGANISMS FOR SUSTAINABLE CROP PRODUCTION B. Hameeda, G. Harini1, B. Keerthi Kiran1, O. P.Rupela1 and Gopal Reddy∗ 1

Department of Microbiology, Osmania University, Hyderabad 500 007 International Crops Research Institute for the Semi-Arid Tropics [ICRISAT], Patancheru, India 502 324

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Microorganisms associated with plants interact in a very complex manner in which the outcome influences plant health and productivity. The interaction may be symbiotic, non symbiotic, parasitic or neutral to the plants. Plant growth–promoting microorganisms [PGPM] occur naturally in soil particularly in the vicinity of roots and are known to stimulate plant growth. Roots release different types of exudates, which act as signal molecules for colonization and multiplication of microorganisms. Several mechanisms of plant growth promotion by microorganisms, involve production of phytohormones, enzymes, antibiotics, organic acids etc. resulting in enhanced nutrient uptake and suppression of crop pests or pathogens. Induction of systemic resistance by microorganisms in host plants is reported to a broad spectrum of pathogens. Selected microorganisms have been commercialized and their external inoculation is practiced in several countries for improving crop productivity. Effectiveness of inoculant microorganisms depends on the survival, colonization and multiplication in the rhizosphere, competition with native microorganisms and level of nutrients in the rhizosphere including abiotic stresses. Developments in molecular biology revealed in mid 1990’s that only small fraction of microorganisms in a given natural niche can be cultured in laboratory. Metagenomics is the answer to identify indirect-culture independent microorganisms that can be characterized for novel mechanisms of PGPM. This fact leads to develop new and appropriate methodologies for identifying and selecting effective microorganisms for sustainable crop production. ∗

Correspondance to: :[email protected]; [email protected]

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4.1. INTRODUCTION Sustainable agriculture is an integration of traditional techniques with modern advances that are appropriate and its progress depends on development of new methodologies and understanding fundamental processes underlying soil fertility. Reduced use of inorganic fertilizers and agro-chemicals together with increased application of organic matter, low cost biological inputs, plant growth promoting microorganisms [PGPM] as biofertilizers have positive effects on nutrient levels, soil biota and crop production [Rupela et al. 2006]. Sustainable agriculture demands a much greater understanding of basic soil microbial processes and plant microbial interactions in the rhizosphere. Rhizosphere is the most complex region in terms of population and community dynamics of symbiotic, free living, parasitic and saprophytic microorganisms. Beneficial free-living soil bacteria in the rhizosphere that promote plant growth were first defined by Kloepper and Scroth and termed as plant growth promoting rhizobacteria [PGPR] [Kloepper and Schroth 1978]. Based on their mode of action PGPR are classified as plant growth promoting bacteria [PGPB] that directly stimulate plant growth and biocontrol PGPB that indirectly stimulate plant growth by reducing deleterious microorganisms in the rhizosphere. PGPM improve plant growth by one or more mechanisms: stimulation of plant growth, augmentation of nutrient uptake, inhibition of plant pathogens and induce alterations in plant physiology by altering host plants defenses towards pathogen attack [i.e. induced resistance] [Vessey 2003; Sharma et al. 2008]. In addition to this, effective colonization of rhizosphere and their interaction with other soil microorganisms also play a key role in plant growth promotion which is quantified by seed vigor, biomass and proliferation of root system and yield. In addition to the mechanisms for growth promotion research on PGPM has advanced to the detailed exploration of cell-cell signaling mechanisms such as biofilm formation, quorum sensing [Stanley and Lazazzera 2004]. Bacterial activity in the rhizosphere can be altered by plants or other microorganisms via quorum sensing molecules. These research findings have provided an insight for strain improvement and developing the effective microorganisms as commercial inoculants and development of suitable delivery systems that can be transferred to the farmers in a viable form for their consistent performance under field conditions. Potential PGPM reported till date are culturable and only these are characterized for their plant growth promoting activity, but it should be noted that less than 1% of microorganisms that survive in soil are culturable [Leveau 2007]. Hence there is need to use molecular approaches for a more representative assessment of the microbial diversity of both culturable and non-culturable microorganisms and their exploitation for crop production. Sustainable crop production is due to diversity and activity of flora and fauna, associations between specific and diverse microbial populations in the rhizosphere, carbon build up, biological nitrogen fixation, mycorrhizal “infection” of roots, phytohormones, induced resistance, diversified root systems to take up nutrients in soil and many more factors [Uphoff et al. 2006]. However we limit this chapter to explain the role of microorganisms and mechanisms of plant growth promotion, factors involved in colonization, formulation development, commercialization and the use of metagenomics to identify novel PGPM.

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4.2. PLANT GROWTH PROMOTING MICROORGANISMS Plant growth promoting microorganisms are reported for their multiple plant growth promoting activity and protection against plant pathogens in several crops whose diversity varies according to plant-soil-nutrient status [Compant et al. 2005; Tilak et al. 2005; Khan 2005]. Other than rhizosphere, plant growth promoting bacteria are reported from endophytic and epiphytic region [Zinniel et al. 2002; Kishore et al. 2005a], composts and vermicomposts [Hardy and Sivasithamparam 1995; Alvarez et al. 1995; Hameeda et al. 2006a, 2006b] and also novel sources such as milk [Nautiyal et al. 2006]. Plant growth promoting microorganisms involves a group of symbiotic or free-living microbes that colonize the rhizosphere and benefit the plant growth. Based on microbial interactions and inhabitance with various plants, PGPMs can be divided into two groups [a] symbiotic microorganisms [iPGPM], living inside the plant cells b] free-living microorganisms [ePGPM], surviving outside the plant cells, but still promote the plant growth [Gray and Smith 2005]. This includes bacteria, fungi and actinomycetes, which comprises of 3-9% of agriculturally useful soil and the rest is predominately organic matter [Magdoff and van Es 2000].

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4.2.1. Bacteria Symbiotic bacteria includes genera of Rhizobium, Bradyrhizobium, Sinorhizobium, Azorhizobium, Mesorhizobium and Allorhizobium that forms root nodules by invading plant root systems [Martinez-Romero and Wang 2000]. Prominent among these organisms are species of genus Rhizobium whose potential and practical use in agriculture is beyond doubt. Diazotrophic PGPB includes the genus Azospirillum, nitrogen fixing bacteria that has been studied for their effect on plant growth [Bashan et al. 2004]. Beneficial free-living soil bacteria termed as PGPR and/or PGPB includes different strains of genera: Achromobacter [Bertrand et al. 2000], Aeromonas [Zhang et al. 1996], Azoarcus [Hurek et al. 2002], Azotobacter [Boddey et al. 2003], Bacillus [Ryu et al. 2003, Jaizme-vega et al. 2004] Burkholderia [Dalton et al. 2004], Enterobacter [Li et al. 2000], Gluconacetobacter [Tejera et al. 2003], Herbaspirillum [James et al. 1997], Klebsiella [Iniguez et al. 2004], Pseudomonas [Raj et al. 2004, Tripathi et al. 2005], Pantoea sp. [Loiret et al. 2004], Serratia [Kishore et al. 2005b, Hameeda et al. 2006a]. Among these, the genera of Pseudomonas and Bacillus are extensively studied both for plant growth promotion and biological control due to their wide distribution patterns and ecological importance [Bano and Musarrat 2003].

4.2.2. Fungi Plant beneficial fungi in association with roots are termed as plant growth promoting fungi [PGPF] or rhizofungi which includes different genera such as, Aspergillus [Atalla et al. 2003], Penicillium [Tan and cheng, 2003; Chandanie et al. 2006] and Trichoderma [Xiao-Yan et al. 2006; Eziashi et al. 2007]. Mycorhizal fungi that interacts symbiotically with plant roots of various agricultural crops are ectomycorrhizae [EM fungi-Basidiomycota, few of Ascomycota] and the arbuscular mycorrhizae [[AM] fungi-Zygomycota] [Johansson et al.

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2005; Artursson et al. 2006; Crusberg 2008]. Ecto mycorrhizal [EM] fungi form a mycelial sheath around root and very few penetrate, where as arbuscular mycorrhizal [AM] fungi penetrate in to roots with out forming any sheath. Arbuscular mycorrhizal fungi are ubiquitous in nature and include different genera of Endogone, Glomus, Entrophosphora, Gigaspora, Acaulospora, Scutellispora [Al-Raddad 1995]. Mycrorrhizal fungi can also act as biocontrol agents through competition for space by virtue of their ecologically obligate association with roots. Ecto mycorrhizal fungi because of physical sheating morphology may well occupy normal pathogen infection sites and AM fungi have potential to occupy space and infection sites in roots. However, biocontrol mechanisms by mycorrhizal fungi is related to IR, improved plant growth and changes in root morphology, rather than competition per se [Whipps, 2001].

4.2.3. Actinomycetes Among soil borne microorganisms, actinomycetes are of special interest since they possess many properties that could benefit plant growth and suppress pathogenic fungi. Many authors have isolated actinomycetes belonging to different genera of Streptomyces and Actinomadura, Curtobacterium, Microbispora, Micromonospora, Nocardia for plant growth promoting activity [Coombs and Franco 2003; Okazaki 2003; Hamdali et al. 2008a]. Actinomycetes are also reported for their biocontrol activity against various phyto-pathogenic fungi, bacteria, and insect-pests by producing highly active antibiotics. Actinomycetes strains Streptomyces griseus [BH7, YH1] and Micromonospora aurantiac [KH7], inhibited Fusarium sp. and Pythium ultimum in plate conditions [Hamdali et al. 2008b].

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4.3. MECHANISMS OF PLANT GROWTH PROMOTION 4.3.1. Direct Plant Growth Promotion Plant growth promoting microorganisms play a vital role in sustainable crop production by regulating the bio-geo-chemical cycles of both organic and inorganic nutrients and organic matter decomposition to metabolize them to available forms of nitrogen, phosphorous, potassium and iron for plants and thereby improving the yield. Apart from nutrient uptake, plant growth is also stimulated by phytohormones, enzymes and volatile metabolites. Details of the mechanisms are given below. Several observations made on the performance of PGPM on crop plants and their mechanisms are presented in Table 1, 2 and 3 and discussed in the following section.

4.3.1.1. Nitrogen Nitrogen [N], major macro nutrient necessary for plant growth, forms the structural element and involves in metabolism of enzymes, proteins, chlorophylls, nucleic acids. Nitrogen availability is the main limiting factor in many agricultural soils and the leaching of N fertilizers in to the ground water causes serious environmental pollution [Mantelin and Touraine 2004].

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Table 1. Plant growth promoting bacteria Plant Growth Promoting Bacteria Acetobacter diazotrophicus Azospirillum brasilense A. irakense Herbaspirillum sp. P. putida GR12-2 P. alcaligenes PsA15 P. dinitrificans PsD6 B. polymyxa BcP26 Mycobacterium phlei Mbp18 B. pumilus B. subtilis B. amyloliquefaciens Bacillus OSU-140 Bacillus OSU-142 Bacillus [M-13] A. brasilense A. amazonense A10 Bacillus pantothenticus P4 Pseudomonas pieketti Psd6 Burkholderia sp. MSSP

Host Sugar cane [Saccharum officinarum] Maize [Zea Mays], Wheat [Triticum aestivum] Rice [Oryza sativa] Mungbean [Vigna radiata] Cotton [Gossypium sp] Pea [Pisum sativum]

Mechanism of action Biological Nitrogen fixation N and P Nutrient uptake

Reference Sevilla et al. [2001] Dobbelaere et al. [2002]

Biological Nitrogen fixation IAA production N and P, K Nutrient uptake IAA production,

Gyaneshwar et al. [2002] Pattren and Glick [2002] Egamberdiyeva et al. [2004]

Cactus [Carnegiea gigantean]

P-solublization

Puente et al. [2004]

Maize [Zea Mays] Sugar beet [Beta vulgaris] Barley [Hordeum vulgare] Rice [Oryza sativa]

IAA production N and P Nutrient uptake

Idris et al. [2004] Sahin et al. [2004]

IAA production

Thakuria et al. [2004]

Mimosa [Mimosa pudica]

Pandey et al. [2006]

B. megaterium sub sp. Phosphaticum strain-PB A.lipoferum 15 Bacillus sp

Chick pea [Cicer arietinum]

Phytohormone, ACC deaminase production N and P Nutrient uptake N and P Nutrient uptake

Rudresh et al. [2005]

Root development P-solubilization

Muratova et al.[ 2005] Canbolat et al. [2006]

Nutrient uptake N and P Nutrient uptake RP and K solubilization

Zaidi et al. [2006] Yao et al. [2006] Han et al. [2006]

B. subtilis SJ-101 B. subtilis FZB24 B. megaterium B. mucilaginosus

Wheat [Triticum aestivum] Wheat [Triticum aestivum] Barley [Hordeum vulgare] Indian mustard plant [Brassica juncea] Cotton [Gossypium sp] Pepper [Capsicum annuum] Cucumber [Cucumis sativus]

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Table 1. (Continued) Plant Growth Promoting Bacteria Bacillus sp . OSU-142, RC07, M13 Paenibacillus polymyxa RC05 P. putida RC06 Bradyrhizobium sp. B. subtilis A. awamori G. fasciculatum P. putida SP21, SP22 B. japonicum TIIIB P. brassicacearum Am3 P. marginalis Dp1 S. marcescens EB 67 Pseudomonas sp. CDB 35 B. megaterium HKP-1 B. mucilaginosus HKK-1 B. mucilaginosus

Host Sugar beet [Beta vulgaris]

Mechanism of action N and P Nutrient uptake

Reference Ramazan et al. [2006]

Green gram[Phaeolus radiatus]

N and P, K Nutrient uptake IAA production

Zaidi and Khan [2006]

Soybean [Glycine max]

P-solubilization siderophore production ACCD Enzyme production

Rosas et al. [2006]

Hameeda et al. [2006b]

Indian mustard plant [Brassica juncea]

Nutrient uptake, Phytoharomone production Root growth

Groundnut [Arachis hypogaea]

K and N, P nutrient uptake

B. subtilis Bacillus sps NR 2, 4 6 B. subtilis SJ-101 Cyanobacteria A. amazonense Herbaspirillum sp. B. tropica A. brasilense Sp.245 B. subtilis 101 Bacillus sp. J119

Chickpea[Cicer arietinum] Pigeon pea [Cajanus cajan] Indian mustard plant [Brassica juncea] Wheat [Triticum aestivum] Sugar cane [Saccharum officinarum]

Root elongation Siderophre production IAA production, P-solubilization Biological Nitrogen fixation Biological Nitrogen fixation

Sugumaran and Janarthanam et al. [2007] Swain and Ray [2007] Geetha et al. [2008] Zaidi et al. [2006] Karthikeyan et al. [2007] Oliveira et al. [2008]

Tomoto [Lycopersicon esculentum]

Nutrient uptake

Felici et al. [2008]

Maize [Zea Mays], Tomato [Lycopersicon esculentum] Canola [Brassica napus]

IAA production Siderophore production ACC deaminase

Sheng et al. [2008]

P. fluorescens P. putida

Pea [Pisum sativum] Pearl millet [Pennisetum glaucum]

Safronova et al. [2006]

Wu et al. [2006b]

Jalili et al. [2008]

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Table 2. Plant growth promoting fungi Plant Growth Promoting Fungi Glomus intraradices G. fasciculatum G .monosporum G. mosseae A. tubingensis A. niger P. rugulosum

Host Soybean [Glycine max]

Mechanism of action N,P Nutrient uptake

Reference Gamal [2001]

Rice [Oryza sativa]

RP solubilization

Maize [Zea Mays]

Rock P solubilisation

G. clarum G. geosporum T. atroviride

Clover [Trifolium grandiflorum] Tomato [Lycopersicon esculentum]

P-solubilisation

Reddy et al. [2002] Reyes and Antoun [2002] Souchie et al. [2006] Gravel et al. [2007]

IAA production

Table 3. Plant growth promoting actinomycetes Actinomycetes S. griseoflavus S. rimosus S. diastaticu Williopsis saturnus

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S. griseus BH7, YH1 S. cavourensis BH2 Micromonospora aurantiaca KH7

Host Bean [Phaseolus vulgaris] Radish [Raphanus raphanistrum]

Mechanism of action IAA and gibberellic acid

Reference Nassar et al. [2003]

Indole-acetic acid and gibberellic acid

El-Tarabily et al. [2003]

Maize [Zea Mays] Wheat [Triticum aestivum]

IAA production

Nassar et al. [2005]

RP solubilization, Siderophore production IAA production

Hamdali et al. [2008a]

Use of high cost nitrogen based chemical fertilizers can be reduced by applying biological nitrogen fixing [BNF] microorganisms in agriculture fields [Kennedy et al. 2004; Ahmed et al. 2006]. Nitrogen fixing microorganisms convert stable nitrogen gas in to biologically useful form. Rhizobia are well known for their nitrogen fixing ability by forming symbiotic association with legumes. In addition, rhizobial strains colonize non-legume roots and also promote plant growth [Antoun et al. 2005] and are considered as PGPR. Growth promotion of maize [Zea mays] and lettuce [Lactuca sativa] by Rhizobium leguminosarum was reported in field conditions due to mechanisms other than nitrogen fixation [Chabot et al. 1996]. Rhizobium sp.sStrain YAS34 with exopolysaccharide production promoted sunflower [Helianthus annuus] growth [Alami et al. 2002].Diazotrophs colonize the rhizosphere by virtue of competence in carbon rich and nitrogen poor environment and the same is reviewed highlighting the BNF and plant growth promotion by various mechanisms by Dobbelaere et al. [2003]. The mechanisms involved in nitrogen nutrition by PGPB such as NO3-uptake capacity, stimulation of root development, NO3- transport system is reported by Mantelin and Touraine [2004]. However, the signaling and transduction pathways elicited by these bacteria still remain unknown. Elucidation of the mechanisms involved and the exploitation of associative diazotrophs along with symbiotic Rhizobium can affect plant growth and act as an alternative to nitrogenous fertilizer [Glick 1995].

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4.3.1.2. Phosphorus Phosphorus [P], second important plant growth-limiting nutrient after N, is abundantly available in soils in both organic and inorganic forms. However, many soils throughout the world are P-deficient because the free P concentration [the form available to plants], even in fertile soils is very low [Stevenson and cole 1999]. These low levels of P are due to high reactivity of soluble P with calcium [Ca-P] in alkaline soils with high buffering capacity, iron [Fe-P] or aluminum [Al-P] in acidic soils that leads to P precipitation [Ae et al. 1991]. Plants utilize fewer amounts of phosphatic fertilizers that are applied and the rest is rapidly converted into insoluble complexes in the soil [Vassilev and Vassileva 2003]. This leads to the need of frequent application of phosphatic fertilizers, but its use on a regular basis has become a costly affair and also environmentally undesirable. Theoretical estimates have suggested that the accumulated P in soil is sufficient to sustain crop yields worldwide for about 100 years [Goldstein et al. 1993]. Microorganisms that dissolve poorly soluble CaP are termed as ‘mineral phosphate solubilizers’ [MPS] [Gyaneswar et al. 2002]. Phosphate-solubilizing microorganisms [PSMs] secrete organic acids [gluconic, citrate, lactate and succinate] and convert insoluble phosphates to soluble monobasic [H2PO4-] and dibasic [HPO42-] ions. Phosphate-solubilizing microorganisms are ubiquitously present and includes bacterial genera, Bacillus, Enterobacter, Erwinia, Pseudomonas, Azotobacter, Serratia, fungal genera, Penicillium, Aspergillus, and Streptomyces sp. [Rodriguez et al. 2004; Chung et al. 2005]. Mineral phosphate solubilizers utilize the direct oxidation pathway to produce gluconic acid and 2 keto-gluconic acid which is catalyzed by glucose dehydrogenase [GDH]. The activity of periplasmic or membrane-bound GDH is one of the best-studied mechanisms by which MPS bacteria liberate P from poorly soluble mineral phosphates. Mutants of E. asburiae, deficient in GDH activity failed to solubilize P from alkaline soils indicating the role of GDH in MPS [Gyaneshwar et al. 1999]. Phosphate solubilizing ability by PSM depends on the carbon and nitrogen sources and also metal ions [Kim et al. 1998a; Nautiyal et al. 2000; Hameeda et al. 2006c]. Reports of PSMs-plant inoculation associations include Azotobacter chroococcum and wheat [Kumar and Narula 1999], Bacillus circulans and wheat [Singh and Kapoor 1999], Enterobacter agglomerans and tomato [Lycopersicon lycopersicum] [Kim et al. 1998b], Penicillium bilaii and wheat [Triticum aestivum] [Asea et al. 1988]. Mycorrhizal association is mediated by specific carbon sources derived from root exudates and in turn plants will get enhanced nutrient supply. AM fungi improve P availability to plants by secreting organic acids such as citrate, malate and oxalate by creating acidic conditions around the rhizosphere which facilitates phosphorus nutrition by solubilization of rock phosphate [Landerweet et al. 2001; Duponnois et al. 2005]. AM fungi also influence the nutrient absorption like carbon and nitrogen, higher exchange of atmospheric gases by increasing soil porosity by altering soil structure for wind and water resistance by forming glycoprotien complex/glomalin [Steinberg and Rillig 2003; Matsumoto et al. 2005]. Around 95% plant species were reported having symbiotic association with AM fungi [Smith and Read 1997]. Phosphatase [acid and alkaline phosphatases] activity by PSM helps in mobilization of the organic P in soil for the benefit of plants. Studies have shown positive correlation between phosphatase activity and phosphate solubilizing ability [Ponmurugan and Gopi 2006]. Microbial phytases significantly increase the efficiency of plants to use organic P by mineralizaton process [Richardson 2001]. Degradation of insoluble phytate to soluble

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phosphorus is reported by microbial phytases produced by Bacillus, Pseudomonas, Klebsiella and Enterobacter sp. [Greiner et al. 1997; Kerovuo et al. 1998, Richardson 2001]. Bacillus amyloliquefaciens FZB45 significantly increased the maize seedling growth in phosphatelimited plant nutrient medium in the presence of phytate. Phytase negative mutant strain FZB45/M2, whose phyA gene is disrupted, did not stimulate plant growth [Idriss et al. 2002]. Phosphorous uptake by Arabidopsis roots was observed by phytase produced from Aspergillus sp. [Richardson et al. 2001]

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4.3.1.3. Potassium The third important plant nutrient potassium [K] is deficient in soils due to the nonavailability, crop uptake, runoff, leaching and soil erosion [Sheng and Huang 2002]. More than 98% of K in soil exists in the form of silicate minerals as microcline, muscovite, orthoclase, biotite, feldspar and not freely available form to plants [Buchholz and Brown 1993]. Potassium solubilizing bacteria [KSB] may produce organic aicds, or chelating substances to enhance the plant mineral uptake [Sugumaran and Janarthanam 2007; Lin et al. 2002]. Bacillus mucilaginosus that produced mucilage and organic acid increased K availability in soils and increased mineral content in plant [Sheng et al. 2002]. Co-inoculation of KSB and PSB with rock K and P application enhanced the mineral uptake and continuous supply of minerals to increase plant growth [Han et al. 2006]. 4.3.1.4. Iron Under aerobic conditions at physiological pH, iron occurs in the Fe+3 state and forms insoluble hydroxides and oxyhydroxide precipitates. The concentration of available iron in the soil under these conditions is as low as 10 -18 M making this crucial element almost unavailable to the soil micro flora [Cuerinot and Yi, 1994]. Iron being the constituent of many enzymes involved in metabolism, most of the aerobically growing organisms synthesize and secrete structurally diverse compounds under iron starved conditions, designated as siderophores, that bind extra cellular Fe+3 with high affinity and this process is vital in absorption of iron by plants [Ratledge and Dover 2000]. Siderophore production and utilization by rhizobia, is of particular interest due to the dominant role of iron in the nitrogen fixation and assimilation process. Burd et al. [2000] found that the iron deficiency plays a part in the toxicity of some heavy metals, and siderophores produced by Kluyvera ascorbata SUD165 reduced Ni toxicity by supplying the plant with Fe. Siderophore producing P. fluorescens promoted growth of mungbean [Vigna radiata [L.] wilczek] and P. chlororaphis ATCC 9446 and Pseudomonas GRP3A, PRS9 promoted maize growth [Sharma and Johri 2003; Katiyar and Goel 2004]. 4.3.1.5. Phytohormones and Enzymes Apart from mineral nutrition, PGPM are reported to produce various phytohormones suh as auxins [indole-3-acetic acid [IAA]] cytokinins, gibberilins, and ethylene [Lebuhn et al. 1997; Antoun and Pre´vost 2005]. Phyto-hormones affect plant growth by regulating root morphology by increasing lateral root length and root hair [Bloemberg and Lugtenberg 2001]. The concentration of hormonal signals is critical to regulate various physiological processes of plants including seed germination, root formation, branching, tillering, fruit ripening and also to increases plant resistance to environmental factors [Tsavkelova et al. 2006]. Of the

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various phytohormones, IAA, most commonly found in plants, is believed to be synthesized by microorganisms but reports have shown variable effect of IAA in plant growth promotion [Kamilova et al. 2006] Endophytic actinomycetes, Streptomyces hygroscopicus TP-A045, Streptomyces sp. MBR-52 has produced pteridic acids A and B, auxin-like plant growth promoters and increased the formation of adventitious roots of kidney beans [Igarashi et al. 2002]. The improvement in plant growth was attributed primarily to the increase in cytokinins production by A. chroococcum in the rhizosphere. Cytokinins may affect protein synthesis by potentiating the activities of RNA polymerases and matrix chromatin, increasing seed germination, increased plant cell division and branching. Gibberillic acid is produced by diazotrophic bacteria, Azospirillum sp., Azotobacter sp., and helps in promotion of root growth, hair density, nutrient uptake and water absorption [Fulchieri et al. 1993]. Aminocyclopropane-1-carboxylate [ACC] is a precursor of the plant hormone ethylene that causes root inhibition. Microorganisms that produce ACC deaminase hydrolyses ACC into ammonia and alpha-keto glutrate to support plant growth by lowering plant ethylene concentration [Glick et al. 1998]. ACC deaminase producers promote plant growth with a net increase in root length [Glick 2004]. ACC deaminase is produced by Enterobacter, Alcaligenes, Burkholderia, Pseudomonas, Bacillus and Penicillium sp. [Jia et al. 2000]. Enterobacter cloacae UW 4, ACC deaminase transformed strain increased root and shoot fresh weight, seed germination and decreased the occurrence of P. ultimum, when compared with non transformed wild strains [Wang et al. 2000]. Bacteria containing ACC deaminase stimulated plant growth in soils with cadmium toxicity [Belimov et al. 2004].

4.3.1.6. Volatiles in Plant Growth Promotion Volatile organic compounds [VOC], 3-hydroxy -2-butanone [acetoin] and 2,3-butanediol produced by PGPR, Bacillus subtilis GB03 and Bacillus amyloliquefaciens stimulated the growth of Arabidopsis thaliana [Ryu et al. 2003]. Signal pathway activated by VOC by PGPR is dependent on cytokinin activation for growth promotion and is dependent on an ethylene-signaling pathway for induced pathogen resistance. Further studies on gene expression profiling of Arabidopsis genes responding to bacterial volatiles will add up more information about the VOC and their mechanisms for plant growth promotion and induced resistance [Ryu et al. 2005].

4.3.2. Indirect Plant Growth Promotion Mechanisms [Biocontrol] Indirect mechanisms involve the use of biocontrol PGPM, able to suppress the phytopathogenic fungi and their relating disease. The broad mechanisms adopted by biocontrol microorgansisms include antibiosis, antimicrobial enzymes, mycoparasitism, niche exclusion, predation and induced resistance [van Loon and Glick 2004; Zahir et al. 2004]. Biocontrol bacteria such as Pseudomonas, Bacillus, Burkholderia, biocontrol fungus such as Trichoderma and actinomycetes such as Streptomyces, Microbiospora, Micromonospora play a dynamic function in management of plant pathogens by the secretion of extracellular metabolites called as antibiotics reported for their broad spectrum activity. Suppression of pathogens by antagonistic microbes reported are due to the production of antibiotics and other metabolites.The antagonistic nature and biocontrol activity of PGPM and their consequent effect on crop plants and their mechanisms involved [Table 4, 5, 6] are discussed.

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Table 4. Biocontrol bacteria Antagonist B. pumilus INR7 Alcaligenes xylosoxydans A. brasilense Curtobacterium flaccumfaciens P. aeruginosa 7NSK2 Pseudomonas GRC2 B. subtilis P. fluorescens A. awamori A. niger P. digitatum P. fluorescens CHA0 P. putida B. amyloliquefaciens IN937a B. pumilus IN937b, SE34, SE49, T4, INR7 P. fluorescens PTB9 P. fluorescens RGAF 19 P. fluorescens RG 26 B. megaterium RGAF 5 P. macerans RGAF 101 B. mycoides

Target pathogen Erwinia tracheiphila Fusarium udum, Rhizoctonia bataticola P. syringae pv.

Host Tomato [Lycopersicon esculentum] Pigeon pea [Cajanus cajan]

Mechanism of action ISR Chitinase

Reference Zehnder et al. [2000] Vaidya et al. [2001]

Tomato [Lycopersicon esculentum]

Bashan and de-Bashan [2005]

Xylella fastidiosa

Citrus [Citrus aurantifolia]

antimicrobial compounds secretion Anitbiosis

Botrytis cinerea Macrophomina phaseolina Fusarium oxisporum

Tomato [Lycopersicon esculentum] Groundnut [Arachis hypogaea ] Tomato [Lycopersicon esculentum]

ISR Siderophore production Antibiosis

Audenaert et al. [2002] Gupta et al. [2002] Khan and Khan [2002]

Macrophomina phaseolina Fusarium wilt

Tomato [Lycopersicon esculentum] Radish [Raphanus raphanistrum]

Antibiosis ISR

Shaukat and Siddiqui [2003] De Boer et al. [2003]

Sclerotium rolfsii Colletotrichum gloeosporioides Cucumber mosaic virus [CMV] Xanthomonas oryzae pv. oryzae [Xoo] Fusarium oxysporum f. sp. ciceris

Cucumber [Cucumis sativus]

ISR

Jetiyanon et al. [2003]

Rice [Oryza sativa]

2,4-DAPG antibiosis

Wheat [Triticum aestivum]

Antibiosis

Velusamy and Gnanamanickam [2003] Landa et al. [2003]

Suger beet [Beta vulgaris]

ISR

Bargabus et al. [2004]

Cercospora beticola

Araujo et al. [2002]

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Table 4. (Continued) Antagonist P. fluorescens PCL1751 P. fluorescens WCS365 B. subtilis UK-9 P. maltophila PM-4

P. fluorescens P. putida Pseudomonas sp Burkholderia MBf21, MBp1, MBf15 Pseudomonas MPp4 B. cereus B. laterosporus, P. fluorescens S. marcescens B. subtilis QST 713

B. cereus BT8, BP24

Target pathogen Fusarium oxysporum

Host Tomato [Lycopersicon esculentum] Cucumber [Cucumis sativus] Mustard [Brassica nigra] Bean [Cyamopsis tetragonoloba ]

Mechanism of action competition for nutrients and niches ISR Chitinase

Reference Kamilova et al. [2006]

Cow pea [Vigna unguiculata]

Antibiosis

Nwaga et al. [2007]

Fusarium verticillioides M1

Maize [Zea Mays]

Antibiosis

Rodriguez et al. [2008]

Pythium ultimum

Sorghum [Sorghum bicolor]

Antibiosis Siderophore production ISR

Idris et al. [2008]

Xanthomonas euvesicatoria Xanthomonas perforans Jones Phytophthora capsici

Tomato [Lycopersicon esculentum]

Antibiosis

Roberts et al. [2008]

Cacao [Theobroma cacao]

Antibiosis

Melnick et al. [2008]

Alternaria Rhizoctonia bataticola, R. solani, Fusarium oxysporum Sclerotinia sclerotiorum Pythium aphanidermaturm

Neeta and Swati [2008] Yadav et al. [2007]

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Table 5. Biocontrol fungi Antagonist T. harzianum Phoma sp . GS8-2 GS8-3 P. simplicissimum GP17-2 G. mosseae T. harzianum PTh18 T.harzianum

Target pathogen Crinipellis perniciosa Colletotrichum orbiculare

Host Cocoa [Theobroma cacao] Cucumber [Cucumis sativus]

Mechanism of action Chitinase ISR

Reference DeMarco et al. [2000] Chandanie et al. [2006]

Rhizoctonia solani Sclerotinia sclerotiorum

Rice [Oryza sativa] Tomato [Lycopersicon esculentum] Squash [Cucurbita moschata] eggplant [Solanum melongena]

Chitinase mycoparasitism and antibiosis

Prabavathy et al. [2006] Abdullah et al. [2008]

Table 6. Biocontrol actinomycetes Antagonist M. carbonacea Streptomyces sp.

Target pathogen Sclerotinia Phytophthora

Streptomyces sp.

Aspergillus sp. Curvularia lunata, Drechslera maydis, Fusarium subglutinans Cephalosporiumacremonium Plectosporium tabacinum Gaeumannomyces graminis var. tritici

A. missouriensis Microbispora sp. Nocardioides sp. Micromonospora sp. Streptomyces sp. g10 M. rosea, M. chalcea A. philippinensis Streptomyces sp. S. sindeneusis 263

Host Lettuce [Lactuca sativa] Alfalfa [Medicago sativa] Soybean [Glycine max] Maize [Zea Mays]

Mechanism of action Chitinase, -1,3-glucanase, Antibiosis

Reference El-Tarabily et al. [2000] Xiao et al. [2002]

Antibiosis

Bressan [2003]

Lupine [Lupinus Arcticus] Wheat [Triticum aestivum]

Chitinase Antibiosis

El-Tarabily [2003] Coombs et al. [2003]

Fusarium wilt P. aphanidermatum

Banana [Musa acuminata] Cucumber [Cucumis sativus]

Antibiosis 1,3, 1,4 and 1,6-glucanases

Getha et al. [2005] El-Tarabily [2006]

Sclerotium rolfsii Magnaporthe oryzae

Sugar beet [Beta vulgaris] Rice [Oryza sativa]

Antibiosis Antibiosis

Errakhi et al. [2007] Zarandi et al. [2009]

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4.3.2.1. 2,4-Diacetyl Phloroglucinol The broad-spectrum antibiotic 2,4-dicaetyl phloroglucinol [2,4-DAPG] is a major determinant involved in the biological control of a wide range of plant diseases by fluorescent Pseudomonas sp. Of the different antibiotics produced by PGPR, DAPG is non-nitrogen containing antibiotic that is used for the control of root diseases. For instance, DAPG produced by Pseudomonas fluorescens CHA0 has been found active against black root rot of tobacco [Thielaviopsis basicola] and take-all of wheat [Gaeumannomyces graminis var. tritici] while P. fluorescens Q2-87 demonstrated antifungal activity against take-all of wheat and P. fluorescens F113 against damping off of sugar beet [P. ultimum] [Raaijmakers et al. 2002]. A DAPG antibiotic-negative mutant strain F113G22 of P. fluorescens had no affect on proliferation of fungus P. ultimum [Shahnahan et al. 1992].

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4.3.2.2. Pyoluteorin Pyoluteorin [Plt], a phenolic polyketide, is reported for its bactericidal, herbicidal and antifungal functions. Structurally Plt is made up of a resorcinol ring linked to a bichlorinated pyrrole moiety [Nowak-Thompsan et al. 1999]. Plt production was first reported by Takeda [1958] from Pseudomonas aeruginosa, followed by P. fluorescens Pf-5 and P. fluorescens CHAO [Bender et al. 1999]. Howell and Stipanovic [1980] found that application of Plt to cotton [Gossypium hirsutum ] seeds has resulted in reduction of cotton damping-off. P. fluorescens strain CHA0 produces pyoluteorin and suppresses damping-off or root rot of P. ultimium. Mutants [CHA660 and CHA661] of strain CHA0 lost the capacity to produce pyoluteorin and consequently, the ability to inhibit this pathogen on King's B agar that favours the production of Plt [Maurhofer et al. 2004]. 4.3.2.3. Phenazine-1-carboxylic acid [PCA] Phenazine is highly active low molecular weight, green pigmented nitrogenous heterocyclic antimicrobial compound produced by species of Pseudomonas, Burkholderia and Brevibacterium [Anjaiah et al. 1998, Tambong and Hofte 2001]. Phenazine-1-carboxylic acid [PCA] antibiotic producing Pseudomonas chlororaphis [strain PA-23] was effective against Sclerotia stem rot of canola [Brassica napus] in greenhouse as well as field evaluations [Zhang and Fernando 2004]. At neutral pH, reduced phenazine-1-carboxamide [PCM] can release Fe2+ ions from Fe3+, which gives the possibility that phenazines might contribute to iron mobilization in soils [Sharma et al. 2008]. P. fluorescens 2-79 is suppressive to take-all, a major root disease of wheat caused by G. graminis var. tritici. Mutants defective in phenazine synthesis [Phz -] were generated by TnS insertion and six independent, prototrophic Phz mutants were noninhibitory to G. graminis var. tritici in vitro and provided significantly less control of take-all than strain 2-79 on wheat seedlings [Thomashow et al. 1990]. 4.3.2.4. Pyrrolnitrin Pyrrolnitrin [PRN] belongs to phenyl pyrrole group and has received greater attention due to its wide range of antibiotic spectrum, produced by pseudomonads such as, Pseudomonas chlororaphis, Pseudomonas aureofaciens, P. fluorescens and Burkholderia cepacia, E. agglomerans, Myxococcus fulvus, and Serratia sp. [Hammer et al. 1999]. Effectiveness of PRN is reported against a wide range of fungal pathogens such as, Rhizoctonia solani, Botrytis cinerea, Verticillium dahlia, Fusarium and Sclerotium sp. [Ligon et al. 2000].

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4.3.2.5. Cyclic Lipopeptides Cyclic lipopeptides [CLPs] are produced by distinctively different groups of bacteria, both Gram-positive and Gram negative strains [Nielsen et al. 2002]. Synthesis of CLPs is non ribosomal and is catalyzed by large peptide synthetase complexes. CLPs produced by Pseudomonas sp. are amphisin, massetolide A, syringomycin, tensin, tolaasin, viscosinamide etc., and Bacillus sp. are surfactin, iturin and fengycin type [Romero et al. 2007]. For a limited number of CLPs, the reported functions include promotion of bacterial swarming and biosurfactant properties. In many cases, CLP compounds are also known to exert a role in antagonistic interactions e.g., plant pathogenicity and antifungal activity [Moffit and Neilan, 2000]. Cyclic lipopeptides are well documented in Pseudomonas sp. and are formed in a peptide ring with 9 or 11 amino acids with C10 fatty acid as one of the amino acids . CLPs [viscosinamide, tensin and amphisin], produced by P. fluorescens strains suppressed the pathogens like P. ultimum and R. solani [Nielsen et al. 2002]. Amphisin is produced at stationary phase of bacteria and performs as biosufactant and antifungal like other CLPs produced by PGPR [Koch et al. 2002]. Cyclic lipopeptide massetolide A produced by P. fluorescens SS101 was effective in biocontrol of tomato late blight by Phytopthora infestans and significantly reduced the expansion of existing late blight lesions while the mutant [Mass A] was less effective [Tran et al. 2007]. Bacillus sp. CY 22 and B. subtilis RB 14 was reported to produce iturin and surfactin and has shown antagonism against R. solani [Asaka and Shoda 1996]. Iturins includes iturin A, bacillomycin L, bacillomycin D, bacillomycin F and mycosubtilins. Iturin A has been reported for its antimicrobial action against P. ultimum, R. solani, Fusarium oxysporum, Sclerotiona sclerotiorum and Macrophomina phaseolina [Constantinescu 2001], bacillomycin D against A. flavus [Moyne et al. 2001]. Other Iturin compounds produced by Bacillus sp. are antagonistic to phytopathogens like Rosellina necatrix, Pyriculria oryzae, Agrobacterium tumefaciens, Xanthomonas campestris pv. campestris Colletotrichum trifolii [Duville and Boland 1992; Yoshida et al. 2001, 2002]. ZwittermicinA, produced by Bacillus sp. is structurally similar to polyketide antibiotics and has wide spectrum of antagonism against plant pathogens [Silo-Suh et al. 1998].

4.2.6. Volatile Metabolites Several genera of PGPR strains were assessed for eliciting growth promotion and ISR by volatiles under in vitro conditions. The volatile secondary metabolite, hydrogen cyanide [HCN], formed from glycine is catalysed by HCN synthase and inhibits cytochrome c oxidases of many organisms [Castric 1994]. HCN is reported to be produced by P. fluorescens, P. aeruginosa, Pseudomonas putida, Chromobacterium violaceum [Faramrzi et al. 2004, Faramarzi and Barandl 2006]. P. fluorescens CHAO suppresses T. basicola, the causative agent of black root rot of tobacco and G. graminis var. tritici the causative agent of take all of wheat by producing HCN [Sacherer et al. 1994].Other volatilic antifungal agents belonging to aldehydes, alcohols, ketones and sulfides identified from P. chlororaphis [PA23], a soyabean root bacterium inhibited Sclerotinia sclerotiorum [Fernando et al. 2005]. Disease severity by Erwinia caratovora sub sp. caratovora was reported in A. thaliana due to the VOC. Mutants of B. subtilis [BSIP1173 and BSIP1174] that were defective in acetoin or

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2,3-butanediol production did not show plant growth promotion or disease protection [Ryu et al. 2005].

4.3.2.7. Siderophore Production Siderophores are iron chelating, highly electronegative low molecular weight compounds that bind ferric form of iron in hexacordinated complex. Efficient utilization of siderophores of pathogenic organisms by rhizospheric bacteria restricts the growth of the pathogen in the rhizosphere due to iron starvation, and hence acting as a good biocontrol agent. Ferrated siderophores are taken up by the microbial cells through specific reorganization by membrane proteins [Hofte 1993]. Pseudomonads are known to produce siderophores like, pyoverdine, pyochelin, pseudobactin and salicylic acids that are found to be restricting the fungal pathogens by sequestering available iron from rhizosphere soil [Loper and Henkels 1999; Yang and Crowley 2000].

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4.2.8. Production of Hydrolytic Enzymes To reduce disease severity in plants biocontrol microorganisms have developed an altered mechanism that hydrolyzes fungal cell and inhibits fungal proliferation [Bashan and de Bashan 2005]. Some PGPM are reported to produce fungal cell wall degrading enzymes such as chitinases [Dunne et al. 1996], proteases [Ross et al. 2000] cellulases [Chatterjee et al. 1995] and glucanases [Jijakli and Lepoivre 1998] and act synergistically with other biocontrol mechanisms [Fogliance et al. 2002].Chitin, a linear polymer of β -[1,4]-N-acetylglucosamine is the constitutent in most of the fungal pathogens and chitinases hydrolyse the same. Chitinolytic enzymes produced by Bacillus cereus, Pantoea agglomerans, Paenibacillus, Serratia marcescens and fluorescent pseudomonads are reported to be involved in biological control of F. oxysporum and R. solani [Someya et al. 2000; Singh et al. 2006]. Another cell wall component β-1,3-glucan can be degraded by β-1,3-glucanases produced by PGPM. β1,3-glucanases contribute to the ability of Lysobacter enzymogenes to control Bipolaris leaf spot [Palumbo et al. 2005]. Such enzymes may be involved in bacterial mycoparasitism, a form of antagonism in which bacteria directly colonize the hyphae. P. cepacia producing β1,3-glucanases showed antagonism against various phytopathogens including R. solani, S. rolfsii and P. ultimum [Fridlender et al. 1993]. Studies reveal that actinomycetes like S. griseus [BH7, YH1], Streptomyces sindeneusis, also facilitate plant growth indirectly by producing chitinases, β-l,3-glucanases, siderophores and antifungal substances [Zarandi et al. 2009; Hamdali et al. 2008b]. In addition, oxidative enzymes synthesized by such organisms also play a significant role in defense reactions against plant pathogens. Bacterization of betelvine cuttings with S. marcescens NBRI1213 induced plant defense enzymes such as, phenylalanine ammonia –lyase, peroxidase and polyphenoloxidase [Lavania et al. 2006].

4.3.2.3. Elicitors and Induced Resistance Elicitors are biofactors or chemicals produced from various sources that can induce physiological and morphological responses in plants [Zhao et al. 2005]. Plant roots must be able to perceive and recognize such elicitors in ways similar to the recognition of elicitors from plant pathogens during biomanagement of pathogens by induced systemic resistance

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[ISR] [van Loon 2007]. A variety of established and putative elicitors are produced by bacteria and fungi. Crude cell wall extracts and purified lipopolysaccharides [LPS] produced by P. fluorescens strain WCS 374 and WCS 417 have shown to induce resistance in radish [Raphanus sativus], while mutants lacking O antigen did not induce resistance. Moreover, oligosaccharides and peptides derived from fungal cell walls can also elicit plant defense response. Such elicitors can widely be used as a tool for improving the yields of secondary metabolites that increases the defense mechanisms and plant growth promotion [Milen et al. 2007]. Plants generally use two defense pathways to resist infections a] systemic acquired resistance [SAR] triggered by pathogen attack and b] induced systemic resistance [ISR] triggered by PGPM [van Loon et al. 1998; Kloepper et al. 2004]. Plant defense mechanisms are characterized by a combination of pre-existing biochemical defense, constitutive and induced resistance. Induced resistance is triggered independently by free living and endophytic root colonizers, biocontrol agents and also by compost amendments with in host plants [Kloepper et al. 1992, Hoitink and Boehm 1999]. Induced systemic resistance is defined as “the process of active resistance to physical or chemical barrier of the host plants activated by biotic or abiotic agents and can be localized or systemic”. The systemic resistance refers to the increased levels of resistance to whole plant or at sites within the plant, distant to those at which the induction had occurred [Bakker et al. 2007]. Induced systemic resistance is mediated by non-pathogenic PGPM, does not involve any damage to plants and requires production of jasmonic acid [JA] and ethylene [Verhagen et al. 2006]. Jasmonic acid has many roles in plant systems, such as activation of plant defense genes onset of senescence, root formation and ethylene synthesis. Presently little information is available about the interaction between PGPM and JA synthesis. However, previous reports has shown that the genes responsible for JA synthesis are activated in barley [Hordeum vulgare] leaves and root tips when the plants are infected by mycorrhizal fungus Glomus intraradicies, supporting symbiotic association [Hause et al. 2002]. Jasmonic acid and ethylene induce genes with antimicrobial proteins [heveine lole protein, chitinase and plant defensin] that have antimicrobial activity [Samac and Shah 1994]. But in SAR, pathogen attack induces and activates resistance in uninfected plant parts where infection inclines the plants to resist further attack. Effectiveness of SAR based plant defense is regulated by accumulation of salicylic acid [SA] and possible involvement of hydrogen peroxide as signal molecules, derived from biotic agents [Hung et al. 2005]. Induction of SA can encode pathogen related proteins and can also promote plant growth Pseudomonas fluorescens Pf4 [Uknes et al. 1992; Singh et al. 2003]. PGPB mediated ISR has been demonstrated in different plant species against different pathogens where pathogens and biocontrol bacteria are spatially separated [Pieterse et al. 2002]. ISR mechanisms induced by Pseudomonads sp. was extensively studied but recently Bacillus strains like B. amyloliquefaciens, B. subtilis, B. pastreuii, B. cereus, B. pumilus, B. mycoides, and B. sphaericus are also reported and reviewed by Kloepper et al. [2004]. Bacillus sp. have also been reported for releasing gaseous volatiles [butanediol/acetoin] involved in ISR by B. pumilus SE 34. Role of siderophores in ISR has also been reported in P. putida WCS358 against bacterial wilt in Eucalyptus urophylla caused by Ralstonia solanacearum [Ran et al. 2005]. Cylic lipopeptide massetolide A produced by P. fluorescens SS101 induced both local and systemic resistance [Tran et al. 2007]. PGPF and most of the non-pathogenic virulent forms of plant pathogen induces ISR in plants. Fungi that produce auxin or auxin precursors

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also stimulate plant defense via auxin regulted ISR pathway. Penicillium janczewskii induced SAR and altered cotton root development [Madi and Katan, 1998]. However, the exact inducing mechanisms are not yet clearly understood and require more attention of the scientists.

4.4. RHIZOSPHERE As local and global market standards have changed, it is a great challenge for the farmers to achieve ever-increasing crop productivity together with improved quality by facilitating adequate nutrient uptake by the plants. Though the nutrient supply is adequate and given to the plant externally, the utilization depends on physical, chemical and biological conditions in the soil surrounding the plant that is closely associated with the roots, which is called rhizosphere. Rhizosphere can be divided in to three different regions- [i] endorhizosphere, which composes of the root tissues [ii] rhizoplane, the root bidimensional surface and [iii] ectorhizophere, which represents the adjacent soil [Kitten et al. 1998; Louws et al. 1994]. Population density and variation in microbial activity has great influence in the rhizosphere as there can be 10 to 100 fold differences between bulk and rhizosphere soil [Semenov et al. 1999]. Roots release various amounts of exudates, which alter physical and chemical characteristics of soil and create a microenvironment that influences the microbial community.

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4.4.1. Competitive Root Colonization For effective plant growth promotion and biological control activity, the inoculated PGPM must colonize the roots by establishing molecular signaling interaction with host plant [Chin-A-Woeng et al. 2000]. Colonization of PGPM in the rhizosphere will be triggered by bacterial two-component system, type III secretion system of bacterial sensor kinase that recognize and respond to the root exudate signal [Preston et al. 1998]. PGPM colonized on roots could act as sink for nutrients available in the rhizosphere, thus reducing the available nutritional status to create starvation and subsequently death of fungal pathogens [Podile and Kishore 2006; Walsh et al. 2001; Fernando et al. 1996]. Phyllosphere as well as rhizosphere bacteria are reported to successfully control pathogens via niche exclusion. One such example for niche exclusion is by biocontrol strain B. subtilis 6051 that competes through biofilm formation against pathogen P. syrinae pv. tomato DC 3000 by colonizing Arabidopsis roots [Bais and Vivanco 2004]. Plant growth promoting psuedomonads have also been reported to colonize root surface by biofilm formation [Ramey et al. 2004].

4.4.2. Factors Affecting Root Colonization Root exudates include organic acids, amino acids, and specific carbohydrates, proteins vitamins and other nutrients that affect the growth and physiology of the inoculated and native rhizosphere population [Welbaum et al. 2004; Somers et al. 2004]. Microorganisms

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that utilize these specific root exudates become primary communities in the rhizosphere. For example, Pseudomonas sp. by virtue of their diverse nutrient utilization and competition for limited carbon sources are believed to be potent root colonizers [Lugtenberg et al. 2001]. Plant roots produce specific chemoattractants and antimicrobials under genetic and environmental control. This specificity implies that the PGPM with ability to change their metabolism according to environmental conditions will only colonize the rhizosphere [Bais et al. 2004]. Studies on rice reveal that rice root exudates induce stronger chemotactic responses of endophytic bacteria than other bacteria in the rhizosphere [Bacilio-Jime´nez et al. 2003]. Bacterial LPS, in particular the O-antigen chain, can also contribute to root colonization and are reported as strain specific [Dekkers et al. 1998]. LPS of Azospirillum brasilense pre treatment significantly promoted plant root attachment [Matora et al. 2001]. O-antigenic aspect of LPS of P. fluorescens WCS417r was not involved on rhizoplane colonization but involved in endophytic colonization [Duijff et al. 1997]. Another determinant of root colonization ability by bacteria is type IV pili, that is involved in cell adhesion, movement and biofilm formation [Steenhoudt et al. 2000]. Root colonization is affected by a number of abiotic and biotic factors, of which important abiotic factors include temperature, pH, nutrients and biotic factors include plant species and the physiological stage of the plant. Some bacterial or fungal populations might be benefited more from the nutrients offered by the plant than others which might affect their numerical dominance and activity. In return, microbes enriched in the vicinity of the root can be beneficial to the plant or cause harm [Brimecombe et al. 2001]. Studies of Udaiyan et al. [1996] revealed that the edaphic factors along with soil moisture negatively influenced root colonization of Acacia planifrons. Further the soil phosphorus and nitrogen were negatively correlated with the density of VAM fungal spores. Schmidt et al. [2004] experiments revealed that antagonistic activity of P. fluorescens B5 decreased with increasing soil temperature and decrease in matric potential. Colonization levels of P. fluorescens SBW25 were substantially increased by the presence of nematodes than in their absence [Oliver et al. 2004].

4.4.3. Perception on Communication in Rhizosphere: Quorum Sensing [Biofilms] Microorganisms respond rapidly to the presence of root exudates in soils and converge at root colonization sites and form biofilms that facilitate a suitable environment for metabolic interactions [Ramey et al. 2004]. Plant associated beneficial bacteria forms biofilms on leaves, root surfaces and also within intracellular spaces of plant tissues [Morris and Monier 2003]. Plant growth promoting microorganisms benefit plants by forming biofilms in different ways-[i] it provides protection against desiccation, [ii] acts as bacteriocides [iii] protection against UV and predation [iv] increased genetic exchange and [v] enhance synergistic interactions by quorum sensing [QS] [Costerton et al. 1995]. Bacterial species employ complex communication mechanisms termed QS that link cell density with gene expression. During this process diffusible signal molecules, autoinducers like acylhomoserine lactones [AHL], accumulate in the extracellular environment, attain a critical threshold concentration and trigger the response which leads to gene expression. Besides QS, it is apparent that some cross-talk between bacterial forms can also occur in PGPM that can influence the operation of QS systems.

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Several QS plant–microbe systems have been investigated in PGPR such as B. cepacia, Pseudomonas corrugata, P. putida, P. chlororaphis, P. fluorescens, Rhizobium elti, R. leguminosarum, Sinorhizobium meliloti, Chromobacter violaceum, Nitrosomonas europaea [Sharma et al. 2003] QS in Rhizobium elti and R. leguminosarum has been characterized, besides the role it plays in symbiotic relationship with legume hosts [Cha et al.1998]. The QS system of R. leguminosarum is regulated by chromosomally encoded CinRI proteins. Phenazine production in fluorescent pseudomonads in rhizosphere is dependent on QS. The regulation of phenazine production is linked to the genes phzR and phzI members of luxI/luxR gene groupand is located upstream of the phenazine operon [Pierson and Pierson 1996]. QS regulatory mechanisms are part of a large complex of regulatory cascades with global regulatory Gac S/ Gac A two component system. Regulatory system influences target gene expression at post-transcriptional level involving two RNA binding proteins [Rsm A and Rsm E] and regulatory RNA [Rsm Z] that binds Rsm A [Haas et al. 2002]. Further advancement and finer understanding of QS in the rhizosphere will facilitate sustained exploitation of bioinoculants in soil health, plant productivity, bioremediation strategies in environmental applications and operation of biodegradation mechanisms that often determine the fate of a microorganism introduced in the natural ecosystems [Maddula et al. 2006]. Molecular genetic approaches can be used to elucidate the metabolic pathways that are activated during root colonization by microorganisms. In vivo expression technology [IVET] provides a useful approach to study bacterial gene expression in the rhizosphere It is also used to identify promoters that are functional and involved in different steps in root colonization. The major advantage of IVET is that it allows the study of microbes where the full repertoire of genes and associated physiological conditions necessary for survival in vivo, are expressed. Microarray studies provides information on gene expression profiling and physiological processes that are involved in response to a particular stimulus. Wang et al. [2005] reported upregulation of 95 genes that were involved in metabolism, signal transduction and stress [defense] response during colonization of endophytic bacterium P. fluorescens using microarray analysis.

4.5. INTERACTIONS WITH OTHER MICROORGANISMS Co-inoculation of symbiotic rhizobia with PGPB play an important role in enhancing available nitrogen to plants through BNF to achieve sustainable agriculture system [Geetha et al. 2008]. Under nitrogen poor environments Rhizobium promotes plant growth by providing a limiting nutrient source of nitrogen [van Loon, 2007]. In case of leguminous plants combined inoculation of Rhizobium with Azospirillum or with Azotobacter has been demonstrated to increase dry matter production, grain yield, and nitrogen content of several legumes when compared with inoculation of Rhizobium alone. These positive results of dually inoculated legumes have been attributed to early nodulation, increased number of nodules, higher N2- fixation rates, and general improvement of root development [Volpin and Kapulnik 1994; Okon and Itzigson 1995]. The greater number of nodules can be expected to contribute fixed nitrogen for higher yields under field conditions. Rhizobium for its part was found to act synergistically with AM fungi to increase lettuce biomass production [Galleguillos et al. 2000]. Zaidi and Khan [2006] have reported a synergistic relationship

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between PSMs [Pseudomonas striata and Penicillium sp.] and A. chroococcum, allowing a better use of poorly soluble P sources and increased grain yield and P uptake of wheat plants. Common mechanisms used by PGPB to alter nodule formation or BNF include the release of auxins, gibberillins, cytokinins and ethylene or the alteration of endogen levels in the plant [Hirsch et al. 1997]. The effects of some phytohormones are indirect, as they stimulate root growth, providing further sites for infection and nodulation. Systemic inductions of secondary metabolites such as flavonoids are implicated due to inoculation of PGPB [Andrade et al. 1998]. However, other compounds with less known functions such as tabtoxinine-beta lactam [Knight and Langston 1988] and B group vitamins [Marek-Kozaczuk and Skorupska 2001], produced by Pseudomonas sp. could also be involved in these bacterial effects. Co-inoculation of Rhizobium, Azospirillum, and Azotobacter with PSMs also showed synergistic effect on plant growth and crop yields [Barea et al. 1983; Kundu and Gaur 1984]. Synergistic effects of PGPR and AM fungus [Glomus fasciculatum] are reported for growth promotion of sorghum under glasshouse and wheat under field conditions [Hameeda et al. 2007; Khan and Zaidi, 2007].

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4.6. MOLECULAR MECHANISM FOR TRAKING BACTERIA IN RHIZOSPHERE Root colonization by the inoculated bacteria depends on root exudates, signal molecules, biotic and abiotic factors. Hence specific and consistent methods are needed for quantification of the inoculated bacteria in rhizosphere. Fluorescently labelled antibodies, immunodiffusion methods, fatty acid profiling, spontaneous or induced antibiotic resistance markers, have long been used for tracking PGPR. Few of these methods are though sensitive and explicit, yet are laborious. Moreover, antibiotic resistance may not be retained after many generations and thus there is a possibility of loss of ecological competence of antibiotic resistant mutants of PGPR [Cavigelli et al. 1995]. To over come this problem, molecular methods have been developed for PGPR tracking.

4.6.1. Marker Based Detection Methods Native organisms form a huge number in the rhizosphere and from this heterogeonous populations, to detect the organisms suitable for inoculation purposes,selectable marker should be designed which should be specific to the organism of interest. In recent years, there has been significant progress to understand the fate of inoculated microbes in rhizosphere using bioreporter strains, in which an environmentally or metabolically responsive promoter is fused to a suitable reporter such as lacZ, gusA, lux, gfp. To monitor the rhizosphere colonization of Pseudomonas sp. the lacZY based expression of E.coli lac operon genes encoding galactosidase and lactose permease, is the sensitive and selectable marker system used extensively [Krishnamurthy and Gnanamanickam 1998]. Marker gene to study ecology of PGPR is gusA gene that encodes β-glucuronidase. Bacterial luciferase, encoded by luxAB genes is used to determine the cellular metabolic activities of bacteria. Rhizosphere colonization by Pseudomonas sp. strains was reported with different crops using this marker

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gene [Beauchamp et al. 1993]. Green fluorescent protein [GFP] gene of the jellyfish, Aequorea victoria, is used commonly for non-destructive monitoring of gene expression, protein localization and various other biological phenomena of PGPR [Chalfie et al. 1994]. Single cell distribution, viability and activity of GFP marked Pseudomonas sp. is reported in barley rhizosphere [Normader et al. 1999].

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4.6.2. Nucleic Acid-Based Detection Methods Both culturable and non culturable microorganisms can be detected with genotyping profiles by nucleic acid based systems [Ward et al. 1990]. These include PCR-independent nucleic acid [DNA and RNA] hybridization and PCR dependent amplification techniques. For DNA hybridization, the standard restriction fragment length polymorphism [RFLP] analysis was improved by DNA fragmentation with infrequently cutting restriction enzymes and the separation of large restriction fragments by pulsed field- or field inversion gel electrophoresis [PFGE; FIGE] followed by hybridization with probes. Fluorescent in situ hybridization [FISH], by use of fluorescent labeled rRNA targeted oligonucleotide probes of 15-20 nucleotide length detects single cells of the target strain, its cellular locations and exact cell numbers [Amann et al. 2001]. FISH technique determined the endophytic root colonization of wheat roots by A. brasilense strains that resulted in plant growth promotion [Rothballer et al. 2003]. The limitations of [FISH] technique are poor cytoplasmic penetration of probes in bacteria with thick cell walls, hindrance of the target site by other ribosomal components and poor detection of metabolically inactive cells. The PCR-dependent methods of PGPR tracking and identification are based on characteristic genomic fingerprints that require only a minute quantity of DNA.Various PCR-based methods that can be used for PGPR identification are arbitrarily primed PCR [AP-PCR], sequence characterized amplified region [SCAR] analysis, repetitive extragenic palindromic [REP]-PCR, reverse transcriptase dependent PCR [RTPCR], amplified fragment length polymorphism [AFLP], amplified ribosomal DNA restriction analysis [ARDRA], amplification of ribosomal intergenic spacer [RIS] regions between the 16s and 23s rRNA genes, terminal restriction fragment length polymorphism [TRFLP], denatured gradient gel electrophoresis [DGGE], temperature gradient gel electrophoresis [TGGE] and single strand conformation polymorphism [SSCP]. Yang et al. [2001] used 16s rDNA fingerprints obtained by PCR-DGGE to monitor bacterial community structures in the rhizosphere of avocado trees during infection by Phytophthora cinnamomi and repeated bioaugmentation with a disease suppressive P. fluorescens st. 513. The majority of DGGE band sequences were related to the genera Acetobacter, Arthobacter, Bacillus, Comamonas, Pseudomonas and Variovorax. Using a pair of SCAR primers specific to P. fluorescens Pf29A, Chapon et al. [2003] observed that the total populations of Pf29A detected in wheat rhizoplane using antibiotic resistance and GFP marker systems was only 13% compared to that quantified using SCAR markers. The occurrence and diversity of endophytic bacteria in relation to plant growth in potato was studied by T-RFLPanalysis of 16S rDNA [Sessitsch et al. 2004]

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4.7. COMMERCIALIZATION OF PGPM In the present scenario bioproducts for crop protection has gained importance because of its ecofriendly nature and economics involved. Also export oriented crops depend on the residue free products which has further increased the importance for mass production and commercialization of PGPM. In this respect, several protocols are being standardized for the formulation development and shelf life improvement. Once this is done industries can get involved and thus the scope for the commercialization can be increased globally. Microbe mediated formulations improve plant growth and yield, check plant pathogens, activate host plant resistance, less expensive compared to chemical fertilizers and pesticides, leave no residues in the products etc. For a good formulation development it has to survive and multiply and should be hardy for better field results [Jeyarajan and Nakeran 2000].

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4.7.1. Carrier Materials Carrier materials play a vital role for working efficiency of the product developed. The frequently used carrier materials include peat, lignite, talc, turf, kaolinite, pyrophyllite, zeolite, montmorillonite, alginate, pressmud, sawdust, compost and vermiculite etc. Beneficial microorganisms are protected from desiccation in presence of carrier materials. Shef life of bacteria depends on the bacterial genera, physical chemical and biological nature of the carrier materials. Studies of Dandurand et al. [1994] revealed that P. fluorescence [279RN10, W4F393] when inoculated in montmorillonite, zeolite and vermiculite with smaller particle size increased its survival rate than in kaolinite, pyrophyllite, and talc with bigger particle size. Plant beneficial microorganisms are multiplied by fermentation technology and the biomass harvested is mixed with suitable carrier material and incubated for the stipulated time and this incubation time differs from organism to organism. Among various carrier materials used peat is considered as ideal and used vastly due to its waterholding capacity, buffering nature and nutrient availability. Peat based formulation had viable populations of B. subtilis AF 1 [log 9.0 CFU g-1] [Manjula and Podile, 2005], Bacillus firmis GRS 123 and Bacillus megaterium GPS 55 and [log 7.0 CFU g-1] [Kishore et al. 2005a], Pseudomonas sp. CDB 35 [log 7.3 CFU g-1], Serratia marcescens EB 67 [log 6.2 CFU g -1] [Hameeda et al. 2008].Compost can also act as a suitable carrier material and supports growth and multiplication of the inoculated organisms. Two PSB, Serratia marcescens EB 67 and Pseudomonas sp. CB 35 inoculated in vermicompost showed log 6.2 CFU g-1 and log 5.8 CFU g-1 after 90 days after inoculation under glass house conditions [Harini, 2005].

4.7.2. Technology Transfer Application of PGPR was done in several forms depending upon the host plant and target pathogen. Few of the reported methods of trasfering inoculants include, seed treatment, biopriming, soil application, seedling dip, foliar spray, fruit spray, hive insert, sucker treatment, sett treatment etc. [Nakkeeran et al. 2005]. Delivery systems can be individual application and/or consortia of microbes. Kloepper and Scroth developed P. fluorescence

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[Pf1] for the first time as talc based formulation for treating potato tubers for improving growth promotion. Talc based formulation of B. subtilis strain Pf1 and Pf2 increased grain yield of pigeon pea besides controlling pigeonpea wilt [Vidhyasekaran et al. 1997]. Treatment of groundnut and pigeonpea seeds with peat based formulation of B.subtilis supplemented with 0.5% chitin or with 0.5% of sterilized Aspergillus mycelium controlled crown rot of groundnut and wilt of pigeonpea respectively [Manjula and Podlie 2001]. Seed treatment with wettable powder formulation of P. putida strain 30 and 180 suppressed wilt of muskmelon to the extent of 63 and 50% after 90 days of tansplanting muskmelon in the field. Some strain mixtures might not work as effectively as individual strains which might be due to the incompatibility between them [Bora et al. 2004]. Powdered formulations of PGPR have been developed and a number of commercially available gums were tested as suitable substrates for PGPR in comparison to methylcellulose. Suslow and Schroth [1982] showed methylcellulose powder formulations were most suitable for pelleting onto sugar-beet. Powered formulations have benefit as the ease of storage, transport, handling with long shelf life. Also, by pelleting seed with a powder formulation can get a higher population of PGPR around the seed than by dipping in bacterial suspensions.

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4.7.3. Multiple Applications Mixtures of the strains employed as a single PGPM may not work all times and under all environments due to their inability to colonize rhizophere and to tolerate stressed environmental conditions. This inconsistency can be circumvented by combined application of several biocontrol agents that mimic the natural environment [Schisler and Sliniger 1997; Raupach and Kloepper 1998]. Strain mixtures have great advantage as broadspectrum of action, enhanced efficiency, reliability etc. [Janisiewicz 1996]. Also when a mixture is applied in the field ensure that at least one of the mechanism to operate under variable environment [Duffy et al. 1996] to get desirable results. Application of the desired microorganism at one site may not give proper results thus application at multiple sites and at different stages of crop period is required for expected results. Seed treatment of pigeonpea with talc based formulation of fluorescent pseudomonads at the rate of 4g/kg of seed followed by soil application at the rate of 2.5 kg/ha at 0, 30, and 60 days after sowing controlled pigeonpea wilt incidence under field conditions. The additional soil application of talc based formulation improved disease control and yield compared to seed treatment alone [Vidhyasekaran et al. 1997]. Rice blast was controlled under field conditions by application of P. fluorescens as seed treatment followed by three foliar applications [Krishnamurthy and Gnanamanickam 1998]. Leaf spot disease of groundnut was reduced under field conditions by foliar application of talc based fluorescent Pseudomonas [Meena et al. 2002]. Enhanced efficacy of consortia application might be due to increase in the population of fluorescent pseudomonads in both rhizosphere and phyllosphere [Viswanathan and Samiyappan 1999]. Application of P. aeruginosa strain 78 as seed treatment and foliar spray reduced root knot incidence of mungbean besides the reduction in the population density of Meloidogyne japanica under field conditions [Ali et al. 2002].

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4.8. METAGENOMIC APPROACH TO EXPLOIT NOVEL PGPM

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The term metagenomics is widely used for the study of non culturable or culture independent application of genomic techniques for the study of microbial communities in their natural environment [Chen and Pachter 2005]. Metagenomics explains the fact that some microorganisms are culturable and some are not but all of them has DNA as genetic material. The metagenomic approach allows accessing, storing, and analyzing this DNA and provide the information of evolution of these organisms without depending on their culturable characters. The unculturable microorganisms often form more than 99% and the study of these include a series of steps as isolation of DNA, cloning, library screening, sequencing of interested clones, and DNA comparison. In metagenomics, three major approaches are recognized-[i] linking phylogeny to function [ii] exploitation for the discovery of novel metabolites with application to agriculture and industry [Lorenz and Eck 2005] and [iii] mass sequencing of environmental samples.Until now, only the culturable organisms are characterized either for plant growth promotion or biocontrol activity. After the introduction of metagenomics emphasis on nonculturable microorganisms having plant growth promoting traits gained importance. Metagenomics can form efficient tool for the study of rhizosphere particularly PGPM. These include-[i] the discovery of novel plant growth promoting genes and gene products and [ii] the characterization of non culturable microorganisms. The application of metagenomics to PGPM in the rhizosphere greatly benefits from the library construction by DNA isolation. The biggest problem in the construction of metagenomic library for PGPM is the low availability of the starting material. Along with the rhizospheric soil many methods for the isolation of genomic material from plant have been established. Jiao et al. [2006] described an indirect method of enzymatic hydrolysis of plant tissues to release associated microbes for DNA isolation and cloning. These tools facilitate screening of large insert DNA libraries for identifying functional phenotypes with plant growth promoting activity.

4.9. SIGNIFICANCE OF PGPM IN SUSTAINABLE AGRICULTURE Sustainable crop production involves the successful management of agriculture resources to meet the changing human needs, while maintaining or enhancing the environment quality and conserving natural resources. Sustainable or alternative agriculture comprises the use of crop production technologies that involves no-tillage, incorporation of crop residues [for recycling of crop nutrients], use of intercropping systems, combination of cereals and legumes, crop rotation, application of low cost biological inputs, composts, green manures, microorganisms as biofertilizers, biopesticides, biostimulators etc [Rupela et al. 2005; Selvamukilan et al. 2006, Martinez and Bernando 2006]. The diversity of microbial populations, flora and fauna diversity, their total number, biomass, respiration rates, enzyme activities have been studied for sustainability in agriculture systems. [Kennedy and Smith, 1995]. Integrated nutrient management [INM] involves the use of low cost biological inputs, such as, Rhizobium, mycorrhizae and PGPM while integrated disease management [IDM] involves the use of composts/organic amendments, biocontrol agents have also been reported for sustainable agriculture management [Miller and Jastrow. 1992; Haggag et al. 2002].

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Although sustainability has become one of the forefront issues faced by agriculture, ‘sustainable agriculture’ continues to remain an ill-defined concept. However, there is fear of low yields in sustainable agriculture when compared to conventional agriculture which is not true and most of the reports show the yields are at par or better than conventional agriculture [Uphoff et al. 2006]. Though different groups are working on these areas but the published reports are scanty. However assessing sustainability is more difficult than evaluating productivity because it depends on future evidence, which by definition cannot be known in present. In order to support the same, studies should be focussed on sustainability of soil systems [nutrient status], that can improve soil fertility which indirectly affects the crop productivity. Application of low cost biological inputs can be made more efficient by applying the scientific knowledge to improve soil health and crop productivity. Various biochemical and molecular methods have been proposed to assess soil quality and productivity [Nannipieri et al. 1990; Suzuki et al. 2005]. Sustainability of rice/corn-wheat cropping system was increased significantly by organic inputs as farm yard manure, rice straw compost or green manure. The organic application increased nutrient status, microbial activity and productive potential of soil which was supported by deriving sustainability indices of soil [Kang et al.2005]. Field that received low cost biological inputs, showed significant population of actinomycetes and siderophore producing bacteria [Hameeda, 2005]. Studies were carried out to determine soil quality and crop productivity in four different crop-husbandry systems; T1 and T2 that represent low-cost systems where crop nutrients are provided from biomass inputs, biofertilizers, biopesticides in addition to what can be mobilized from the soil through biotic activity, T3 is the treatment most similar to conventional current cropping systems, i.e., relying for its nutrient inputs on inorganic fertilizers,. T4 is a combination of conventional and alternative systems as it receives the same organic inputs that are provided for T2 plus the T3. Sustainability indices of the the different crop-husbandry systems mentioned above were derived using biological, microbial, nutrient and crop index. From this study it is apparent that plant biomass the engine of crop productivity mediated by biological process can enhance soil fertility and crop productivity [Hameeda et al. 2006d].

CONCLUSION AND FUTURE OUTLOOKS The global necessity to increase agricultural production from a steadily decreasing and degrading land resource base has placed considerable strain on the fragile agro-ecosystems. These concerns are driving the search for more environmentally friendly methods to promote plant growth that will contribute to sustainable agriculture. Understanding the complex soilplant-microbe interaction in the rhizosphere, the mechanisms of action of PGPMs, and signaling in the rhizosphere provides strong incentive for developing inoculant formulation and develop these as commercial biofertilizers and biofungicides. However, success of these products will depend on our ability to manage the rhizosphere to enhance survival and competitiveness of these beneficial microorganisms. PGPMs that show in vitro plant growth and antagonistic traits should not only perform in microenvironment but also should show in situ efficacy when inoculated. The major challenges to be resolved prior to widespread commercial exploitation are the performance of these strains in agriculture, horticulture and

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agroforestry conditions. However, there should be continuous efforts to characterize effective microbial inoculants and development of economic and suitable formulations and delivery mechanisms that should be successful. Application of molecular tools enhances the understanding and managing the rhizosphere for plant beneficial affects. In the agro-chemical industry, thousands of prospective compounds are screened annually in efficient highthroughput assays to select the best one or two compounds for further development. Similar approaches are not yet in place for PGPMs. Effective strategies for initial selection and screening of rhizobacterial isolates are required. It may be important to consider host plant specificity or adaptation to a particular soil, climatic conditions or pathogen in selecting the isolation conditions, and screening assays. Fortunately, interest in these PGPM has provided more information about the genetics, physiology and their ecology that have paved the way for strain improvement through selection or genetic engineering. Genetic manipulation of host crops for root-associated traits to enhance establishment and proliferation of beneficial microorganisms is being pursued. However, regulatory issues and public acceptance of genetically engineered organisms may delay their commercialization. The use of consortia of PGPM with known functions is of interest as these formulations my increase consistency in field. They offer the potential to address multiple modes of action, multiple pathogens and temporal or spatial variability. Prior to registration and commercialization of these PGPM, hurdles must be overcome. These include scale up, production of the organism under commercial fermentation conditions while maintaining quality, stability and efficacy of the product. Formulation development must consider factors such as shelf life, compatability with current practices, cost and ease of application. Health and safety testing may be required to address such issues as non-target effects on other organisms including toxigenicity, allergenecity and pathogenecity, persistence in the environment and potential of horizontal gene transfer. Use of metagenomics for discovering novel PGPM either by functional screening or based on DNA sequence information will add information on varied mechanisms. Novel molecular approaches such as genome sequencing of new PGPM isolates and transcriptional profiling will undoubtedly lead to the discovery of novel mechanism of PGPM and new types of PGPM. Soil health in a holistic manner involves physical, chemical and microbiological parameters. As quoted by MS Swaminathan “Soil Breeding” is the initiative for sustainable agriculture and it should receive as much attention as crop breeding has been given to promote advances in productivity in perpetuity without having adverse ecological consequences. In view of these, sustainable agriculture which involves the application of these potential microorganisms is a challenge to the microbiologists.

REFERENCES Abdullah, M.T. Ali, N.Y. and Suleman, P. [2008]. Biological control of Sclerotinia sclerotiorum [Lib.] de Bary with Trichoderma harzianum and Bacillus amyloliquefaciens. Crop Protc, 27, 1354-1359. Ae, N. Arihara, J. and Okada, K. [1991]. Phosphorus response of chick pea and evaluation of phosphorus availability in Indian alfisols and vertisols. In C. Johansen, and K.K. Lee, and

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

90

B. Hameeda, G. Harini, B. Keerthi Kiran et al.

K.L. Sharawat [Eds.], Phosphorus Nutrition of Grain Legumes [pp. 33–41]. ICRISAT, India. Ahmed, F. Ahmed, I. and Khan, M.S. [2006]. Screening of free-living rhizospheric bacteria for their multiple plant growth activities. Microbiol. Res, 163, 173-181. Alami, Y. Achouak, W. Marol, C. and Heulin, T. [2002]. Rhizosphere soil aggregation and plant growth promotion of sunflowers by an exopolysaccharide-producing Rhizobium sp. strain isolated from sunflower roots. Appl. Environ. Microbiol, 66, 3393-3398. Ali, N.I. Siddiqui, I.A. Shahid, J. Shaukat, S. and Zaki, M.J. [2002]. Nematicidal activity of some strains of Pseudomonas sp. Soil Biol Biochem, 34, 1051-1058. Al-Raddad, A. [1995]. Mass production of Glomus mossae spores. Mycorrhiza, 5, 229-231. Alvarez, M.A.B. Gagne, S. and Antoun, H. [1995]. Effect of compost on rhizosphere microflora of the tomato and on the incidence of plant growth-promoting rhizobacteria. Appl Environ Microbiol, 61,194-199. Amann, G. Stetter, K.O. Llobete-Brossa, E. Amann, R. and Anton, J. [2001]. Direct proof for the presence and expression of two 5% different 16S rRNA genes in individual cells of Haloarcula marismortui. Extremeophiles, 4,373-376. Andrade, G. Deleij, F.A. and Lynch, J.M. [1998]. Plant mediated interactions between Pseudomonas fluorescens, Rhizobium leguminosarum and arbuscular mycorrhizae on pea. Lett. Appl. Microbiol, 26, 311-316. Andrews, J.H. [1992]. Biological control in the phyllosphere. Annu. Rev. Phytopathol, 30, 603 - 635. Anjaiah, V. Koedam, N. Nowak-Thompson, B. Loper, J.E. Hofte, M. Tambong, J.T. and Cornelis, P. [1998]. Involvement of phenazines and anthranilate in the antagonism of Pseudomonas aeruginosa PNA1 and Tn5 derivatives towards Fusarium sp. and Pythium sp. Mol Plant Microb Interact, 11, 847-854. Antoun, H. and Pre´vost, D. [2005]. Ecology of plant growth promoting rhizobacteria. In Z.A. Siddiqui [Eds,], PGPR: Biocontrol and Biofertilization [pp. 1–38]. Araujo, W.L. Marcos, A. Acheron, W. Van Verde, J.W.L. and Azevedo, L. [2002]. Diversity of endophytic bacteria populations and their interaction with Xyletta fastidiosa in citrus plants. App. Environ. Microbiol., 68, 4906-4914. Artursson, V. Finlay, R.D. and Jansson, J.K. [2006]. Interactions between arbuscular mycorrhizal fungi and bacteria and their potential for stimulating plant growth. Environ. Microbiol, 8, 1–10. Asaka, O. and Shoda, M. [1996]. Biocontrol of R. solani damping off of tomato with B. subtilis RB14. Appl. Environ. Microbiol, 11, 4081- 4085. Asea, P.E.A. Kucey, R.M.N. and Stewart, J.W.B. [1988]. Inorganic phosphate solubilization by two Penicillium species in solution culture and in soil. Soil Biol. Biochem., 20, 459464. Atalla, M.M. Naziha, M.H. Ahmed, A. and Youssef, A.Y. [2003]. Mycotoxin production in wheat grains by different Aspergilli in relation to different relative humidities and storage periods. Food, 47, 6-10. Audenaert, K. Pattery, T. Cornelis, P. and Hofte, M. [2002]. Induction of systemic resistance to Botrytis cinerea in tomato by Pseudomonas aeruginosa 7NSK2: role of salicylic acid, pyochelin, and pyocyanin. Mol. Plant Microbe Interact,11, 1147-1156. Bacilio-Jime´nez, M. Aguilar-Flores, S. Ventura-Zapata, E. Pe´rez-Campos, E. Bouquelet, S. and Zenteno, E. [2003]. Chemical characterization of root exudates from rice [Oryza

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Role of Plant Growth Promoting Microorganisms for Sustainable Crop Production

91

sativa] and their effects on the chemotactic response of endophytic bacteria. Plant Soil, 249, 271–277. Bais, H.P. and Vivanco, J.M. [2004]. Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production. Plant Physiol, 134, 307-319. Bais, H.P. Park, S.W. Weir, T.L. Callaway, R.M. and Vivanco, J.M. [2004]. How plants communicate using the underground information superhighway. Trends Plant Sci, 9, 26– 32. Bakker, P.A.H.M. Pieterse, C.M.J. and Van Loon, L.C. [2007].Induced systemic resistance by fluorescent Pseudomonas sp. Phytopathology, 97, 239-243. Bano, N. and Musarrat, J. [2003]. Characterization of a new Pseudomonas aeruginosa strain NJ-15 as a potential biocontrol agent. Curr. Microbiol., 46, 324-28. Bargabus, R.L. Zidack, N.K. Sherwood, J.E. and Jacobsen, B.J. [2004]. Screening for the identification of potential biological control agents that induce systemic acquired resistance in sugar beet. Biological Control, 30, 342-350. Bashan, Y. and deBashan, L.E. [2005]. Plant growth-promoting bacteria. In D. Hillel [Eds.], Encyclopedia of Soil in the Environment [vol 1, pp. 103-115]. Oxford, Elsevier. Bashan, Y. Gina, H. and de-Bashan, L.E. [2004]. Azospirillum-plant relationships: physiological, molecular, agricultural, and environmental advances [1997-2003]. Can. J. Microbiol., 50, 521-577. Belimov, A.A. Hontzeas, N. Safronova, V.I. Demchinskaya, S.V. Piluzza, G. Bullitta, S. and Glick, B.R. [2004]. Cadmium-tolerant plant growth-promoting rhizobacteria associated with the roots of Indian mustard [Brassica juncea L. Czern.]. Soil Biol. Biochem., 37, 241-250. Bender, C.L. Rangaswamy, V. and Loper, J.E. [1999]. Polyketide production by plant associated pseudomonads. Annu. Rev. Phytopathol., 37, 175-196. Barea, J.M. Bonis, A.F. and Olivaries, J. [1983]. Interactions between Azospirillum and VA mycorrhiza and their effects on growth and nutrition of maize and ryegrass. Soil Biol. Biochem., 15, 705-709. Bertrand, H. Plassard, C. Pinochet, X. Touraine, B. Normand, P. and Cleyet-Marel, J.C. [2000]. Stimulation of the ionic transport system in Brassica napus by a plant growthpromoting rhizobacterium [Achromobacter sp.]. Can. J. Microbiol., 46, 229-236. Beauchamp, C.J. Kloepper, J.W. Lemke, P.A. [1993]. Luminometric analyses of plant root colonization by bioluminescent pseudomonads. Can. J. Microbiol., 39, 434-441. Bloemberg, G.V. and Lugtenberg, B.J. [2001]. Molecular basis of plant growth promotion and biocontrol by rhizobacteria. Curr Opin Plant Biol, 4, 343-350. Boddey, R.M. Urquiaga, S.B. Alves, J.R. and Reis, V. [2003]. Endophytic nitrogen fixation in sugarcane: present knowledge and future applications. Plant Soil, 252,139-149. Bora, T. Ozaktan, H. Gore, E. and Aslan, E. [2004]. Biological control of Fusarium oxysporum f. sp. melonis by wettable powder formulations of the two strains of Pseudomonas putida. Phytopathology, 152, 471-475. Bressan, W. [2003]. Biological control of maize seed pathogenic fungi by use of actinomycetes. Biocontrol, 48, 233-240. Brevin, N. [1991]. Development of the legume root nodule. Annu. Re Cell Biol, 7, 191-226. Brimecombe, M.J. De Leij, F.A. and Lynch, J.M. [2001]. The effect of root exudates on

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

92

B. Hameeda, G. Harini, B. Keerthi Kiran et al.

rhizosphere microbial population. In R. Pinton [Eds.], The Rhizosphere.[pp. 95-140], Inc. New York, Marcel Dekker. Buchholz, D.D. Brown, J.R. [1993]. Potassium in Missouri soils. Agricultural publication, 9185. Burd, G.I. George, D. Bernard, D. and Glick, R. [2000].Plant growth-promoting bacteria that decrease heavy metal toxicity in plants. Can. J. Microbiol., 46, 237-245. Burdman, S. Jurkevitch, E. and Okon, Y. [2000]. Recent advances in the use of plant growth promoting Rhizobacteria [PGPR] in agriculture. In N.S. Subba Rao and Dommergues, Y.R. [Eds.], Microbial Interactions in Agriculture and Forestry [vol 2, pp. 229-250]. Plymouth, UK, Science Publishers Inc. Canbolat, M. Bilen, S. Akmak, R. Fiahin, F. and Aydın, A.[2006]. Effect of plant growth promoting rhizobacteria and soil compaction on barley seedling growth, nutrient uptake, soil properties and rhizosphere microflora. Biol. Fertil Soils, 42, 350-357. Castric, P.[1994]. Influence of oxygen on the Pseudomonas aeruginosa hydrogen cyanide synthase. Curr. Microbiol., 29, 19-21. Cavigelli, M.A. Robertson, G.P. and Klug, M.J. [1995]. Fatty acid methyl ester [FAME] profiles as a measures of soil microbial community structure. Plant Soil, 170, 99-113. Cha, C. Gao, P. Chen, Y.C. Shaw, P.D. and Farrand, S.K. [1998]. Production of AcylHomoserine Lactone Quorum-Sensing Signals by Gram-Negative Plant-Associated Bacteria. Mol. Plant Microb. Interact, 11,1119–1129. Chabot, R. Antoun, H. and Cescas, P.M. [1996]. Growth promotion of maize and lettuce by phosphate solubilizing Rhizobium leguminosarum biovar. Phaseoli. Plant Soil, 184, 311– 321. Chalfie, M. Tu, Y. Euskirchen, G. Ward, W.W. and Prasher, D.C. [1994]. Green fluorescent protein as a marker for gene expression. Science, 263, 802-805. Chandanie, W.A. Kubota, M. and Hyakumachi, M. [2006]. Interactions between plant growth promoting fungi and arbuscular mycorrhizal fungus Glomus mosseae and induction of systemic resistance to anthracnose disease in cucumber. Plant Soil, 286, 209–217. Chapon, A. Boutin, M. Rime, D. Delalande, L. Guillerm, A.Y. and Sarniguet, A. [2003]. Direct and specific assessment of colonization of wheat rhizoplane by Pseudomonas fluorescens Pf29A. Eur. J. Plant Pathol., 109, 61-70 Chatterjee, A. Cui, Y. Liu, Y. Dumenyo, C.K. and Chatterjee, A.K. [1995]. Inactivation of rsm A lead to over production of extracellular pectinases, cellulases, and proteases in Erwinia carotovora sub sp carotovora in the absence of the starvation/cell densitysensing signal N- [3-oxohexanoyl] - L - homoserine lactone. Appl. Environ. Microbiol., 61,1959-1967. Chen, K. and Pachter, L. [2005]. Bioinformatics for wholegenome shotgun sequencing of microbial communities. PLoS. Computational Biology,1,106–112. Chin-A-Woeng, T.F.C. Bloemberg, G.V. Mulder, I.H.M. Dekkers, L.C. and Lugtecnberg, B.J.J. [2000]. Root colonization by phenazine-I-carboxamide-producing bacterium Pseudomonas chlororaphis PCL 1391 is essential for bicontrol of tomato foot and root rot. Mol. plant-microbe Interact,13,1340-1345. Chung, H. Park, M. Madhaiyan, M. Seshadri, S. Song, J. Cho, H. and Sa, T. [2005]. Isolation and characterization of phosphate solubilizing bacteria from the rhizosphere of crop plants of Korea. Soil Biol. Biochem., 37,1970-1974.

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Role of Plant Growth Promoting Microorganisms for Sustainable Crop Production

93

Compant, S. Duffy, B. Nowak, J. Christophe, C. and Barka, E.A. [2005]. Use of Plant Growth-Promoting Bacteria for Biocontrol of Plant Diseases: Principles, Mechanisms of Action, and Future Prospects. Appl. Environ. Microbiol., 71,4951–4959. Constantinescu, F. [2001]. Extraction and identification of antifungal metabolites produced by some B. subtilis strains. Analele Institutului de Cercetari Pentru Cereale Protectia Plantelor, 31, 17-23. Coombs, J.T.and Franco, C.M. [2003]. Isolation and identification of actinobacteria from surface-sterilized wheat roots. Appl. Environ. Microbiol., 69, 5603–5608. Costerton, J.W. Lewandowski, Z. Caldwell, D.E. Korber, D.R. and Lappinscott, H.M. [1995]. Microbial biofilms. Annu Rev Microbiol, 49, 711-745. Crusberg, T.C. [2008]. Fungi and nutrient cycling: An overview. In G. George, G. Khachatourians, D.K, Arora, Rajendran, T.P. and Srivastava A.K. [Eds.], Agriculturally important microorganisms [vol 1,pp. 133-152], India, Academic world international. Cuerinot, M. and Yi, Y. [1994]. Iron: Nutritious, Noxious, and Not Readily Available. Plant Physiol, 815-820. Dalton, D.A. Kramer, S.B. Azios, N. Fusaro, S. Cahill, E. and Kennedy, C. [2004]. Endophytic nitrogen fixation in dune grasses [Ammophila arenaria and Elymus mollis] from Oregon. FEMS Microbiol Ecol, 49, 469-479. Dandurand, L.M. Morra, M.J. Chaverra, M.H. and Orser, C.S. [1994]. Survival of Pseudomonas sp in air-dried mineral powders. Soil Biol. Biochem., 26, 1423-1430. De Boer, M. Bom, P. Kindt, F. Keurentjes, J.J.B. van der Sluis, I. Van Loon, L.C. and Bakker, P.A.H.M. [2003]. Control of Fusarium wilt of radish by combining Pseudomonas putida strains that have different disease-suppressive mechanisms. Phytopathology, 93, 626-632. Dekkers, L.C. van der Bij, A.J. Mulders, I.H.M. Phoelich, C.C. Wentwoord, R.A.R. Glandorf, D.C.M. Wijffelman, C.A. and Lugtenberg, B.J.J. [1998]. Role of the O-antigen of lipopolysaccheride, and possible roles of growth rate and of NADH:ubiquinone oxidoreductase [nuo] in competitive tomato root-tip colonization by Pseudomonas fluorescens WCS365. Mol. Plant-Microbe Interact, 11,763–771. DeMarco, J.L. Lima, C.L.H. Desousa, M.V. and Felix, C.R. [2000]. A Trichoderma harzianum chitinase destroys the cell walls of the phytopathogen Crinipellis pernicosa, the casual agent of the witches broom disease of cocoa. World J. Microbiol Biotechnol, 16,383–386. Dobbelaere, S. Vanderleyden, J. and Okon, Y. [2003]. Plant growth promoting effects of diazotrophs in the rtizosphere. Crit Rev. Plant Sci, 22, 107-149. Dobbelaere, S. Croonborghus, A. Thys, A. Ptacek, D. Okon, Y. and Vanderleyden, J. [2002]. Effect of inoculation with wild type Azospirillum brasilense and A. irakense strains on development and nitrogen uptake of spring wheat and grain maize. Biol. Ferti Soils, 36, 284-197. Duffy, B.K. Simon, A. and Weller, D.M. [1996]. Combination of Trichoderma koningii with fluorescent pseudomonads for control of take all of wheat. Phytopathology, 86, 188-194. Duijff, B.J. Gianinazzi-Pearson, V. and Lemanceau, P. [1997]. Involvement of the outer membrane lipopolysaccharides in the endophytic colonization of tomato roots by biocontrol Pseudomonas fluorescens strain WCS417r. New Phytol, 135, 325–334. Dunne, C. Delany, I. Fenton, A. and Gara, F.O. [1996]. Mechanisms involved in biocontrol by microbial inoculants. Agronomie, 16.721-729.

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

94

B. Hameeda, G. Harini, B. Keerthi Kiran et al.

Duponnois, R. Colombet, A. Hien, V. and Thioulouse, J. [2005]. The mycorrhizal fungus Glomus intraradices and rock phosphate amendment influence plant growth and microbial activity in the rhizosphere of Acacia holosericea. Soil Sci. Biochem., 37,14601468. Duville, Y. and Boland, G.L. [1992]. A note on the antibiotic properties of B. subtilis against Colletotrichum trifoli. Phytoprotection, 73, 31-36. Egamberdiyeva, D. and Höflich, G. [2004]. Effect of plant growth promoting bacteria on growth and nutrient uptake of cotton and pea in semiarid region of Uzbekistan. J. Arid. Environ., 56, 293-301. El-Tarabily, K.A. [2006]. Rhizosphere-competent isolates of streptomycete and nonstreptomycete actinomycetes capable of producing cell wall degrading enzymes to control Pythium aphanidermatum damping-off disease of cucumber. Can. J. Bot,84, 211222. El-Tarabily, K.A. [2003]. An endophytic chitinase-producing isolate of Actinoplanes missouriensis, with potential for biological control of root rot of lupine caused by Plectosporium tabacinum. Aus. J. Bot., 51,257–266. El-Tarabily, K.A. Soliman, M.H. Nassar, A.H. Al-Hassani, H.A. Sivasithamparam, K. McKenna, F. and Hardy, G.E. [2000]. Biological control of Sclerotinia minor using a chitinolytic bacterium and actinomycetes. Plant Pathol., 49,573-583. El-Tarabily, K.A. Nassar, A.H. Hardy, G.E.S.J. and Sivasithamparam, K. [2003]. Fish emulsion as a food base for rhizobacteria promoting growth of radish [Raphanus sativus L. var. sativus] in a sandy soil. Plant Soil, 252,397–411. Errakhi, R. Bouteau, F. Lebrihi, A. and Barakate, M. [2007]. Evidences of biological control capacities of Streptomyces sp. against Sclerotium rolfsii responsible for damping-off disease in sugar beet [Beta vulgaris L.]. World J. Microbiol. Biotechnol., 23,1503– 1509. Eziashi, E.I. Omamor, I.B. and Odigie, E.E. [2007]. Antogonism of Trichoderma viride and effects of extracted water soluble compounds from Trichoderma species and benlate solution on Ceratocystis paradoxa. African J. Biotechnol., 6,388-392. Faramarzi, M.A. and Brandl, H. [2006]. Formation of water-soluble metal cyanide complex from solid minerals by Pseudomonas plecoglossida. FEMS Microbiol Lett, 259,47-52. Faramarzi, M.A. Stagars, M. Pensini, E. Krebs, W. and Brandl, H. [2004]. Metal solubilization from metal-containing solid materials by cyanogenic Chromobacterium violaceum. J. Biotechnol., 113,321–326. Felici, C. Vettori, L. Giraldi, E. Costantina, L.M.F. Toffanin, A. Tagliasacchi, A.M. and Nuti, M. [2008]. Single and co-inoculation of Bacillus subtilis and Azospirillum brasilense on Lycopersicon esculentum: Effects on plant growth and rhizosphere microbial community. Appl Soil Ecol, 40, 260-270. Fernando, W.G.D. and Nakkeeran, S.Z. [2005]. Biosynthesis of antibiotics by pgpr and its relation in biocontrol of plant diseases. In Z.A. Siddiqui [Ed.], PGPR: Biocontrol and Biofertilization [pp 67-100] , The Netherlands, Dordrecht, Springer. Fernando, W.G.D. Ramarathnam, R. Krishnamoorthy, A.S. and Savchuk, S. [2005]. Identification and use of bacterial organic volatiles in biological control of Sclerotinia sclerotiorum. Soil Biol Biochem, 36,955-964. Fernando, W.G.D. Watson, A.K. and Paulitz, T.C. [1996]. The role of Pseudomonas sp. and competition for carbon, nitrogen and iron in the enhancement of appressorium formation by Colletotrichum coccodes on velvet leaf. Eur. J. Plant. Pathol, 102,1-7.

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Role of Plant Growth Promoting Microorganisms for Sustainable Crop Production

95

Fogliane, V. Ballio, A. Gallo, M. Woo, S. Scala, F. and Lorita, M. [2002]. Pseudomonas lipodepsipeptides and fungal cell-wall degrading enzymes acts synergistically in biological control. Mol Plant Microbe Interact, 15,323-333. Fridlender, M. Inbarm J, and Chet, I. [1993]. Biological control of soilborne plant pathogens by a β-1,3 glucanase – producing Pseudomonas cepacia. Soil Biol Biochem, 19,12111221. Fulchieri, M. Lucangeli, C. and Bottini, R. [1993]. Inoculation with Azospirillum lipoferum affects growth and gibberellins status of corn seedling roots. Plant Cell Physiol. ,34, 1305-1309. Galleguillos, C. Aguirre, C. Barea, J.M. and Azcon, R. [2000]. Growth promoting effect of two Sinorhizobium meliloti strains [a wild type and its genetically modified derivative] on a non legume plant species in specific interaction with two arbuscular mycorrhizal fungi. Plant Sci, 159,57-63. Gamal, M.A.F. [2001]. Measurement of the viability of arbuscular-mycorrhizal fungi using three different stains; relation to growth and metabolic activities of soybean plants. Microbiol Res, 156,359-367. Geetha, R. Sing, F. Anjana, J. Desai, G. and Archana. [2008]. Enhanced growth and nodulation of pigeon pea by co-inoculation of Bacillus strains with Rhizobium sp. Bioresour Technol., 99, 4544-4550. Getha, K. Vikineswary, S. Wong, W.H. Seki, T. Ward, A. and Goodfellow, M. [2005]. Evaluation of Streptomyces sp. strain g10 for suppression of Fusarium wilt and rhizosphere colonization in pot-grown banana plantlets. J. Ind. Microbiol Biotech, 32,2432. Glick, G.R. [2004]. Bacterial ACC deaminase and alleviation of plant stress. Adv. Appl. Microbiol., 56,291-312. Glick, B.R. Penrose, D.M. and Li, J. [1998]. A model for the lowering of plant ethylene concentrations by plant growth-promoting bacteria. J. Theor. Biol., 190,63-68. Glick, B.R. [1995]. The enhancement of plant growth by free-living bacteria. Can. J. Microbiol, 41,109-117. Goldstein, A.H. Rogers, R.D. and Mead, G. [1993]. Mining by microbe. Bio Technol, 11,1250-1254. Gravel, V. Antoun, H. and Tweddell, R.J. [2007]. Growth stimulation and fruit yield improvement of greenhouse tomato plants by inoculation with Pseudomonas putida or Trichoderma atroviride: Possible role of indole acetic acid [IAA]. Soil Biol. Biochem., 39,1968–1977. Gray, E.J. and Smith, D.L. [2005]. Intracellular and extracellular PGPR: commonalities and distinctions in the plant–bacterium signaling processes. Soil Biolo Biochem., 37,395–412. Greiner, R. Haller, E. Konietzny, U. and Jany, K.D. [1997]. Purification and characterization of a phytase from Klebsiella terrigena. Arch Biochem Biophys, 341, 201-206. Gupta, C.P. Dubey, R.C. and Maheshwari, D.K. [2002]. Plant growth enhancement and suppression of Macrophomina phaseolina causing charcoal rot of peanut by fluorescent Pseudomonas. Biol Fert Soils, 35,399-405. Gyaneshwar, P. Naresh Kumar, G. Parekh, L.J. and Poole, P.S. [2002]. Role of soil microorganisms in improving P nutrition of plants. Plant Soil., 245, 83–93. Gyaneshwar, P. Parekh, L.J. Archana. G. Poole, P.S. Collins, M.D. Hutson, R.A. and Naresh, K.G. [1999]. Involvement of a phosphate starvation inducible glucose dehydrogenase in

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Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

96

B. Hameeda, G. Harini, B. Keerthi Kiran et al.

soil phosphate solubilization by Enterobacter asburiae. FEMS Microbiol Lett, 171, 22329. Haggag, W.M. [2002]. Sustainable agriculture management of plant diseases. J. Biol. Scie, 2,280-284. Hamdali, H. Bouizgarne, B. Hafidi, M. Lebrihi, A. Virolle, M.J. and Ouhdouch, Y. [2008a]. Screening for rock phosphate-solubilizing Actinomycetes from Moroccan phosphate mines. Appl. Soil Ecol, 38,12–19. Hamdali, H. Hafidi, M. Marie, J.V. and Yedir, O. [2008b]. Rock phosphate-solubilizing Actinomycetes: screening for plant growth-promoting activities. World J. Microbiol. Biotechnol., 24, 2565–2575. Hameeda, B. Harini, G. Rupela, O.P. and Gopal Reddy. [2008]. Growth promotion of maize by phosphate solubilizing bacteria isolated from composts and macrofauna. Microbiol Res, 163,234-242. Hameeda, B. Srijana, M. Rupela, O.P. and Gopal Reddy. [2007] Effect of bacteria isolated from composts and macrofauna on sorghum growth and mycorrhizal colonization. World J. Microbiol. Biotechnol, 23,883–887. Hameeda, B. Rupela. O.P. Gopal Reddy, and Satyavani, K. [2006a]. Application of plant growth promoting bacteria associated with composts and macrofauna for growth promotion of Pearl millet [Pennisetum glaucum L.]. Biol. Fertil Soil, 43,221-227. Hameeda, B. Rupela, O.P. and Gopal Reddy. [2006b]. Evaluation of bacterial isolates from composts and macrofauna for their biocontrol activity against soil-borne plant pathogenic fungi. Indian J. Microbiol, 46, 389-396. Hameeda, B. Harish Kumar Reddy, Y. Rupela, O.P. Kumar, G.N. and Gopal Reddy. [2006c]. Effect of carbon substrates on rock phosphate solubilization by bacteria from composts and macrofauna. Current Microbiol, 53, 298-302. Hameeda, B. Rupela, O.P. Wani, S.P. and Gopal Reddy. [2006d]. Indices to assess soil quality and sustainability in systems involving low cost biological inputs and conventional system. Inter J. Soil Scien, 3, 196-206. Hameeda, B. [2005] Studies on plant growth promoting microorganisms and recycling of crop residues for sustainable agriculture. PhD Thesis, ICRISAT. Hammer, P.E. Burd, W. Hill, D.S. Ligon, J.M. and van Pee, K.H. [1999]. Conservation of the pyrrolnitrin gene cluster among six pyrrolnitrin-producing strains. FEMS Microbiol Lett, 180, 39-44. Han, H.S. Supanjani. Lee, K.D. [2006]. Effect of co-inoculation with phosphate and potassium solubilizing bacteria on mineral uptake and growth of pepper and cucumber. Plant Soil Environ, 52,130–136. Hardy, G.E.S.J. and Sivasithamparam, K. 1995. Antagonism of fungi and actinomycetes isolated from composted eucalyptus bark to Phytophthora drechsleri in a steamed and non-steamed composted eucalyptus bark-amended container medium. Soil Biol. Biochem. 27: 243-246. Harini, G. [2005]. Effect of vermicomposts enriched with phosphate solubilizing bacteria [PSB] on growth of maize and mycorrhizal colonization. [M Sc. dissertation work].ICRISAT. Hause, B. Maier, W. Miersch, O. Kramell, R. and Strack, D. [2002]. Induction of Jasmonate Biosynthesis in Arbuscular Mycorrhizal Barley Roots. Plant Physiology, 130,1–8.

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Role of Plant Growth Promoting Microorganisms for Sustainable Crop Production

97

Hass, D. Keel, C. Reimman, C. [2002]. Signal transduction in plant beneficial rhizobacteria with biocontrol properties. Antonie van Leeuwenhoek, 81, 385-395. Hirsch, A.M. Fang, Y. Asad, S. and Kapulnik, Y. [1997]. The role of phytohormones in plant microbe symbioses. Plant Soil,194, 171-184. Höfte, M. [1993]. Classes of microbial siderophores. In L.L. Barton and B.C. Hemming [Eds.], Iron chelation in plants and soil microorganisms.[ pp. 3–26], San Diego, Calif, Academic Press. Hoitink, H.A.J. and Boehm, M.J. [1999]. Biocontrol within the context of soil microbial communities: a substrate-dependent phenomenon. Annu. Rev. Phytopathol, 37, 427-446. Howell, C.R. and Stipanovic, R.D. [1980]. Suppression of Pythium ultimum-induced damping-off of cotton seedlings by Pseudomonas fluorescens and its antibiotic, pyoluteorin. Phytopathology, 70,712-715. Hung, S.H. Yu, C.W. and Lin, C.H. [2005]. Hydrogen peroxide function as a stress signal in plants. Bot Bull Acad Sin, 46, 1-10. Hurek, T. Handley, L.L. Reinhold-Hurek, B. and Piche, Y. [2002]. Azoarcus grass endophytes contribute fixed nitrogen to the plant in an unculturable state. Mol Plant Micro Interact, 15,233-242. Idris, H.A. Labuschagnea, N. Korstena, L. [2008]. Suppression of Pythium ultimum root rot of sorghum by rhizobacterial isolates from Ethiopia and South Africa. Biocontrol, 45,7284. Idris, E.E.S. Bochow, H. Ross, H. and Borriss, R. [2004]. Use of Bacillus subtilis as biocontrol agent. VI. Phytohormone like action of culture filtrates prepared from plant growth-promoting Bacillus amyloliquefaciens FZB24, FZB42, FZB45 and Bacillus subtilis FZB37. J. Plant Dis. Prot, 111,583-597. Idris, E.E.S. Makarewicz, O. Farouk, A. Rosner, K. Greiner, R. Bochow, H. Richter, T. and Borriss, R. [2002]. Extracellular phytase activity of Bacillus amyloliquefaciens FZB 45 contributes to its plant growth- promoting effect. Microbiology, 148,2097-2109. Igarashi, Y. Iida, T. Sasaki, Y. Saito, N. Yoshida, R. and Furumai, T. [2002]. Isolation of actinomycetes from live plants and evaluation of anti phytopathogenic activity of their metabolites. Actinomycetologica, 16, 9–13. Iniguez, A.L. Dong, Y. and Triplett, E.W. [2004]. Nitrogen fixation in wheat provided by Klebsiella pneumoniae 342. Mol. Plant Microb Interact, 17,1078-1085. Jaizme-Vega, M.D.C. Rodriguez-Romero, A.S. and Guerra, M.S.P. [2004]. Potential use of rhizobacteria from the Bacillus genus to stimulate the plant growth of micropropagated bananas. Fruits, 59, 83-90. Jalili, F. Khavazi, K. Pazira, K. Nejat, A. Rahman, H.S. Sadaghiani, H.S.and Miransari, M. [2008]. Isolation and characterization of ACC deaminase-producing fluorescent pseudomonads, to alleviate salinity stress on canola [Brassica napus L.] growth . J. Plant physiol, doi:10.1016/j.jplph.2008.08.004. James, E.K. Olivares, F.L. Baldani, J.I. and Döbereiner, J. [1997]. Herbaspirillum, an endophytic diazotroph colonizing vascular tissue in leaves of Sorghum bicolor L. Moench. J. Experi Bot., 48,785-797. Janisiewicz, W.J. and Jeffers, S.N. [1997]. Efficacy of commercial formulation of two biofungicides for control of blue mold and gray mold of apple in cold storage. Crop Protect, 16, 629 – 633.

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

98

B. Hameeda, G. Harini, B. Keerthi Kiran et al.

Janisiewicz, W.J. [1996]. Ecological diversity, niche overlap, and coexistence of antagonists used in developing mixtures for biocontrol of post harvest diseases of apples. Phytopathology, 86, 473-479. Jetiyanon, K. Fowler, W.D. and Kloepper, J.W. [2003]. Broad-spectrum protection against several pathogens by PGPR mixtures under field conditions in Thailand. Plant Dis, 87,1390-1394. Jeyarajan, R. and Nakkeeran, S. [2000]. Exploitation of microorganisms and viruses as biocontrol agents for crop disease mangement. In Upadhyay [Ed.] Biocontrol Potential and their Exploitation in Sustainable agriculture [pp. 95-116], USA, Kluwer Academic/ Plenum Publishers. Jia, J. Ito, H. Matsui, H. and Honma, M. [2000]. 1-Aminocyclopropane-1-carboxylate [ACC] deaminase induced by ACC synthesized and accumulated in Penicillium citrinum intracellular spaces. Biosci. Biotech Biochem., 64,299-305. Jiao, J.Y. Wang, H.X. Zeng, Y. and Shen, Y.M. [2006]. Enrichment for microbes living in association with plant tissues. J App Microbiol, 100, 830–837. Jijakli, M.H. and Lepoivre, P. [1998]. Characterization of an exo-beta-1, 3-gluconase produced by Pichia anomala strain K, antagonist of Botrytis cinerea on apples. Phytopathology, 88,335-343. Johansson, J.F. Paul, L.R. and Finlay, R.D. [2005]. Microbial interactions in the mycorrhizosphere and their significance for sustainable agriculture. FEMS Microbiol Eco,l 48,1–13. Kamilova, F. Kravchenko, L.V. Shaposhnikov, A.I. Azarova, T. Makarova, N. and Lugtenberg, B. [2006]. Organic acids, sugars, and L-tryptophan in exudates of vegetables growing on stone wool and their effects on activities of rhizosphere bacteria. Mol. Plant– Microbe Interact, 19,250–256. Kang, G.S. Beri, V. Rupela, O.P. and Sidhu, B.S. [2005]. A new index to assess soil quality and sustainability of wheat based cropping systems. Bio. Fertil Soils, 41, 389-398. Karthikeyan, N. Prasanna, R. Nain, L. Kaushik, B.D. [2007]. Evaluating the potential of plant growth promoting cyanobacteria as inoculants for wheat. Eur J Soil Biol, 43,23-30. Katiyar, V. and Goel, R. [2004]. Siderophore-mediated plant growth promotion at low temperature by mutant of fluorescent pseudomonad. Plant Growth Regul, 42,239-244. Kennedy, I.R. Choudhury, A.T.M.A.and Kecskés, M.L. [2004]. Non-symbiotic bacterial diazotrophs in crop-farming systems: can their potential for plant growth promotion be better exploited?. Soil Biol Biochem, 36,1229-44. Kennedy, A.C. and Smith, K.L. [1995]. Soil Microbial Diversity and Sustainability of Agricultural Soils. Plant Soil, 170,75–86. Kerovuo, J. Lauraeus, M. Nurminen, P. Kalkkinen, N. and Apajalahti, J. [1998]. Isolation, characterization, molecular gene cloning, sequencing of a novel phytase from Bacillus subtilis. Appl Environ Microbiol, 64,2079-85. Khan, A.G.[2005]. Role of soil microbes in the rhizospheres of plants growing on trace metal contaminated soils in phytoremediation. J Trace Elem Med Biol, 18,355–364. Khan, M.R. and Khan, S.M. [2002]. Effects of root-dip treatment with certain phosphate solubilizing microorganisms on the fusariam wilt of tomato. Bioresour technol, 85,213215.

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Role of Plant Growth Promoting Microorganisms for Sustainable Crop Production

99

Khan, M.S. and Zaidi, A. [2007]. Synergistic Effects of the Inoculation with Plant GrowthPromoting Rhizobacteria and an Arbuscular Mycorrhizal Fungus on the Performance of wheat. Turk J. Agric. For, 31, 355-362. Kim, K.Y. Jordan, D. McDonald, G.A. [1998a]. Enterobacter agglomerans, phosphate solubilizing bacteria and microbial activity in soil: Effect of carbon sources. Soil Biol. Biochem, 30, 995-1003. Kim, K.Y. Jordan, D. and McDonald, G.A. [1998b]. Effect of phosphate solubilizing bacteria and vesicular-arbuscular mycorrhizae on tomato growth and soil microbial activity. Biol. Fertil Soils, 26, 79-87. Kishore, G.K. Pande, S. and Podile, A.R. [2005a]. Phylloplane bacteria increase seedling emergence, growth and yield of field-grown groundnut [Arachis hypogeae L.]. Lett. Appl. Microbiol, 40, 260-268. Kishore, G,K. Pande, S. and Podile, A.R. [2005b]. Biological control of late leaf spot of peanut [Arachis hypogaea L.] with chitinolytic bacteria. Phytopathology, 95,1157-1165. Kitten, T. Kinscherf, T. McEvoy, G. and Willis, D.K. [1998]. A newly identified regulator is required for virulence and toxin production in Pseudomonas syringae. Mol Microbiol, 28, 917–929. Kloepper, J.W. Ryu, C.M. and Zhang, S. [2004]. Induced systemic resistance and promotion of plant growth by Bacillus sp. Phytopathology, 94,1259-1266. Kloepper, J.W. Tuzun, S. and Kuc, J. [1992]. Proposed definitions related to induced disease resistance. Biocont Sci Technol, 2, 347-349. Kloepper, J.W. and Schroth, M.N. [1978]. Plant growth promoting rhizobacteria on radishes. Proc IVth Int Conf Plant Pathogenic Bacteria, Angers, France 2:879-882. Knight, T.J. and Langston, U.P.J. [1988]. Enhancement of symbiotic dinitrogen fixation by a toxin releasing plant pathogen. Science, 241, 951-954. Koch, B. Nielsen, T.H. Sorensen, D. Andersen, J.B. Christophersen, C. Molin, S. Givskov, M. Sorensen, J. and Nybroe, O. [2002]. Lipopeptide production in Pseudomonas sp. strain DSS73 is regulated by components of sugar beet exudates via the Gac twocomponent regulatory system. Appl. Environ. Microbiol., 68, 4509-4516. Krishnamurthy, K. and Gnanamanickam, S.S. [1998]. Biological control of rice blast by Pseudomonas fluorescens strain Pf7-14: evaluation of a marker gene and formulations. Biocontrol, 13,158-165. Kumar, V. and Narula, N. [1999]. Solubilization of inorganic phosphates and growth emergence of wheat as affected by Azotobacter chroococcum mutants. Biol Fertil Soil, 28,301-305. Kundu, B.S. and Gaur, A.C. [1984]. Rice response to inoculation with N2-fixing and Psolubilizing microorganisms. Plant Soil, 79,227-234. Landa, B.B. Mavrodi, D.M. Thomashow, L.S. and Weller, D.M. [2003]. Interactions between strains of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens in the rhizosphere of wheat. Phytopathology, 93, 982–994. Landerweert, R. Hoffland, E. and Finlay, R.D. [2001]. Linking plants to rocks, ectommycorrhizal fungi mobilize nutrients from minerals. Trends Ecol Evol, 389,682683. Lavania, M. Chauhan, P.S. Chauhan, S.V.S. Singh, H.B. and Nautiyal, C.S. [2006]. Induction of plant defense enzymes and phenolics by treatment with plant growth-promoting rhizobacteria Serratia marcescens NBRI1213. Curr Microbiol, 52, 363-368.

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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100

B. Hameeda, G. Harini, B. Keerthi Kiran et al.

Lebuhn, M. Heulin, T. and Hartmann, A. [1997]. Production of auxin and other indolic and phenolic compounds by Paenibacillus polymyxa strains isolated from different proximity to plant roots. FEMS Microbiol Ecol, 22,325-34. Leveau, J.H.J. [2007]. The magic and menace of metagenomics: prospects for the study of plant growth-promoting rhizobacteria. Eur. J. Plant Pathol, 119,279–300. Li, J. Ovakim, D.H. Charles, T.C. and Glick, B.R. [2000]. An ACC deaminase minus mutant of Enterobacter cloacae UW4 no longer promotes root elongation. Curr. Microbiol, 41,101-105. Ligon, J.M. Hill, D.S. Hammer, P.E. Torkewitz, N.R. Hofmann, D. Kemph, H.J.and Van Pee, K.H. [2000]. Natural products with antifungal activity from Pseudomonas biocontrol bacteria. Pest management science, 56, 688-695. Lin, Q.M. Rao, Z.H. Sun, Y.X. Yao, J. and Xing, L.J. [2002]. Identification and practical application of silicate-dissolving bacteria. Agri Sci in China, 1, 81-85. Loiret, F.G. Ortega, E. Kleiner, D. Ortega-Rodes, P. Rodes, R. and Dong, Z. [2004]. A putative new endophytic nitrogen-fixing bacterium Pantoea sp. from sugarcane. J. Appl. Microbiol, 97,504-511. Loper, J.E. and Henkels, M.D. [1999]. Utilization of heterologous siderophores enhances levels of iron availability to Pseudomonas putida in the rhizosphere. Appl. Environ Microbiol, 65,5357-5363. Lorenz, P. and Eck, J. [2005]. Metagenomics and industrial applications. Nature Reviews Microbiol, 3,510–516. Louws, F.J. Fullbright, D.W. Stephens, C.T. and de Bruijn, F.J. [1994]. Specific genomic fingerprintings of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR. Appl. Environ. Microbiol, 60, 2286-2295. Lugtenberg, B.J.J. Dekkers, L. and Bloemberg, G.V. [2001]. Molecular determinants of rhizosphere colonization by Pseudomonas. Ann. Rev. Phytopathology, 39, 461-490. Lugtenberg, B.J.J. and Dekkers, L.C. [1999]. What make Pseudomonas bacteria rhizosphere competent?. Environ. Microbiol., 1,9–13. Maddula, V.S.R.K. Zhang, Z. Pierson, E.A. and Pierson, III L.S. [2006]. Quorum sensing and phenazines are involved in biofilm formation by Pseudomonas chlororaphis [aureofaciens] strain 30-84. Microbiol. Ecol., 52, 289-301. Madi, L. and Katan, L. [1998]. Penicillium janczewskii and its metabolites, applied to leaves, elicit systemic acquired resistance to stem rot caused by Rhizoctonia solani. References and further reading may be available for this article. To view references and further reading you must purchase this article. Physiology Mole Plant Pathol, 53,163-175. Magdoff, F. and van Es, H. [2000]. Building Soils for Better Crops. In Sustainable Agriculture Network. Beltsville, MD USA, Handbook Series Book 4. Manjula, K. and Podile, A.R. [2005]. Increase in seedling emergence and dry weight of pigeon pea in the field with chitin-supplemented formulations of Bacillus subtilis AF 1. World J. Microbiol. Biotech., 21,1057-1062. Manjula, K. and Podile, A.R. [2001]. Chitin-supplemented formulations improve biocontrol and plant growth promoting efficiency of Bacillus subtilis AF 1. Can. J. Microbiol, 47, 618-625. Mantelin, S. and Touraine, B. [2004]. Plant growth-promoting bacteria and nitrate availability: impacts on root development and nitrate uptake. J. Exp. Bot., 55, 27-34.

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Role of Plant Growth Promoting Microorganisms for Sustainable Crop Production 101 Marek-Kozaczuk, M. and Skorupska, A. [2001]. Production of B-group vitamins by plant growth promoting Pseudomonas fluorescens strain 267 and the importance of vitamins in the colonization and the nodulation of red clover. Biol. Fertil Soils, 33, 146-151. Martinez, R.V. and Bernando, D.A. [2006]. Practical application of bacterial biofertilizers and biostimulants. In N. Uphoff, S.A. Ball, E. Fernandes, H. Herren, O. Husson, M. Laing, C.Palm, J. Pretty, N. Sanchez, and J. Thies [Eds.], Biological approaches to sustainable soil systems [pp. 467-478], Florida, USA, CRC Press. Martinez-Romero, E., and Wang, E.T. [2000]. Sesbania herbacea–Rhizobium huautlense nodulation in flooded soils and comparative characterization of S. herbaces nodulating rhizobia in different environments. Microbe Ecol, 41,25–32. Matora, L.Y. Serebrennikova, O.B. and Shchyogolev, S.Y. [2001]. Structural effects of the Azospirillum lipopolysaccharides in cell suspensions. Biomacromolecules, 2, 402-406. Matsumoto, L.S. Martines, A.M. Avanzi, M.A. Albino, U.B. Brasil, C.B. Saridakis, D.P. Rampazo, L.G.L. Zangaro, W. and Andrade, G. [2005]. Interactions among functional groups in the cycling of carbon, nitrogen and phosphorous in the rhizosphere of three successional species of tropical woody trees. Appl. Soil Ecol., 28, 57-65. Meena, B. Radhajeyalakshmi, R. Marimuthu, T. Vidhyasekaran, P. and Velazhahan, R. [2002]. Biological control of groundnut late leaf spot and rust by seed and foliar applications of a powder formulation of Pseudomonas fluorescens. Biocont Sci. Technol, 12, 195-204. Melnick, R.L. Zidack. N.K. Bailey, B.A. Maximova, S.N. Guiltinan, M. and Backman, P.A. [2008]. Bacterial endophytes: Bacillus sp. from annual crops. Biocontrol doi: 10.1016/j.biocontrol.2008.01.022. Milen, I. Georgiev, Atanas, I. Pavlov, and Bley, T. [2007]. Hairy root type plant in vitro systems as sources of bioactive substances. Appl. Microbiol. Biotechnol., 74, 1175–1185. Miller, R.M. and J. D. Jastrow. [1992]. The role of mycorrhizae in soil conservation. In G. Bethlenfalvay and R. Linderman [Eds.], Mycorrhizae in Sustainable Agriculture pp. 2944. Special Publ. 54 of the ASA, CSSA, and SSSA. Moffitta, M.C. and Neilana, B.A. [2000]. The expansion of mechanistic and organism diversity associated with non-ribosomal peptides. FEMS Microbiol letters, 191, 159-167. Morris, C.E. and Monier, J.M. [2003]. The ecological significance of biofilm formation by plant associated bacteria. Annu. Rev. Phytopathol, 41,429-453. Moyne, A.L. Shalby, R. Cleveland, T.E.and Tuzun, S. [2001]. Bacillomycin, D, an iturin with antifungal activity against Aspergillus flavus. J. Appl. Microbiol., 90, 622-629. Muratova, A.Y.U. Turkovskaya, O.V. Antonyuk, L.P. Makarov, O.E. Pozdnyakova, L.I. and Ignatov, V.V. [2005]. Oil-oxidizing potential of associative rhizobacteria of the genus Azospirillum. Microbiology [Moscow] [Engl Transl], 74,210–215. Maurhofer, M. Baehler, E. Notz, R. Martinez, V. and Keel, C. [2004] Cross talk between 2,4Diacetylphloroglucinol – producing biocontrol pseudomonads on wheat roots. Appl. Environ Microbiol, 70,1990-1998. Nakkeeran, S. Fernando, W.G.D. and Siddiqui, Z.A. [2005] Plant Growth Promoting Rhizobacteria Formulations and its Scope in Commercialization for the Management of Pests and Diseases. In Z.A. Siddiqui [Eds.], PGPR: Bicontrol and Biofertilization [pp. 257-296]. Nautiyal, C.S. Sangeeta, M. and Harikesh, B.S. [2006]. Biological Control and Plant-Growth Promotion by Bacillus Strains from Milk. J. Microbiol. Biotechnol., 16, 184–192.

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102

B. Hameeda, G. Harini, B. Keerthi Kiran et al.

Nautiyal, C.S. Bhadauria, S. Kumar, P. Lal, H. Mondal, R. and Verma, D. [2000]. Stress induced phosphate solubilization in bacteria isolated from alkaline soils. FEMS Microbiol Lett, 182,291–296. Nassar, A.H. El-Tarabily, K.A. and Sivasithamparam, K. [2005]. Promotion of plant growth by an auxin-producing isolates of the yeast Williopsis saturnus endophytic in maize [Zea mays L.] roots. Biol Fertil Soil, 42, 97-108. Nassar, A.H. El-Tarabily, K.A. and Sivasithamparam, K. [2003]. Growth promotion of bean [Phaseolus vulgaris L.] by a polyamine-producing isolate of Streptomyces griseoluteus. Plant Growth Regul, 40,97–106. Nannipieri, P. Grego, S. and Ceccanti, B. [1990]. Ecological significance of biological activity in soil biochemistry. J.M. Bollang, and G. Stotzky [Eds.], Marcel Dekker [pp. 293-355]Inc. New York. Neeta, S. and Swati, S. [2008]. Control of foliar diseases of mustard by Bacillus from reclaimed soil. Microbiol. Res. 163,408-413. Nielsen, T.H. Sorensen, D. Tobiasen, C. Andersen, J.B. Christophersen, C. Givskov, M., and Sorensen, J. [2002]. Antibiotic and biosurfactant properties of cyclic lipopeptides produced by fluorescent Pseudomonas sp. from the sugar beet rhizosphere. Appl. Environ. Microbiol, 68, 3416-3423. Normander, B. Hendriksen, N.B. and Nybroe, O. [1999]. Green Fluorescent Protein-Marked Pseudomonas fluorescens: Localization, Viability, and Activity in the Natural Barley Rhizosphere. Appl. and Environ. Microbiol., 65, 4646-4651. Nowak-Thompson, B. Chaney, N. Wing, J.S. Gould, S.J. and Loper, J.E. [1999]. Characterization of the pyoluteorin biosynthetic gene cluster of Pseudomonas fluorescens Pf-5. J. Bacteriol., 181,2166-2174. Nwaga, D. Henri, F. Germain, E.O. Laurette, N.K.and Jean Stephan, R. [2007]. Pseudomonads and symbiotic micro-organisms as biocontrol agents against fungal disease caused by Pythium aphanidermatum. Afr J. Biotech., 6,190-197. Okazaki, T. [2003]. Studies on actinomycetes isolated from plant leaves. In I. Kurtboke [Eds.], Selective Isolation of Rare Actinomycetes [pp. 102–121]. Okon, Y. and Itzigsohn, R. [1995]. The development of Azospirillum as a commercial inoculant for improving crop yields. Biotechnol Adv., 13,415-24. Oliveira, A.L.M. Stoffels, M. Schmid, M. Reis, V.M. Baldani, J.I. and Hartmann, A. [2008]. Colonization of sugarcane plantlets by mixed inoculations with diazotrophic bacteria . Eur J Soil Biol doi: 10. 1016 /j.ejsobi.2008.09.004. Oliver, R.P. and Ipcho, S.V.S. [2004]. Arabidopsis pathology breathes new life into the necrotrophs-vs. biotrophs classification of fungal pathogens. Mol. Plant Pathol, 5,347352. Palumbo, J.D. Yuen, G.Y. Jochum, C.C. Tatum, K. and Kobayashi, D.Y. [2005]. Mutagenesis of beta-1,3-glucanase genes in Lysobacter enzymogenes strain C3 results in reduced biological control activity toward Bipolaris leaf spot of tall fescue and Pythium dampingoff of sugar beet. Phytopathology, 95,701-707. Pandey, A. Trivedi, P. Kumar, B. and Palni, L.M.S. [2006]. Characterization of a phosphatesolubilizing and antagonistic strain of Pseudomonas putida [B0] isolated from a subalpine location in the Indian central Himalaya. Curr Microbiol, 53,102–107. Pattern, C.L. and Glick, B.R. [2002]. Role of Pseudomonas putida Indoleacetic acid in development of the host plant root system. Appl. Environ. Microbiol., 68,3795-3801.

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Role of Plant Growth Promoting Microorganisms for Sustainable Crop Production 103 Parmer, N. and Dadarwal, K.R. [1999]. Stimulation of nitrogen fixation and induction of flavonoid like compounds by rhizobacteria. J. Appl. Microbiol., 86, 36-44. Pieterse, C.M.J. Van Wees, S.C.M. Ton, J. Van Pelt, J.A. and van Loon, L.C. [2002]. Signaling in rhizobacteria-induced systemic resistance in Arabidopsis thaliana. Plant Biol, 4,535- 544. Podile, A.R. and Kishore, G.K. [2006]. Plant growth promoting rhizobacteria. In S.S. Gnanamanickam SS [Eds.], Plant associated bacteria[pp.195-230], Springer, Dordrecht. Ponmurugan, P.and Gopi, C. [2006]. In vitro production of growth regulators and phosphatase activity by phosphate solubilizing bacteria. Afr. J. Biotech, 5, 4-12. Pierson, L.S. and Pierson, E.A. [1996]. Phenazine antibiotic production in Pseudomonas aureofaciens: role in rhizosphere ecology and pathogen suppression. FEMS Microbiol Lett, 136,101–108. Prabavathy, V.R. Mathivanan, N. and Murugesan, K. [2006]. Control of blast and sheath blight diseases of rice using antifungal metabolites produced by Streptomyces sp. PM5. Biol Control,39,313-319. Preston, G.M. Houbold, B. and Rainey, P.B. [1998]. Bacterial geonomics and adaptation to life on plants: implications for the evolution of pathogencity and symbiosim. Curr opinion microbial,1,598-597. Puente, M.E. Bashan, Y. Li, C.Y. and Lebsky, V.K. [2004]. Microbial populations and activities in the rhizoplane of rock-weathering desert plants. I. Root colonization and weathering of igneous rocks. Plant Biol.,6,629-642. Raaijmakers, J.S. Vlami, M. and de Souza, J.T. [2002]. Antibiotic production by bacterial biocontrol agents. Antonie van Leeuwenhoek, 81,537-547. Raj, N.S. Shetty, N.P. and Shetty, H.S. [2004]. Seed bio-priming with Pseudomonas fluorescens isolates enhances growth of pearl millet plants and induces resistance against downy mildew. Int. J. Pest. Manage, 50,41-48. Ramazan, C. Dönmez, F. Aydın, A. and Şahin, F. [2006]. Growth promotion of plants by plant growth-promoting rhizobacteria under greenhouse and two different field soil conditions. Soil Biol. Biochem., 38,1482-1487. Ramey, B.E. Koutsoudis, M. Bodman, S.B and Fuqua, C. [2004]. Biofilm formation in plant– microbe associations. Curr. Opi. Microbiol.,7, 602-609. Ran, L.X. Li, Z.N. Wu, G.J. Van Loon, L.C. and Bakker, P.A.H.M. [2005]. Induction of systemic resistance against bacterial wilt in Eucalyptus urophylla by fluorescent Pseudomonas sp. Eur J Plant Pathol, 113,59-70. Ratledge, C. and Dover, L.G. [2000]. Iron metabolism in pathogenic bacteria. Annu. Rev. Microbiol, 54,881–941. Raupach, G.S. and Kloepper, J.W. [1998]. Mixtures of plant growth promoting rhizobacteria enhance biological control of multiple cucumber pathogens. Phytopathology, 88,11581164. Reddy, M.S. Kumar, S. Babita, K. and Reddy, M.S. [2002]. Biosolubilization of poorly soluble rock phosphates by Aspergillus tubingensis and Aspergillus niger. Biores Technol, 84,187–189. Reyes, Bernier, L. and Antoun, H.[2002]. Rock Phosphate solubilization and colonization of maize rhizosphere by wild and genetically modified strains of Penicillium rugulosum. Microb Ecol, 44,39-48.

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104

B. Hameeda, G. Harini, B. Keerthi Kiran et al.

Richardson, A. [2001]. Prospects for using soil microorganisms to improve the acquisition of phosphorus by plants. Aust. J. Plant Physiol., 28,897-907. Richardson, A.E. Hadobas, P.A. and Hayes, J.E. [2001]. Extracellular secretion of Aspergillus phytase from Arabidopsis roots enables plants to obtain phosphorus from phytate. Plant, 25,641-649. Roberts, P.D. Momol, M.T. Ritchie, L. Olson, S.M. Jones, J.B. and Balogh, B. [2008]Evaluation of spray programs containing famoxadone plus cymoxanil, acibenzolar-S-methyl, and Bacillus subtilis compared to copper sprays for management of bacterial spot on tomato. Crop. Protec, 27,1519-1526. Rodríguez, A.H. Heydrich-Pérez, M. Acebo-Guerrero, Y. Velazquez-del Valle, M.G. and Hernández-Lauzardo, A.N. [2008]. Antagonistic activity of Cuban native rhizobacteria against Fusarium verticillioides [Sacc.] Nirenb. in maize [Zea mays L.]. Appl. Soil. Ecol., 39,180-186. Rodriguez, A.H. Gonzalez, T. Goire, I. and Bashan, Y. [2004]. Gluconic acid production and phosphate solubilization by the plant growth promoting bacterium Azospirillum sp. Nature, 91,552–555. Romero, D. Vicente, A.D. Rakotoaly, R.H. Dufour, S.E. Veening, J.D. Arrebola, E. Cazorla, F.M. Kuipers, O.P. Paquot, M. and Garcial, A.P. [2007]. The Iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca. Mol. Plant Microbe Interact, 20, 430-440. Rosas, S.B. Andrés, J.A. Rovera, M. and Correa, N.S. [2006]. Phosphate-solubilizing Pseudomonas putida can influence the rhizobia–legume symbiosis. Soil Biol. Biochem., 38, 3502-3505. Ross, I.L. Alami, Y. Harvey, P.R. Achouak, W. and Ryder, M.H. [2000]. Genetic diversity and biological control activity of novel species of closely related pseudomonads isolated from wheat field soils in South Australia. Appl. Environ. Microbiol., 66,1609-1616. Rothballer, M. Schmid, M. and Hartmann, A. [2003]. In situ localization and PGPR-effect of Azospirillum brasilense strains colonizing roots of different wheat varieties. Symbiosis, 34, 261-279. Rudresh, D.L. Shivaprakash, M.K. and Prasad, R.D. [2005]. Tricalcium phosphate solubilizing abilities of Trichoderma sp. in relation to P uptake and growth and yield parameters of chickpea [Cicer arietinum L.] Can. J. Microbiol., 51,217–222. Rupela, O.P. Gowda, C.L.L. Wani, S.P. and Hameeda, B. [2006]. Evaluation of cropproduction systems based on locally available biological inputs. In N. Uphoff, S.A.Ball, E. Fernandes, H. Herren, O. Husson, M. Laing, C. Palm, J. Pretty, N. Sanchez, and Thies. J [Eds.], Biological approaches to sustainable soil systems[pp.501-515], Florida, USA CRC Press. Rupela, O.P. Wani, S.P. and Rego, T.J. [2005]. Managing and harnessing soil flora-fauna biodiversity in the tropics, for sustianbale crop production. In Water, Soil and Agrobiodiversity management for ecosystem health ICRISAT. Ryu, C.M. Farag, M.A. Hu, C.H. Reddy, M.S. Wei, H.X. Pare, P.W. and Kloepper, J.W. [2003]. Bacterial volatiles promote growth in Arabidopsis. Proc. Nati. Acad. Scie. USA, 100, 4927-4932. Ryu, C.M. Farag, M.A. Paul, W.P. and Kloepper, J.W. [2005]. Invisible Signals from the Underground: Bacterial Volatiles Elicit Plant Growth Promotion and Induce Systemic Resistance. Plant Pathol,21,7-12.

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Role of Plant Growth Promoting Microorganisms for Sustainable Crop Production 105 Sacherer, P. Défago, G. and Haas, D.[1994]. Extracellular protease and phosholipase C are controlled by the global regulatory gene gacA in the biocontrol strain Pseudomonas fluorescens CHA0. FEMS Microbiol Lett,116,155–160. Safronova, V.I. Stepanok, V.V. Engqvist, G.L. Alekseyev, Y.V. and Belimov, A.A. [2006]. Root-associated bacteria containing 1-aminocyclopropane-1-carboxylate deaminase improve growth and nutrient uptake by pea genotypes cultivated in cadmium supplemented soil. Biol Fertil Soil, 42,267–272. Sahin, F. Çakmakçi, F. and Kantar, F. [2004]. Sugar beet and barley yields in relation to inoculation with N2-fixing and phosphate solubilizing bacteria. Plant soil, 265,123-129. Samac, D.A. and Shah, D.M. [1994]. Effect of chitinase antisense RNA expression on disease susceptibility of Arabidopsis plants. Plant Mol Biol, 25, 587-596. Schisler, D.A. and Slininger, P.J. [1997]. Microbial selection strategies that enhance the likelihood of developing commercial biological control products. J. Ind. Microbiol. Biotechnol,19,172–179. Schmidt, C.S. Agostini, F. Leifert, C. Killham, K. and Mullins, C.E. [2004]. Influence of inoculum density of the antagonistic bacteria Pseudomonas fluorescens and Pseudomonas corrugata on sugar beet seedling colonization and suppression of Pythium damping off. Plant Soil, 265,111-122. Semenov, A.M. van Bruggen, A.H.C. and, Zelenev, V.V. [1999]. Moving Waves of Bacterial Populations and Total Organic Carbon along Roots of Wheat. Microbiol Ecol,37,116128. Selvamukilan, B. Rengalakshmi, S. Tamizoli, P. and Sudha, N. [2006]. Village-level production and use of biological agents and biofertilizers.In Uphoff et al. [Eds.], Biological approaches to sustainable soil systems[pp 647-645], Florida, USA CRC Press. Sessitsch, A. Reiter, B. and Berg, G.[2004]. Endophytic bacterial communities of field-grown potato plants and their plant-growth-promoting and antagonistic abilities. Can. J. Microbiol,50,239-249. Sevilla, M. Burris, R.H. Gunapala, N. and Kennedy, C. [2001]. Comparison of benefit to sugarcane plant growth and 15N2 incorporation following inoculation of sterile plants with Acetobacter diazotrophicus wild-type and nif- mutant strains. Mol Plant-Microbe Interact, 14,358-366. Shanahan, P. Daniel, J. O'Sullivan, Paul Simpson, Glennon, J.D. and Fergal, G. [1992]. Isolation of 2,4-Diacetylphloroglucinol from a Fluorescent Pseudomonad and Investigation of Physiological Parameters Influencing Its Production. Appl. Environ. Microbiol., 58, 353-358. Sharma, A. and Johri, B.N. [2003]. Growth promoting influence of siderophore-producing Pseudomonas strains GRP3A and PRS9 in maize [Zea mays L.] under iron limiting conditions. Microbiol. Res., 158, 243-248. Sharma, A. Sahgal, M. and Johri, B.N. 2003. Microbial communication in the rhizosphere: Operation of quorum sensing. Current Science, 85, 1164-1172. Sharma, S.K. Gupta. G.K. Ramesh, A. Joshi, O.P. and Sharma, M.P. [2008]. Mechanisms of Control of Soil-borne Plant Pathogens by Rhizobacteria: An Overview. In G. George, G. Khachatourians, D.K. Arora, T.P. Rajendran, A.K. Srivastava [Eds.], Agriculturally important microorganisms [vol 1, pp133-152], Academic world international.

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106

B. Hameeda, G. Harini, B. Keerthi Kiran et al.

Shaukat, S.S. and Siddiqui, I.A. [2003]. The influence of mineral and carbon sources on biological control of charcoal rot fungus, Macrophomina phaseolina by fluorescent Pseudomonads in tomato. Lett. Appl. Microbiol.,36,392-398. Sheng, X. Linyan, He. Wang, Q. Ye, and Jiang, C.[2008]. Effects of inoculation of biosurfactant-producing Bacillus sp. J119 on plant growth and cadmium uptake in a cadmium-amended soil. J. Haz Mat.,155,17-22. Sheng, X.F. and Huang, W.Y.[2002]. Mechanism of potassium release from feldspar affected by the strain NBT of silicate bacterium. Acta Pedol Sin,39,863–871. Sheng, X.F. He, L.Y. and Huang, W.Y. [2002]. The conditions of releasing potassium by a silicate-dissolving bacterial strain NBT. Agr Sci China,1,662–666. Silo-suh, L.A. Stab, V.E. Raffel, S.R. and Handelsman, J.[1998]. Target range of Zwittermicin A, an Aminopolyol antibiotic from Bacillus cereus. Curr Microbiol, 37,611. Singh, A.N. Verma, R. and Shanmugam, V.[2006]. Extracellular chitinase of fluorescent pseudomomads antifungal to Fusarium oxysporum f. sp. dianthi causing carnation wilt. Curr Microbiol,52,310-316. Singh, U.P. Sarma, B.K.and Singh, D.P.[2003]. Effect of plant growth-promoting rhizobacteria and culture filtrate of Sclerotium rolfsii on phenolic and salicylic acid contents in chickpea [Cicer arietinum]. Curr. Microbiol., 46,131-140. Singh, S. and Kapoor, K.K. [1999]. Inoculation with phosphate solubilizing microorganisms and a vesicular arbuscular mycorrhizal fungus improves dry matter yield and nutrient uptake by wheat grown in sandy soil. Biol. Fertil. Soils, 28,139-144. Smith, S.E.and Read, D.J.[1997]. Mycorrhizal Symbiosis. Academic Press San Diego USA. Somers, E. Vanderleyden, J. and Srinivasan, M.[2004]. Rhizosphere bacterial signaling: A love parade beneath our feet. Crit. Rev. Microbiol.,30,205-240. Someya, N. Kataoka, N. Komagata, T. Hirayae, K. Hibi, T.and Akutsu, K.[2000]. Biological control of cyclamen soilborne diseases by Serratia marcescens strain B2. Plant Dis,84,334-340. Soo Jeong, C. Sang Ryeol, P. Minkeun, K. Woojin, L. Sungkee, R. Changlong, A. Suyoung, H. Younghan, L. Seoncii, J. Yongun, C. and Han Dae, Y.[2002]. Endophytic Bacillus sp. isolated from the interior of balloon flower root. Biosci. Biotech. Biochem., 66,12701275. Souchie, E.L. Orivaldo, J. Saggin-Júnior, O.J. Silva, E.M.R. Campello, E.F.C. and Azcón, R. and Barea, J.M. [2006]. Communities of P-solubilizing bacteria, fungi and arbuscular mycorrhizal fungi in grass pasture and secondary forest of Paraty, RJ-Brazil. An. Acad. Bras. Cienc., 78, 183-193. Stanley, N.R. and Lazazzera, B.A. [2004]. Environmental signals and regulatory pathways that influence biofilm formation. Mol. Micrbiol., 52,917-924. Steenhoudt, O. and Vanderleyden, J.[2000]. Azospirillum, a free-living nitrogen- fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects. FEMS Microbiol. Rev., 24,487–506. Steinberg, P.D. and Rillig, M.C.[2003]. Differential decomposition of arbuscular mycorrhizal fungi hyphae and glomalin. Soil Biol. Biochem., 35,191-194. Stevenson, F.J.and Cole, M.A.[1999]. Cycles of soil: carbon, nitrogen, phosphorus, sulfur, micronutrients. 2nd edition. Wiley, New York, USA.

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Role of Plant Growth Promoting Microorganisms for Sustainable Crop Production 107 Sugumaran, P. and Janarthanam, B. [2007]. Solubilzation of Potassium containing minerals by bacteria and their effect on plant growth. Wor. J. Agr. Scie., 3,350-355. Suslow, T.V. and Schroth, M.N.[1982]. Rhizobacteria of sugar beets: effects of seed application and root colonization on yield. Phytopathology, 72,199-206. Suzuki, C. Kunito, T. Aono, T. Liu, C.T. and Oyaizu, H.[2005]. Microbial indices of soil fertility. J Applied Microbiol, 98,1062-1074. Swain, M.R. and Ray, R.C.[2007]. Biocontrol and other beneficial activities of Bacillus subtilis isolated from cowdung microflora. Microbiol Res,doi:10.1016/ j.micres.2006.10.009. Takeda, R.[1958]. Pseudomonas pigments. I. Pyoluteorin, a new chlorine-containing pigment produced by Pseudomonas aeruginosa. Hako Kogaku Zasshi, 36,281-290. Tambong, J.T. and Hofte, M.[2001]. Phenazines are involved in biocontrol of Pythium myriotylum on cocoyam by Pseudomonas aeruginosa PNA1. Eur. J. Plant Pathol., 107,511-521. Tan, T.and Cheng, P. [2003]. Biosorption of metal ions with Penicillium chrysogenum. Appl. Biochem. Biotechnol.,104,119-128. Tejera, N.A. Oetega, E. Gonzalez-Lopex, J. and Lluch, C.[2003]. Effect of some abiotic factors on the biological activity of Gluconacetobacter diazotrophicus. J. App. Microbiol., 95,528-535. Thakuria, D. Talukdar, N.C. Goswami, C. Hazarika, S. Boro, R.C.and Khan, M.R.[2004]. Characterization and screening of bacteria from rhizosphere of rice grown in acidic soils of Assam. Curr. Sci., 86,7-16. Thomashow, L.S. and David, M.W. [1990]. Role of antibiotics and siderophores in biocontrol of take-all disease of wheat. Plant Soil, 129,93-99. Tilak, K.V.B.R. Raganayaki, N. Pal, K.K. De, R. Saxena, A.K. Nautiyal, C.S. Shilpi Mittal, Tripathi, A.K. and Johri, B.N.[2005]. Diversity of plant growth and soil health supporting bacteria. Curre Scien, 89,136-150. Tran, H. Ficke, F. Asiimwe, T. Höfte, M. and Raaijmakers, J.M. [2007]. Role of the cyclic lipopeptide massetolide A in biological control of Phytophthora infestans and in colonization of tomato plants by Pseudomonas fluorescens. New Phytol, 175,731-742. Tripathi, M. Munot, H.P. Schouche, Y. Meyer, J.M. and Goel, R. [2005]. Isolation and functional characterization of siderophore-producing lead- and cadmium-resistant Pseudomonas putida KNP9. Current Microbiol.,50,233-237. Tsavkelova, E.A. Klimova, S.Yu. Cherdyntseva, T.A. and Netrusov, A.I.[2006]. Microbial Producers of Plant Growth Stimulators and Their Practical Use: A Review. Mikrobiologiya,42,133–143. Udaiyan, K. Karthikeyan, A. and Muthukumar, T. [1996]. Influence of edaphic and climatic factors on dynamics of root colonization and spore density of vesicular-arbuscular mycorrhizal fungi in Acacia farnesiana Wild. and A. planifrons. Trees, 11,65–71. Uknes, A. Mauch-Mani, B. Moyer, M. Potter, S. Williams, S. Dincher, S. Chandler, D. Slusarenko, A. Ward, E. and Ryals, J. [1992]. Acquired resistance in Arabidopsis. Plant Cell, 4,645-656. Uphoff, N. Ball, S.A. Fernandes, E. Herren, H. Husson, O. Laing, M. Palm, C. Pretty, J. Sanchez, N. and Thies, J. [2006] Biological approaches to sustainable soil systems. Florida, USA ,CRC Press.

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108

B. Hameeda, G. Harini, B. Keerthi Kiran et al.

Vaidya, R.J. Shah. I.M. Vyas, P.R. and Chhatpar, H.S. [2001]. Production of chitinase and its optimization from a novel isolate Alcaligenes xylosoxydans: potential in antifungal biocontrol. World J. Microbiol. Biotechnol.,17,691-696. van Loon, L.C.[2007]. Plant responses to plant growth-promoting rhizobacteria. Eur. J. Plant Pathl, 119,243-254. van Loon, L.C. and Glick, B.R. [2004]. Increased plant fitness by rhizobacteria. In H. Sandermann [Ed.], Ecological studies- Molecular Ecotoxicoloy of Plant [vol 170, pp 177205],Berlin, Springer-Verlag. van Loon, L.C. Bakker, P.A.H.M. and Pieterse, C.M.J. [1998]. Systemic resistance induced by rhizosphere bacteria. Annu. Rev. Phytopathol., 36, 453–483. Vassilev, N. and Vassileva, M. [2003]. Biotechnological solubilization of rock phosphate on media containing agroindustrial wastes. Appl. Microbiol. Biotechnol., 61,435–444. Velusamy, P. and Gnanamanickam, S.S.[2003]. Identification of 2,4-diacetylphloroglucinol production by plant-associated bacteria and its role in suppression of rice bacterial blight in India. Curr. Sci., 85,1270-1273. Verhagen, B.W.M. Van Loon, L.C. and Pieterse, C.M.J. [2006]. Induced disease resistance signaling in plants, In J.A. Teixeira de Silva [Ed.], Floriculture, Ornamental and Plant Biotechnology [vol III. pp 334-343]. Vessey, J.K. [2003]. Plant growth promoting rhizobacteria as biofertilizers. Plant Soil, 255,571–586. Vidhyasekaran, P. Sethuraman, K. Rajappan, K. and Vasumathi, K. [1997]. Powder formulation of Pseudomonas fluorescens to control pigeonpea wilt. Biol. Contr, 8,166171. Vidhyasekaran, P. and Muthamilan, M.[1995]. Development of formulations of Pseudomonas fluorescens for control of chickpea wilt. Plant Dis., 79,782–786. Viswanathan, R. and Samiyappan, R. [2002]. Induced systemic resistance by fluorescent pseudomonads against red rot disease of sugarcane caused by Colletotrichum falcatum. Crop Protect, 21, 1-10. Volpin, H. and Kapulnik, Y.[1994]. Interactions of Azospirillum with beneficial soil microorganisms. In Y. Okon [Ed.], Azospirillum Plant Associations [pp111-118], Florida,CRC Press, Boca Raton. Walsh, U.F. Morrissey, J.P. and O’Gara, F. [2001]. Pseudomonas for biocontrol of phytopathogens: from functional genomics to commercial exploitation. Curr. Opinion Biotechnol., 12, 289-295. Wang, C. Ohara, Y. Nakayashiki, H. Tosa, Y. and Mayama, S.[2005]. Microarray analysis of the gene expression profile induced by the endophytic plant growth-promoting rhizobacteria, Pseudomonas fluorescens FPT9601-T5 in Arabidopsis. Mol Plant-Microb Interact, 18,385-396. Wang, C. Knill, E. Glick, B.R. and Defago, G. [2000]. Effects of transferring 1aminocyclopropane-1-carboxylic acid [ACC] deaminase gene into Pseudomonas fluorescens strain CHA0 and its gac A derivatives CHA96 on their growth promoting and disease- suppressive capacities. Can. J. Microbiol., 46,898-907. Ward, D.M. Weller, R. Bateson, M.M.[1990]. 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature, 345, 63-65. Welbaum, G. Sturz, A.V. Dong, Z. and Nowak, J.[2004]. Fertilizing soil microorganisms to improve productivity of agroecosystems. Crit. Rev. Plant Sci., 23,175–193.

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Role of Plant Growth Promoting Microorganisms for Sustainable Crop Production 109 Whipps, J.M. [2001]. Microbial interactions and biocontrol in the rhizosphere. J. Exper. Bot,52, 487-511. Wu, S.C. Cheung, K.C. Luo, Y.M. and Wong, M.H. [2006]. Effects of inoculation of plant growth-promoting rhizobacteria on metal uptake by Brassica juncea. Environ. Pollut, 140,124– 135. Xiao, K. Samac, D.A. and Kinkel, L.L.[2002]. Biological control of Phytophthora root rots on alfalfa and soybean with Streptomyces. Biol Control,23,285–295. Xiao-Yan, S. Qing-Tao, S. Shu-Tao, X. Xiu-Lan, C. Cai-Yun, S.and Yu-Zhong, Z. [2006]. Broad-spectrum antimicrobial activity and high stability of Trichokonins from Trichoderma koningii SMF2 against plant pathogens. FEMS Microbiol. Lett., 260,119125. Yadav, E. Pathak, D.V. Sharma, S.K. and Sharma, P.K.[2007]. Isolation and characterization of mutants of Pseudomonas maltophila PM-4 altered in chitinolytic activity and antagonistic activity against root rot pathogens of clusterbean [Cyamopsis tetragonoloba ]. Ind J Microbiol,47,64-71. Yang, C.H. Crowley, D.E. and Menge, J.A. [2001]. 16S rDNA fingerprinting of rhizosphere bacterial communities associated with healthy and Phytophthora infected avocado roots. FEMS Microbiol Ecol, 35,129–136. Yang, C.H.and Crowley, D.E. [2000]. Rhizosphere microbial community structure in relation to root location and plant iron nutrition status. Appl. Environ. Microbiol., 66,345-351. Yao, A.V. Bochow, H. Karimov, S. Boturov, U. Sanginboy, S. and Sharipov, K. [2006]. Effect of FZB24 Bacillus subtilis as a biofertilizers on cotton yields in field tests. Arch Phytopathol Plant Prot, 39,1-6. Yoshida, S. Shirata, A.and Hiradate, S. [2002]. Ecological characteristics and biological control of mulberry anthracnose. Jap. Agri. Res. Quer, 36, 89-95. Yoshida, S. Hiradate, S. Tsukamoto, T. Hatakeda, K. and Shilata, A. [2001]. Antimicrobial activity of culture filtrate of B. amyloquefaciens RC2 isolated from mulberry leaves. Phytopathol,91,181-187. Zaidi, A. and Khan, M.S.[2006]. Co-inoculation effects of phosphate solubilizing microorganisms and Glomus fasciculatum on green gram-Bradyrhiz obium symbiosis. Turk .J. Agric., 30,223-230. Zahir, Z.A. Arshad, M. and Frankenberger, W.T. Jr. [2004]. Plant growth promoting rhizobacteria: applications and perspectives in agriculture. Adv. Agron., 81,97-168. Zaidi, S. Usmani, S. Singh, B.R.and Musarrat, J. [2006]. Significance of Bacillus subtilis strain SJ-101 as a bioinoculant for concurrent plant growth promotion and nickel accumulation in Brassica juncea . Chemosphere, 64,991-997. Zarandi, M. Ebrahimi, Bonjar, Shahidi, G.H. Dehkaei, F. and Padasht. [2009].Biological control of rice blast [Magnaporthe oryzae] by use of Streptomyces sindeneusis isolate 263 in greenhouse. Eur J Soil Biol doi :10.1016/j.ejsobi.2008.09.004. Zehnder, G.W. Yao, C. Murphy, J.F. Sikora, E.R. and Kloepper, J.W. [2000]. Induction of resistance in tomato against cucumber mosaic cucumovirus by plant growth-promoting rhizobacteria. BioControl 45,127-137. Zhang, F. Dashti, N. Hynes, R.K. and Smith, D.L.[1996]. Plant growth promoting rhizobacteria and soybean [Glycine max [L.] Merr.] nodulation and nitrogen fixation at suboptimal root zone temperatures. Annals of Bot,77,453-459.

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Zhang, Y. and Fernando, W.G.D. [2004]. Presence of biosynthetic genes for phenazine-1carboxylic acid and 2,4-diacetylphloroglucinol and pyrrolnitrin in Pseudomonas chlororaphis strain PA-23. Can. J. Plant Pathol., 26,430 Zhao, J. Davis, L.C. and Verpoorte, R. [2005]. Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol. Adv., 23,283–333. Zinniel, D.K. Lambrecht, P. Harris, N.B. Feng, Z. Kuczmarski, D. and Higley, P.[2002]. Isolation and characterization of endophytic colonizing bacteria from agronomic crops and prairie plants. Appl. Environ. Microbiol., 68,2198-2208.

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Chapter 5

GENETIC AND FUNCTIONAL DIVERSITY OF PHOSPHATE SOLUBILIZING FLUORESCENT PSEUDOMONADS AND THEIR SIMULTANEOUS ROLE IN PROMOTION OF PLANT GROWTH AND SOIL HEALTH M. Jaharamma, K. Badri Narayanan and N. Sakthivel∗ Department of Biotechnology, Pondicherry University, Kalapet, Puducherry 605014, India

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ABSTRACT Soil microbes that solubilize the insoluble phosphates play a vital role in maintaining soil fertility, plant health and subsequent enhancement of crop yield. Fluorescent pseudomonad group of bacteria are often predominant among bacterial species associated with the plant rhizosphere. Due to their innate capability for plant growth promotion, plant disease suppression and their potential for biodegradation of agricultural chemical pollutants, fluorescent pseudomonads have been a major focus for investigators around the world. In recent years, rich knowledge has been generated on diversity and functional potential of fluorescent pseudomonads. This chapter describes the genetic and functional diversity of fluorescent pseudomonads and their role in phosphate solubilization, biological control and soil fertility.

5.1. INTRODUCTION Phosphorous is the major essential macronutrient of plants and its deficiency is a severe constraint to crop production. Plants absorb only inorganic form of phosphorous and the level of inorganic phosphorous is very low in the soil because most of the phosphorous is present ∗

Corresponding authoe: [email protected]

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in insoluble mineral forms such as hydroxyapatites, oxyapatites and apatites. In ferralite soils mineral phosphates are associated with the poorly soluble and unassimilated forms of hydrated oxides of iron, aluminum and manganese. In acid soils, phosphorous is fixed by free oxides and hydroxides of Al and Fe but in alkaline soils, phosphorous is fixed by Ca and therefore, resulting poor solubility of phosphorous fertilizers. The low level of available phosphorous in soil is reported to be less than 1 ppm [Goldstein, 1994]. Phytopathogens reduce crop yield all over the world. Chemical control of phytopathogens is one of the major approaches for disease control. The overuse of chemical pesticides and chemical fertilizers in agricultural soils has led to the lethal consequences to useful arthropods and other beneficial microbes as well as led to soil pollution and accumulation of phosphorous as insoluble form [Rodriguez and Fraga, 1999]. Regular application of chemical pesticides and fertilizers escalated soil problems such as structural degradation, reduction of organic matter, soil colloidal content and accumulation of agricultural residues and phosphorous in soil. Crop residues consisting of cellulose and hemicellulose contain 53-75% carbohydrate. Cellulose is a polymer of glucose and hemicellulose consists of xylose, arabinose, glucose, galactose and mannose [Mosier et al. 2005]. Phosphate solubilizing bacteria are known to utilize carbohydrates of crop residues. Therefore, phosphate solubilizing bacteria with multiple functional traits such as plant growth promotion and crop protection are preferred to enhance mineralization and decomposition of crop residues. Fluorescent pseudomonad group of bacteria with such innate ability to promote growth, control diseases and degrade pollutants may play a vital role in promoting agricultural yields.

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5.2. MICROBIAL PHOSPHATE SOLUBILIZATION Soil microorganisms have the ability to solubilize the insoluble phosphates and to improve the quality of soil health and its fertility. Phosphate solubilizing microbes have been reported for promoting plant growth and enhancing yield [Kapoor et al. 1989; Rodriguez and Fraga, 1999; Wani et al. 2007]. Evaluation of their potential to mobilize soil phosphate has been the subject of interest for soil microbiologist [Rodriguez and Fraga, 1999; Whitelaw, 2000]. Microorganisms play an essential role to facilitate the availability of soil phosphate to the root system and enhance the mobilization of phosphate in soil [Richardson, 2001]. Efficacy of phosphate solubilizing microorganisms has been identified on the basis of kinetics and phosphorous accumulation. Fungal species such as Aspergillus, Penicillum and Rhizopus have been reported for phosphate solubilization [Rodriguez and Fraga, 1999; Whitelaw, 2000; Vassilev and Vassileva, 2003]. The phosphate uptake by plants and subsequent growth promotion in plantsoil systems are more pronounced when phosphate solubilizing microorganisms are coinoculated with arbuscular mycorrhizal fungi that offer beneficial symbiosis with plant roots [Smith and Read, 1997]. Phosphate solubilizing fungi have been reported to solubilize 9.0 to 34.6% of total phosphate in synthetic medium [Narsian et al. 1994]. These fungi are capable of mobilizing soluble inorganic phosphate by the excretion of H+ after the utilization of ammonium by the hyphae [Yao et al. 2001]. Several bacterial genera such as, Pseudomonas, Azospirillum, Bacillus, Rhizobium, Burkholderia, Alcaligenes, Serratia, Enterobacter, Acinetobacter, Flavobacterium and Erwinia have been reported to solubilize tricalcium

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phosphate, dicalcium phosphate, hydroxyapatite and rock phosphates [Rodriguez et al. 1996; Goldstein, 1986]. Among these phosphate solubilizing bacteria, plant rhizosphere associated fluorescent pseudomonads are considered important due to their simultaneous potential of plant growth promotion, biocontrol of pathogens and biodegradation of soil pollutants [Bano and Musarrat, 2004; O’Sullivan and O’Gara, 1992; Ravindra Naik and Sakthivel, 2006].

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5.3. MICROBIAL MECHANISMS MEDIATING PHOSPHATE SOLUBILIZATION Phosphate mineralization from non-specific substrates by several bacterial genera such as Pseudomonas, Burkholderia, Enterobacter, Citrobacter, Proteus and Serratia and phosphate mineralization from inositol phosphate by Bacillus and Pseudomonas as well as mineralization of phosphate from phosphonoacetate by Pseudomonas has been reported. The production of organic acids by these bacteria seems to be the main cause of phosphate solubilization [Khan et al. 2007]. These bacteria are found to produce monocarboxylic [acetic, formic], dicarboxylic [oxalic, succinic], tricarboxylic hydroxyl [citric] acids in liquid media. The role of organic acids in dissolving mineral phosphates and phosphorylated minerals can be attributed to the lowering of pH, which helps in the formation of stable complexes with cations such as Ca2+, Mg2+, Fe3+ and Al3+. Phosphatase and phytase enzymes secreted by these bacteria play an essential role in phosphate solubilization because of the predominant presence of their substrates in soil. Phosphorous can be released from organic compounds in soil by three groups of enzymes such as nonspecific phosphatases [To-O et al. 2000] that perform dephosphorylation of phospho-ester or phospho-anhydride bonds in organic matter, phytases that specifically cause the release of phosphorous from phytic acid and phosphonoacetases and carbon-phosphate [C-P] lyase enzymes that perform C–P bond cleavage in organophosphonates [Rodriguez et al. 2006]. Secretions of organic acids and phosphatase enzymes have been reported as common mechanisms to facilitate the conversion of insoluble forms of phosphorous to accessible forms like orthophosphate. Although several phosphate solubilizing bacteria occur in soil, usually their numbers are not sufficient enough to compete with other rhizobacteria. Therefore, inoculation of plants by a target microorganism at a much higher concentration is necessary. It has been reported that phosphate mineralization of fluorescent pseudomonads from the substrates has been mediated by the enzymes, acid phosphatase and phosphonoacetatehydrolase of Pseudomonas fluorescens strains and phytase enzyme of P. putida. At least 30-48% of culturable soil microbes utilize phytase [Greaves and Webley, 1965]. This is evidently suggested that phytase producing P. putida and phosphatase enzyme producing fluorescent pseudomonad strains may play an efficient role in phosphate solubilization. In agricultural fields, the pH of most soils are acidic to neutral range and therefore, acid phosphatase producing fluorescent pseudomonads are considered as important microbial inoculants for enhancing soil fertility.

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5.4. MICROBIAL DIVERSITY OF PHOSPHATE SOLUBILIZING FLUORESCENT PSEUDOMONADS

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Plant growth promoting bacteria such as fluorescent pseudomonads and rhizobia are able to solubilize both organic phosphates [Abd-Alla, 1994] and inorganic phosphates [Antoun et al. 1998]. The advantage of using this group of bacteria with other innate biocontrol and biofertilizing traits will be their dual beneficial nutritional effect resulting both from phosphate mobilization, nitrogen fixation and suppression of phytopathogens. These bacteria are documented well for their synergistic interactions with arbuscular mycorrhizal fungi [Peix et al. 2001]. Several species of fluorescent pseudomonads such as P. fluorescens NJ-101 [Bano and Musarrat, 2004], P. fluorescens EM85 [Dey et al. 2004], P. aeruginosa [Bano and Musarrat, 2004], Pseudomonas sp. [Lehinos and Vacek, 1994; Lehinos, 1994], P. chlororaphis, P. savastanoi, P. pickettii [Cattelan et al. 1999], P. lutea OK2 [Peix et al. 2004], P. rhizophaerae LMG21640, P. graminis DSM11363 [Peix et al. 2003], P. striata [Gaind and Gaur, 2002] and P. corrugata [Pandey and Palni, 1998] have been reported as efficient phosphate solubilizers. It has been reported that 18% of fluorescent pseudomonad strains were positive for the solubilization of tri-calcium phosphate [Ca3[PO4]2 and formed a visible dissolution halos on Pikovskaya agar plates [Ravindra Naik et al. 2008]. A high degree of genetic diversity among phosphate solubilizing fluorescent pseudomonad strains have been reported on the basis of phenotypic characterization and 16S rRNA gene phylogenetic analyses. All these phosphate solubilizing fluorescent pseudomonads strains have been identified as P. aeruginosa, P. mosselii, P. monteilii, P. plecoglossicida, P. putida, P. fulva and P. fluorescens [Ravindra Naik et al. 2008].

5.4.1. Functional Diversity of Phosphate- Solubilizing Fluorescent Pseudomonads 5.4.1.1. Plant Growth Promoting Traits 5.4.1.1.1. Siderophore Siderophores by fluorescent pseudomonads are determined using the FeCl3 test [Neilands, 1995], and the chrome azurol S agar [CAS] assays [Schyn and Neilands, 1987]. Bacterial suspensions are dropped onto the center of a CAS plate and after incubation at 25°C for three days; siderophore production is assessed on the basis of change in colour of the medium from blue to orange. Fluorescent pseudomonads are known to produce hydroxamate type of siderophores [Neilands, 1995; Bano and Musarrat, 2004; Ravindra Naik et al. 2008]. 5.4.1.1.2. Protease Proteases by fluorescent pseudomonads are determined using skim milk agar medium. Bacterial cells are spot inoculated and after two days incubation at 28°C and proteolytic activities are identified by formation of clear zones around the cells [Smibert and krieg, 1994]. Several species of fluorescent pseudomonads have been reported to produce protease [Blumer et al. 1999; Sacherer et al. 1994].

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5.4.1.1.3. Indole-3-Acetic Acid The production of phytohormone, indole-3-acetic acid [IAA] is determined by using standard method [Bric et al. 1991]. Single colony is streaked onto Luria-Bertani [LB] agar amended with 5 mM L-tryptophan, 0.06% sodium dodecyl sulphate and 1% glycerol. Bioassay plates are overlaid with Whatman no. 1 filter paper and the bacterial strain is allowed to grow for a period of three days. After the incubation period, the paper is removed and treated with Salkowski’s reagent [Gordon and Weber, 1951] with the formulation, 2% of 0.5 M ferric chloride in 35% perchloric acid. Membranes are saturated in a Petri dish by soaking directly in Salkowski’s reagent and the production of IAA is identified by the formation of a characteristic red halo within the membrane immediately surrounding the colony. Quantification of IAA is done by following colorimetric method [Patten and Glick, 2002]. A single colony of the bacterium is propagated overnight in 5 ml of DF minimal salt medium and an aliquot of bacterial suspension is transferred into 5 ml of DF minimal salt medium amended with 500 μg/ml of L-tryptophan. After 40 h of growth at 25oC in a rotary shaker at 180 rpm, the cells are removed by centrifugation. To the supernatant, Salkowski’s reagent is added, mixed well and allowed to stand at room temperature for 20 min. The absorbance is measured in a spectrophotometer at 535 nm. An un-inoculated control with Salkowski’s reagent is used as reference. The concentration of IAA is determined by comparing with the standard curve. Selective strains of phosphate solubilizing fluorescent pseudomonads have also been reported for the production of IAA [Patten and Glick, 2002; Barazani and Friedman, 1999; Bric et al. 1991]. 5.4.1.1.4. 1-Aminocyclopropane-1-carboxylate [ACC] Deaminase The ACC deaminase activity of fluorescent pseudomonads is estimated by using DF salts medium [Dworkin and Foster, 1958]. The solution of ACC [3 mM] is filter sterilized through a 0.2 µm membrane [Millipore] and spread over the agar plates, allowed to dry for 10 min. and inoculated with bacteria. Observation is made after two days incubation at 28°C. Growth on the medium is considered as positive for ACC deaminase production [Penrose and Glick, 2002]. Strains of fluorescent pseudomonads have been reported for ACC deaminase activity. 5.4.1.1.5. N-acyl homoserine Lactone [AHL] Production of AHL by fluorescent pseudomonads is screened as described by Molina et al. [2003]. Briefly, AHL biosensor, Chromobacterium violaceum CV026 is streaked in a line on plates of LB agar. The AHL donor Erwinia carotovara is used as a control and applied in the spots 16 mm from the C. violaceum CV026 line. A test strain is spotted in between the biosensor and the AHL donor at a distance 6 mm from the C. violaceum CV026 line. Plates are incubated at 28°C for two days. Migration of AHL from the donor E. carotovara and test bacterium is confirmed by the production of purple-pigmented antibiotic violacein in the biosensor. 5.4.1.2. Biocontrol Traits 5.4.1.2.1. Antagonism Phosphate-solubilizing strains of fluorescent pseudomonads are tested for in vitro antagonism towards plant pathogens by following standard co-inoculation technique on

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potato dextrose agar or nutrient agar [Sakthivel and Gnanamanickam, 1987; Ayyadurai et al. 2007]. Briefly, bacterial plugs are removed from a 48 h culture and are transferred to the centre of potato dextrose agar or nutrient agar plates, which had been inoculated with fungal spore or bacterial cell suspension. Assay plates are incubated at 28°C to 42°C for two to three days and growth-inhibition appeared around the plugs are measured and the strains are identified as antagonists.

5.4.1.2.2. Fungal Cell Wall Degrading Enzyme The production of fungal cell wall degrading enzyme, chitinase by fluorescent pseudomonads can be tested on chitin agar medium [Renwick et al. 1991]. Briefly, overnight grown bacterial cells are spot inoculated onto chitin agar plates and after five days of incubation at 30°C, chitinase activity are identified by formation of clear zones around the cells. In a recent study from our laboratory, a total of 9% strains of phosphate solubilizing fluorescent pseudomonads showed chitinase activity [Ravindra Naik et al. 2008]. 5.4.1.2.3. Production of Enzymes for Decomposition of Crop Residues

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5.4.1.2.3.1. Cellulase Strains are screened for cellulase production by plating onto M9 medium agar amended with 10 g of cellulose and 1.2 g of yeast extract per litre. After eight days of incubation at 28oC, colonies surrounded by clear halos are considered positive for cellulase production [Cattelan, 1999]. Cellulase production of selective strains of phosphate solubilizing fluorescent pseudomonads has been reported [Ravindra Naik et al. 2008]. 5.4.1.2.3.2. Pectinase Pectinase production is determined using M9 medium amended with 4.8 g of pectin per litre. After two days of incubation at 28oC, plates are flooded with 2 mol/l HCl and strains surrounded by clear halos are considered positive for pectinase production [Cattelan, 1999]. Pectinase production of selective strains of phosphate solubilizing fluorescent pseudomonads has been reported [Ravindra Naik et al. 2008]. 5.4.1.2.4. Rapid Detection of Antibiotic Genes and Antimicrobial Metabolites Detection of the genes that encode the known antimicrobial metabolites of fluorescent pseudomonads such as 2,4-diacetylphloroglucinol [DAPG], phenazine-1-carboxylic acid [PCA], phenazine-1-carboxamide [PCN], pyrrolnitrin [PRN], pyoluteorin [PLT] and hydrogen cyanide [hcnBC] is done by PCR using gene-specific primers pairs such as, Phl2a [5’-GAGGACGTCGAAGACCACCA-3’] and Phl2b [5’-ACCGCAGCATCGTGT ATGAG3’] for DAPG [Mavrodi et al. 2001b], PCA2a [5’-TGCCAAGCCTCGCTCCA AC-3’] and PCA3b [5’-CCCGTTTCAGTAAGTCTTCCATGATGCG-3’] for PCA [Raaijmakers et al. 1997], PhzHup [5’-CGCACGGATCCTTTCAGAATGTTC-3’] and PhzHlow [5’GCCACGCCAAGCTTCACGCTCA-3’] for PCN [Mavrodi et al. 2001a], Prncf [5’CCACAAGCCCGGCCAGGAGC-3’] and Prncr [5’-GAGAAGAGCGGGTCG ATGAAGCC-3’] for PRN, PltBf [5’-CGGAGCATGGACCCCCAGC-3’] and PltBr [5’GTGCCCGATATTGGTCTTGACCGAG-3’] for PLT [Mavrodi et al. 2001b], and ACa [5’ACTGCCAGGGGCGGATGTGC-3’] and ACb [5’-ACGATGTGCTCGGCGTAC-3’] for

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hcnBC [Ramette et al. 2003]. In order to test the production of antifungal metabolites, strains are inoculated in the fermentation media. Production of DAPG and PCA is tested by growing bacteria in pigment producing medium [g/l: peptone 20, 20 ml glycerol, NaCl 5 and KNO3 1, pH 7.2] for five days at 28°C [Gurusiddaiah et al. 1986]. Production of PLT is tested by growing bacteria in KB broth for 14 days at 25°C [de Souza and Raaijimakers, 2003]. Production of PRN is tested by growing bacteria in a minimal medium [g/l: glycerol 30, K2HPO4 3, KH2PO4 0.5, NaCl 5, MgSO4.7H2O 0.5, 0.35 mM ZnSO4, 0.5 mM MO7 [NH4]6O24.H2O and D-tryptophan 0.61] for 24 h at 25°C and subsequently incubated at 25°C in dark for four days [de Souza and Raaijimakers, 2003]. The culture supernatants are extracted with ethyl acetate and the production of antibiotics is verified by analytical HPLC [Sunish kumar et al. 2005; Ayyadurai et al. 2007].

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5.4.2. Phenotypic and Genotypic Diversity of Phosphate SolubilizingFluorescent Pseudomonads Phosphate solubilizing fluorescent pseudomonad strains exhibited positive traits for cytochrome oxidase, arginine dihydrolase, and showed variability for traits such as gelatin hydrolysis, levan production and growth at 4°C and 42°C. All these strains have been reported to utilize dextrose, galactose, mannose and citrate but exhibited varying degree of utilization towards other carbon sources such as lactose, xylose, fructose, melibiose, Larabinose, glycerol, ribose, α-methyl-D-mannoside, xylitol, esculin, D-arabinose, malonate, sorbose, trehalose, sorbitol, mannitol, adonitol and glucosamine [Ravindra Naik et al. 2008]. These strains did not utilize maltose, sucrose, inulin, salicin, dulcitol, inositol, α-methyl-Dgluconate, rhamnose, cellobiose, melazitose, xylitol and ONPG. Numerical analysis of phenotypic characteristics revealed a high degree of polymorphism into three major phenons [Ravindra Naik et al. 2008]. Cluster analysis of phosphate solubilizing fluorescent pseudomonads based on the pair-wise coefficient similarity with UPGMA of BOX-PCR has resulted into 3 distinct genomic clusters and 26 distinct BOX profiles [Ravindra Naik et al. 2008]. All phosphate solubilizing fluorescent pseudomonads strains showed wide variations in fingerprinting pattern due to their high degree of genetic variability and distributed into different clusters. These results identified a high degree of genetic variability among different species of phosphate solubilizing fluorescent pseudomonads.

5.5. GENES INVOLVED IN PHOSPHATE SOLUBILIZATION The ability of mineral phosphate solubilization [mps] has been shown to be related to the production of organic acid [Rodrıguez and Fraga, 1999]. Direct glucose oxidation to gluconic acid [GA] has been reported as a major mechanism for mps [Goldstein, 1994]. In fluorescent pseudomonads, gluconic acid biosynthesis is mediated by glucose dehydrogenase [GDH] and the co-factor, pyrroloquinoline quinone [PQQ]. Although some genes involved in mps in different bacterial species have been reported, the genetic basis of mps is not well understood [Goldstein and Liu, 1987]. Any gene involved in organic acid synthesis might

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have an effect on mps. The gene mps from Erwinia herbicola that is involved in mineral phosphate solubilization has been cloned [Goldstein and Liu, 1987]. The expression of mps gene mediated the production of gluconic acid and mineral phosphate solubilization activity in E. coli HB101. Sequence analysis of mps gene [Liu et al. 1992] suggested its role in the biosynthesis of pyrroloquinoline quinone [PQQ] synthase, which directs the synthesis of PQQ, a co-factor necessary for the formation of the holoenzyme glucose dehydrogenase [GDH]-PQQ. This enzyme catalyzes the formation of gluconic acid from glucose by the direct oxidation pathway. Mineral phosphate solubilization gene gabY from P. cepacia was isolated [Babu-Khan et al. 1995]. Expression of gabY gene has led to the mineral phosphate solubilization via gluconic acid production in Escherichia coli JM109. The gabY gene could play an alternative role in the expression and regulation of the direct oxidation pathway in P. cepacia. This gabY gene showed no apparent homology with the previous cloned PQQ synthetase gene [Liu et al. 1992; Goosen et al. 1989]. Many acid phosphatase genes from Gram-negative bacteria have been identified [Rossolini et al. 1998]. These genes are the important source of genetic materials for the gene transfer to other plant growth promoting strains. In general, genes coding for acid phosphatases are capable of performing well in soil. The acpA gene from Francisella tularensis expresses an acid phosphatase with optimum action at pH 6, with a wide range of substrate specificity [Reilly et al. 1996] and genes encoding non-specific acid phosphatases class A [PhoC] and class B [NapA] from Morganella morganii are also promising genetic materials [Thaller et al. 1994 and 1995b].

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5.6. MOLECULAR TOOLS USED FOR ISOLATION AND CHARACTERIZATION OF PHOSPHATE SOLUBILIZING FLUORESCENT PSEUDOMONADS 5.6.1. Isolation and Screening of Phosphate-Solubilizing Strains Phosphate-solubilizing bacteria are isolated from rhizospheric samples by plating serial dilution of rhizospheric soil extracts in Pikovskaya agar medium [Pikovskaya, 1948]. The medium contains insoluble tri- or bi-calcium phosphate, allowing the detection of phosphate solubilizing bacteria by the formation of “halo” around their colonies. The addition of bromophenol blue, which produces yellow colored halos around the colonies in response to the pH drop by the release of organic acids, or protein release in exchange for cation uptake, generates more accurate results than those observed with the simple halo method [Gupta et al. 1994]. Although phosphate solubilizing capability remains stable in most strains, few other strains also show instability after several cycle of inoculation [Halder et al. 1990; Illmer and Schinner, 1992] possibly due to microbial attenuation process.

5.6.2. Estimation of Phosphate Solubilization Strains are inoculated into phosphate solubilization estimation medium [g/l: yeast extract 0.5, dextrose 10, CaCl2 5, [NH4]2SO4 0.5, Ca3[PO4]2 5, KCl 0.2, MgSO4 0.1, MnSO4 0.0001

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and FeSO4 0.0001, pH 7] and grown at 28°C with 180 rpm on rotary shaker [King, 1936]. At different time intervals, samples are drawn and used for the estimation of soluble phosphate and for checking the pH of the culture medium. For estimation of soluble phosphate in culture medium, one millilitre culture filtrate is added to 4.5 ml of chloromolybdic acid [1.5 g of ammonium molybdate dissolved in 40 ml of warm water, 34.2 ml of 12 N HCl is added, allowed to cool and made up to 100 ml with distilled water] in each test tube and vortexed. To this, 0.025 ml of chlorostannous acid [2.5 g of SnCl2.H2O dissolved in 10 ml of 12 N HCl, made up to 100 ml with distilled water] is added and immediately made up to 5 ml and optical density [OD] is measured at 600 nm. Standard curve is prepared using different concentrations of potassium dihydrogen phosphate.

5.6.3. Evaluation of Strains for Efficient Phosphate Solubilization The effectiveness of phosphate solubilizing fluorescent pseudomonads may be evaluated by testing their response towards crop plants of various species and genotypes. Differential rhizosphere effects of these bacteria may be due to the variability in their root exudates which in turn play an important role in microbial phosphate solubilization. Efficacy of phosphate solubilization by microbes should also be tested under field conditions of varying phosphate content, soil pH and other soil characteristics.

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5.6.4. Phenotypic Characterization Phenotypic characterization of phosphate solubilizing bacteria relies upon biochemical tests and carbon assimilation profiles. For rapid identification of soil bacteria including Pseudomonas, Bacilli and Acinetobacter commercial kits such as BIOLOG system [BIOLOG, California, USA], API 20NE system [BioMerieux, France] and HiCarbohydrate kits [Himedia, Mumbai, India] are available.

5.6.5. Molecular Characterization 5.6.5.1. 16S rRNA, gyrB and rpoD amplifications Amplification of 16S rRNA gene is performed from the genomic DNA using universal primers fD1 [5΄-GAGTTTGATCCTGGCTCA-3΄] and rP2 [5΄-ACGGCTACCTTGTTA CGACTT-3΄] [Weisburg et al. 1991]. The gyrB and rpoD genes are amplified using the primer pairs GYRBF [5’-CAGGAAACAGCTATGACCAYGSNGGNGGNAARTTYR A-3’] and GYRBR [5’-TGTAAAACGACGGCCAGTGCNGGRTCYTTYTCYTGRCA-3’] for gyrB and RPODF[5’-ACGACTGACCCGGTACGCATGTAYATGMGNGARA TGGGNACNGT-3’] and RPODR [5’-ATAGAAATAACCAGACGTAAGTTNGCYTC NACCATYTCYTTYTT-3’] for rpoD [Yamamoto et al. 2000].

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5.6.5.2. Sequencing and Phylogenetic Tree Analyses Sequences of 16S rRNA, gyrB and rpoD genes are subjected to BLAST search from the NCBI database for bacterial strain identification. The reference sequences required for comparison are downloaded from the European Molecular Biology Laboratory [EMBL] database available on the site http://www.ncbi.nlm.nih.gov/GenBank. Sequences are aligned by the aid of multiple sequence alignment program CLUSTAL V [Higgins et al. 1992]. The aligned sequences are then checked for gaps manually, arranged in a block of 600bp in each row and saved as molecular evolutionary genetics analysis [MEGA] format in software MEGA v3.0. The pairwise evolutionary distances are computed with the help of Kimura 2parameter [Kimura, 1980]. To obtain the confidence values, the original data set is resampled 1000 times using the bootstrap analysis method. The bootstrapped data set is used directly for constructing the phylogenetic tree by the MEGA v3.0 program for calculating the multiple distance matrixes. The multiple distance matrix obtained is then used to construct phylogenetic trees using neighbor-joining [NJ] method [Saitou and Nei, 1987]. All these analyses are performed with the aid of MEGA v3.0 [Kumar et al. 2004].

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5.6.5.3. Amplified Ribosomal DNA Restriction Analysis [ARDRA] ARDRA is done by digestion of 16S RNA gene with discriminative restriction endonucleases such as AluI, DdeI, HinfI and MspI [Laguerre et al. 1994]. The digested PCR products are separated by horizontal electrophoresis and gels are stained with ethidium bromide and photographed under UV illumination. Depending on their ARDRA profiles, the strains are distributed into genotypic groups [Laguerre et al. 1994; Frey et al. 1997]. 5.6.5.4. Electrophoresis Single dimensional [1-D] electrophoresis has permitted optimum separation of stable low molecular weight RNA [Cruz-Sanchez et al. 1997] that includes 5S rRNA and tRNA of bacteria [Velazquez et al. 1988, 1998 and 2001; Palomo et al. 2000]. These RNA molecules are of great interest for taxonomic affiliation due to their highly conserved nature and have been successfully used to identify bacterial strains. 5.6.5.5. Random Amplified Polymorphic DNA [RAPD] RAPD profiles can be used to identify bacteria at species and subspecies level. At least three different families of short intergenic repeated sequences such as REP [repetitive extragenic palindromic elements], ERIC [enterobacterial repetitive intergenic consensus] and BOX elements have been reported in the genome of eubacteria [Louws et al. 1994]. PCR based techniques using specific primers of these repetitive DNA sequences [REP, ERIC and BOX] are collectively known as rep-PCR. This rep-PCR based RAPD analysis is a universal tool for assessing genomic variation and microbial biodiversity.

CONCLUSION Soil fertility is one of the most important factors for plant growth and yield [Richardson, 2001]. The excess use of chemical pesticides in agricultural fields minimizes the solubility of chemical fertilizers and making it unavailable to plants. The availability of soil phosphorous

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is largely controlled by biologically mediated process such as gross mineralization and immobilization rates. Microorganisms play a major role in phosphate solubilization and the use of phosphate solubilizing microorganism in agriculture is a low-cost technology as well as environment friendly approach without disturbing ecological balance. It is believed that microbial-mediated solubilization of insoluble phosphates in soil is through the release of organic acids and microbial metabolites [Jones and Darrah, 1994; Gyaneshwar, 1998; Carrillo et al. 2002; Rodriguez et al. 2004]. However, in addition to acid production, other mechanisms can also cause phosphate solubilization [Nautiyal et al. 2000]. Also, phosphate solubilization has been reported to depend on the structural complexity, particle size of phosphates and the quantity of organic acid secreted by microbes [Gaur, 1990]. Fluorescent pseudomonads are predominant group of rhizobacteria that enhance plant growth by improving soil nutrient status, producing plant growth hormones, enzymes and suppressing the growth of phytopathogenic fungi [Glick et al. 1995; Antoun and Kloepper, 2001; Albert and Anderson, 1987]. Plant growth promoting rhizobacterial types of fluorescent pseudomonads exhibit an array of mechanisms such as solubilizing of inorganic phosphate and iron, production of vitamins, phytohormones and antimicrobial metabolites in improving plant growth. These mechanisms can probably be active simultaneously or sequentially at different stages of plant growth and improve plant nutrients uptake, tolerance to stress, salinity, metal toxicity and pesticide. Production of antimicrobial metabolites such as 2,4diacetyphloroglucinol [DAPG], phenazine-1-carboxylic acid [PCA], phenazine-1carboxamide [PCN], pyrrolnitrin [PRN] and pyoluteorin [PLT] by fluorescent pseudomonads is considered as a key mechanism for the suppression of phytopathogens. Several enzymes by fluorescent pseudomonads are also involved in lysis and fragmentation of fungal cell wall and suppression of phytopathogenic fungi. Utilization of variety of carbon sources by phosphate solubilizing fluorescent pseudomonads may also play an important role in adapting to a variety of crop plants and soil types. The phytohormone by fluorescent pseudomonads are known to have dual role in influencing plant growth, by involving in the biocontrol together with glutathione-stransferases in defense-related plant reactions and inhibits the germination of spore and growth of mycelium of different pathogenic fungi [Brown and Hamilton, 1993; Strittmatter, 1994]. Chitinases are known to be involved in antagonistic activity against phytopathogenic fungi and insects [Chernin, 1995; Dunn et al. 1997]. Many phosphate solubilizing antagonistic fluorescent pseudomonad strains exhibit multiple traits such as production of IAA, proteases, chitinases and cellulases. Selective microbial producers of chitinases are also reported to be efficient phosphate solubilizers [Greaves and Webley, 1965]. Considering the complexity of soil conditions, the environment factors that affect phosphate solubilization and other functional traits of fluorescent pseudomonads in soils should be studied in detail. Fluorescent pseudomonad bacteria with phosphate solubilizing activity, pesticide degradation potential and ability to excrete phytohormones and antimicrobial metabolites may be exploited to develop potent biological inoculants for sustainable agriculture.

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ACKNOWLEDGMENTS We thank the Department of Science and Technology, Government of India, New Delhi, for financial support through a FIST project.

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REFERENCES Abd-alla, M. H. [1994]. Phosphates and the utilization of organic phosphorous by Rhizobium leguminosarum biovar viceae. Lett. Appl. Microbiol., 18, 294-296. Albert, F. and Anderson, A. J. [1987]. The effect of Pseudomonas putida colonization on root surface peroxidases. Plant Physiol., 85, 535-541. Antoun, H. Beauchamp, C. J. Goussard, N. Chabot, R., Lalande, R. [1998]. Potential of Rhizobium and Bradyrhizobium species as growth promoting bacteria on non-legumes; effect on radishes [Raphanus sativus L.], Plant and Soil, 204, 57-67. Antoun, H; Kloepper, JW. Plant growth promoting rhizobacteria. In: Brenner S, Miller JF editors. Encyclopedia of Genetics. San Diego, Academic Press; 2001; 1477-1480. Ayyadurai, N. Ravindra Naik, P. and Sakthivel, N. [2007]. Functional characterization of antagonistic fluorescent pseudomonads associated with rhizospheric soil of rice [Oryza sativa L.]. J. Microbiol. Biotechnol., 17, 919-927. Babu-Khan, S. Yeo, T. C. Martin, W. L. Duron, M. R Rogers, R. D. and Goldstein, A. H. [1995]. Cloning of a mineral phosphate-solubilizing gene from Pseudomonas cepacia. Appl. Environ. Microbiol. 61, 972–978. Bano, N. and Musarrat, J. [2003]. Characterization of a new Pseudomonas aeruginosa strain NJ-15 as a potential biocontrol agent. Curr. Microbiol., 46, 324-328. Bano, N. and Musarrat, J. [2004]. Characterization of a novel carbofuran degrading Pseudomonas sp. with collateral biocontrol and plant growth promoting potential. FEMS Microbiol. Lett., 231, 13-17. Barazani, O. and Friedman, J. [1999]. Is IAA the major root growth factor secreted from plant-growth-mediating bacteria. J. Chem. Ecol., 25, 2397-2406. Bashan, Y. and Holguin, G. [1998]. Proposal for the division of plant growth-promoting rhizobacteria into two classifications: biocontrol-PGPB [plant growth-promoting bacteria] and PGPB. Soil Biol. Biochem., 30, 1225-1228. Blumer, C. Heeb, S. Pessi, G. and Haas, D. [1999]. Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites. Proc. Natl. Acad. Sci. USA, 96, 14073-14078. Bric, J. M. Bostock, R. M. and Silverstone, S. E. [1991]. Rapid in situ assay for indoleacetic acid production by bacteria immobilized on nitrocellulose membrane. Appl. Environ. Microbiol., 57, 535–538. Brown, A. E. and Hamilton, J. T. G. [1993]. Indole-3-ethanol produced by Zygorrhynchusmoelleri, and indole-3-acetic acid analogue with antifungal activity. Mycol. Res., 96, 71-74. Carrillo, A. E. Li, C. Y. and Bashan, Y. [2002]. Increased acidification in the rhizosphere of cactus seedlings induced by Azospirillum brasilense. Naturwissenschaften, 89, 428-432.

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Genetic and Functional Diversity of Phosphate...

123

Cattelan, A. J. Hartel, P. G. and Furhmann, F. F. [1999]. Screening for plant growth promoting rhizobacteria to promote early soybean growth. Soil Sci. Soc. Am. J., 63, 16701680. Chernin, I. Ismailov, Z. Haran, S. and Chet, I. [1995]. Chitinolytic Enterobacter agglomerans antagonistic to fungal plant pathogens. Appl. Environ. Microbiol., 61, 1720-1726. Cruz-Sanchez, J. M. Velazquez, E. Mateos, P. F. and Martinez-Molina, E. [1997]. Enhancement of resolution of low molecular weight RNA profiles by staircase electrophoresis. Electrophoresis, 18, 1909-1911. Dalla, G. C. [1986]. Esperienze di lotta biologicacontrola fusariosi vascolare del garofano [Trials on the biological control of vascular kilt of carnations]. Ann. Inst. Sper. Floric, 17, 3012. de Souza, J. T. and Raaijmakers, J. M. [2003]. Polymorphisms within the PrnD and PltC genes from pyrrolnitrin and pyoluteorin-producing Pseudomonas and Burkholderia spp. FEMS Microbiol. Ecol., 43, 21–34. Dey, R. Pal, K. K. Bhatt, D. M. and Chauha, S. M. [2004]. Growth promotion and yield enhancement of peanut [Arachis hypogaea L.] by application of plant growth-promoting rhizobacteria. Microbiol. Res., 159, 371-39. Dunn, C. Crowley, J. J. Moenne-Loccoz, Y. Dowling, D. N. de Bruijn, F. J. and O’Gara, F. [1997]. Biological control of Pythium ultinum by Stenotrophomonas maltophilia W18 is mediated by an extracellular proteolytic activity. Microbiology, 143, 3921-3931. Dworkin, M. and Foster, W. J. [1958]. Experiments with some microorganisms which utilize ethane and hydrogen. J. Bacteriol., 75, 592-601. Frey, P. Frey-Klett, P. Garbaye, J. Berge, O. and Heulin, T. [1997]. Metabolic and genotypic fingerprinting of fluorescent pseudomonads associated with the Gouglas-fir--Laccaria bicolor mycorrhizosphere. Appl. Environ. Microbiol., 63, 1852-1860. Gaur, A. C. [1990]. Phosphate solubilizing microorganisms as bio-fertilizers. New Delhi, Omega Scientific Publication. Gaind, S. and Gaur, A. C. [2002]. Impact of fly ash and phosphate solubilizing bacteria on soybean productivity. Biores. Tech., 58, 313-315. Glick, B. R. Karaturovic, D. M. and Newell, P. C. [1995]. A novel procedure for rapid isolation of plant growth promoting Pseudomonas. Can. J. Microbiol., 41, 533-536. Goldstein, A. H. [1986]. Bacterial solubilization of mineral phosphates: historical perspective and future prospects. Am. J. Altern. Agri, 1, 51–57. Goldstein, A. H. and Liu, S. T. [1987]. Molecular cloning and regulation of a mineral phosphate solubilizing gene from Erwinia herbicola. Bio/Technology, 5, 72–74. Goldstein, AH. Involvement of the quinoprotein glucose dehydrogenase in the solubilization of exogenous phosphates by gram-negative bacteria. In: Torriani-Gorini A, Yagil E, Silver S, editors. Phosphate in Microorganisms: Cellular and Molecular Biology. Washington, DC: ASM Press; 1994; 197–203. Goosen, N. Horsman, H. P. Huinen, R.G. and van de Putte, P. [1989]. Acinetobacter calcoaceticus genes involved in biosynthesis of the coenzyme pyrrolo-quinolinequinone: nucleotide sequence and expression in Escherichia coli K-12. J. Bacteriol., 171, 447–455. Gordon, S. A. and Weber, R. P. [1951]. Colorimetric estimation of Indoleacetic acid. Plant Physiol., 26, 192–195.

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124

M. Jaharamma, K. Badri Narayanan and N. Sakthivel

Greaves, M. P. and Webley, D. M. [1965]. A study of the breakdown of organic phosphates by microorganisms from the root region of certain pasture grasses. J. Appl. Bacteriol., 28, 454–465. Gupta, R. Singal, R. Sankar, A. Chander, R. M. and Kumar, R. S. [1994]. A modified plate assay for screening phosphate solubilizing microorganisms. J. Gen. Appl. Microbiol., 40, 255-260. Gurusiddaiah, S. Weller, M. D. Sarkar, A. and Cook, J. R. [1986]. Characterization of an antibiotic produced by a strain of Pseudomonas fluorescens inhibitory to Gaeumannomyces graminis var. tritici and Pythium spp. Antimicrob Agents Chemother, 29, 488-495. Gyaneshwar, P. Kumar, G. N. and Parekh, L. J. [1998]. Effect of buffering on the phosphatesolubilizing ability of microorganisms. World J. Microbiol. Biotechnol., 14, 669-673. Halder, A. K. Mishra, A. K. Bhattacharyya, P. and Chakrabartty, P. K. [1990]. Solubilization of inorganic phosphate by Rhizobium and Bradyrhizobium. J. Gen Appl. Microbiol., 36, 81-92. Higgins, D. G. Bleashy, A. T. and Fuchs, R. [1992]. Clustal V: Improved multiple sequence alignment. Comput Appl. Biosci., 8, 189-191. Illmer, P. and Schinner, F. [1992]. Solubilization of inorganic phosphates by microorganisms isolated from forest soil. Soil Biol. Biochem., 24, 389-395. Jones, D. L. and Darrah, P. R. [1994]. Role of root derived organic acids in the mobilization of nutrients from the rhizosphere. Plant soil, 166, 247-257. Kapoor, K. K. Mishra, M. M. and Kuhkreja, K. [1989]. Phosphate solubilization by soil microorganisms-a review. Indian J Microbiol, 29, 119-127. Khan, M. S. Zaidi, A. Wani, P. A. [2007]. Role of phosphate solubilizing microorganisms in sustainable agriculture- A review. Agron Sustain Dev, 27, 29-43. Kimura, M. [1980]. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol., 16, 111-120. King, J. E. [1936]. The colorimetric determination of phosphorous. Biochem. J., 26, 292-295. Kumar, S. Tamura, K. and Nei, M. [2004]. MEGA3: Integrated software for molecular evolutionary genetic analysis and sequence alignment. Brief Bioinform, 5, 1596-1599. Laguerre, G. Rigottier-Gois, L. and Lemanceau, P. [1994]. Fluorescent Pseudomonas species categorized by using polymerase chain reaction [PCR]/restriction fragment analysis of 16S rRNA. Mol. Ecol., 3, 479-487. Lehinos, V. and Vacek, O. [1994]. Biosynthesis of auxin by phosphate-solubilizing rhizobacteria from wheat [Triticum aestivum] and rye [Secale cereale]. Microbiol. Res., 149, 31-35. Lehinos, V. [1994]. Effects of pH and glucose on auxin production of phosphate-solubilizing rhizobacteria in vitro. Microbiol. Res., 149, 135-138. Liu, T. S. Lee, L. Y. Tai, C. Y. Hung, C. H. Chang, Y. S. Wolfram, J. H. Rogers, R. and Goldstein, A. H. [1992]. Cloning of an Erwinia herbicola gene necessary for gluconic acid production and enhanced mineral phosphate solubilization in Escherichia coli HB101: Nucleotide sequence and probable involvement in biosynthesis of the coenzyme pyrroloquinoline quinone. J. Bacteriol., 174, 5814–5819. Louws, F. J. Fulbright, D. W. Stephens, C. T. and de Bruijn, F. J. [1994]. Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR. Appl. Environ. Microbiol., 60, 2286-2295.

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Mavrodi, D. V. Bonsall, R. F. Delaney, S. M. Soule, M. J. Phillips, G. and Thomashow, L. S. [2001a]. Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1carboxamide from Pseudomonas aeruginosa PAO1. J. Bacteriol., 183, 6454-6465. Mavrodi, O. V. Gardener, B. B. M. Mavrodi, D. V. Bonsall, R. F. Weller, D. M. and Thomashow, L. S. [2001b]. Genetic diversity of PhlD from 2,4-diacetylphloroglucinolproducing fluorescent Pseudomonas spp. Phytopathol, 91, 35-43. Molina, L. Constantinescu, F. Michel, L. Reimmann, C. Duffy, B. and Defago, G. [2003]. Degradation of pathogen quorum sensing molecule by soil bacteria: a preventive and curative biocontrol mechanism. FEMS Microbiol. Ecol., 45, 71-81. Mosier, N. Wyman, C. Dale, B. Elander, R. Lee, Y. Y. Holtzapple, M. and Ladisch, M. [2005]. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresource Technol., 96, 673-686. Narsian, V. Thakkar, J. and Patel, H. H. [1994]. Isolation and screening of phosphate solubilizing fungi. Indian J. Microbiol., 34, 113-118. Nautiyal, C. S. Bhadauria, S. Kumar, P. Lal, H. Mondal, R. and Verma, D. [2000]. Stress induced phosphate solubilization in bacteria isolated from alkaline soils. FEMS Microbiol. Lett., 182, 291-296. Neilands, J. B. [1995]. Siderophore: structure and function of microbial iron transport compounds. J. Biol. Chem., 270, 26723-26726. O’Sullivan, D. J. and O’Gara, F. [1992]. Traits of fluorescent Pseudomonas spp. involved in suppression of plant root pathogens. Microbiol. Rev., 56, 662-676. Palomo, J. L. Velazquez, E. Mateos, P. F. Garcia-Benavides, P. and Martinez-Molina, E. [2000]. Rapid identification of Clavibacter michiganensis subspecies sependonicus based of the stable Low Molecular weight RNA [LMW RNA] profiles, Eur. J. Plant Pathol., 106, 789-793. Pandey, A. and Palni, L. M. S. [1998]. Isolation of Pseudomonas corrugata from Sikkim Himalaya. World J Microbiol Biotechnol, 14, 411-413. Patten, C. L. and Glick, R. B. [2002]. Role of Pseudomonas putida in indoleacetic acid in development of the host plant root system. Appl. Environ. Microbiol., 68, 3795-3801. Peix, A, Rivas-Boyero, A. A. Mateos, P. F. Rodriguez-Barrueco, C. Martinez-Molina, E. and Velazquez, E. [2001]. Growth promotion of chickpea and barley by a phosphate solubilizing strain of Mesorhizobium mediterraneum under growth chamber conditions. Soil Biol. Biochem., 33, 103-110. Peix, A. Rivas, R. Mateos, P. F. Martinez-Molina, E. Rodriguez-Barrueco, C. and Velazquez, E. [2003]. Pseudomonas rhizosphaerae sp. nov., a novel species that actively solubilizes phosphate in vitro. Int. J. syst. Evol. Microbiol., 53, 2067-2072. Peix, A. Rivas, R. Santa-Regina, I. Mateos, P. F. Martinez-Molina, E., Rodriguez-Barrueco, C. and Velazquez, E. [2004]. Pseudomonas lutea sp. nov., a novel phosphate-solubilizing bacterium isolated from the rhizosphere of grasses. Int. J. syst Evol. Microbiol., 54, 847850. Penrose, D. M. and Glick, B. [2002]. Methods for isolating and characterizing ACC deaminase containing plant growth promoting rhizobacteria. Physiol Plant, 118, 10-15. Pikovskaya, R. I. [1948]. Mobilization of phosphorous in soil in connection with vital activity of some microbial species. Mikrobiologiya, 17, 363−370.

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126

M. Jaharamma, K. Badri Narayanan and N. Sakthivel

Raaijmakers, J. Weller, D. M. Thomashow, and L. S. [1997]. Frequency of antibiotic producing Pseudomonas spp. in natural environments. Appl. Environ. Microbiol., 63, 881-887. Ramette, A. Frapolli, M. Defago, G. Monenne, Y. [2003]. Phylogeny of HCN synthaseencoding hcnBC genes in biocontrol fluorescent pseudomonads and its relationship with host plant species and HCN synthesis ability. Mol. Plant-Microbe Interact, 16, 525-535. Ravindra Naik, P. and Sakthivel, N. [2006]. Functional characterization of a novel hydrocarbonoclastic Pseudomonas sp. strain PUP6 with plant-growth-promoting traits and antifungal potential. Res. Microbiol., 157, 538−546. Ravindra Naik, P. Raman, G. Badri Narayanan, K. and Sakthivel, N. [2008]. Assessment of genetic and functional diversity of phosphate solubilizing fluorescent pseudomonads isolated from rhizospheric soil. BMC Microbiol, 8, 230 In Press. DOI No. 10.1186/14712180-8-230. Reilly, T. J. Baron, G. S. Nano, F. and Kuhlenschmidt, M. S. [1996]. Characterization and sequencing of a respiratory burst-inhibiting acid phosphatase from Francisella tularensis. J. Biol. Chem., 271, 10973–10983. Renwick, A. Campbell, R. and Coe, S. [1991]. Assessment of in vivo screening systems for potential biocontrol agents of Gaeumannomyces graminis. Plant Pathol, 40, 524-532. Richardson, A. E. [2001]. Prospects for using soil microorganisms to improve the acquisition of phosphorous by plants. Aust. J. Plant Physiol., 28, 8797-8906. Rodriguez, H. and Fraga, R. [1999]. Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnol. Adv., 17, 319-339. Rodríguez, H. Goire, I. and Rodríguez, M. [1996]. Characterización de cepas de Pseudomonas solubilizadoras de fósforo. Rev ICIDCA, 30, 47–54. Rodriguez, H. Gonzalez, T. Goire, I. and Bashan, Y. [2004]. Gluconic acid production and phosphate solubilization by the plant growth promoting bacterium Azospirillum spp. Naturwissenschaften, 91, 552-555. Rodriguez, H. Fraga, R. Gonzalez, T. and Bashan, Y. [2006]. Genetics of phosphate solubilization and its potential applications for improving plant growth-promoting bacteria. Plant Soil, 287, 15–21. Rossolini, G. M. Shipa, S. Riccio, M. L. Berlutti, F. Macaskie, L. E. and Thaller, M. C. [1998]. Bacterial non-specific acid phosphatases: physiology, evolution, and use as tools in microbial biotechnology. Cell Mol. Life Sci., 54, 833–850. Sacherer, P. Defago, G. and Haas, D. [1994]. Extracellular protease and phospholipase C are controlled by global regulatory gene gacA in the biocontrol strain Pseudomonas fluorescens CHA0. FEMS Microbiol Lett, 116, 155-160. Saitou, N. and Nei, M. [1987]. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol, 4, 406-425. Sakthivel, N. and Gnanamanickam, S. S. [1987]. Evaluation of Pseudomonas fluorescens for suppression of sheath-rot disease and for enhancement of grain yields in rice [Oryza sativa L]. Appl Environ Microbiol, 53, 2056–2059. Schyn, B. and Neilands, J. B. [1987]. Universal chemical assay for the detection and determination of siderophores. Anal. Biochem., 160, 47-56. Smibert, RM; Krieg, NR. [1994]. Phenotypic characterization. In: Gerhardt P, Murray RGE, Wood WA, Krieg NR editors. Methods for General and Molecular Bacteriology. American Society of Microbiology, Washington DC: 607-654.

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Smith, S. E. and Read, D. J. [1997]. Mycorrhizal symbiosis: [2nd edition]. San Diego, Academic Press. Strittmatter, H. K. [1994]. Pathogen-defense gene prp1-1 from potato encodes an auxinresponsive glutathione-s-transferase. Eur. J. Biochem., 226, 619-626. Sunish Kumar, R. Ayyadurai, N. Pandiaraja, P. Reddy, A. V. Venkateswarlu, Y. Prakash, O. Sakthivel, N. [2005]. Characterization of antifungal metabolite produced by a new strain Pseudomonas aeruginosa PUPa3 that exhibits broad-spectrum antifungal activity and biofertilizing traits. J. Appl. Microbiol., 98, 145-154. Thaller, M. C. Berlutti, F. Schippa, S. Lombardi, G. and Rossolini, G. M. [1994]. Characterization and sequence of PhoC, the principal phosphateirrepressible acid phosphatase of Morganella morganii. Microbiol, 140, 1341– 1350. Thaller, M. C. Lombardi, G. Berlutti, F. Schippa, S. and Rossolini, G. M. [1995b]. Cloning and characterization of the NapA acid phosphatase/phosphotransferase of Morganella morganii: identification of a new family of bacterial acid phosphatase encoding genes. Microbiol, 140, 147–151. To-O K. Kamasaka, H. Kuriki, T. and Okada, S. [2000]. Substrate selectivity in Aspergillus niger KU-8 acid phosphatase II using phosphoryl oligosaccharides. Biosci. Biotechnol. Biochemi, 64, 1534-1537. Vassilev, N. and Vassileva, M. [2003]. Biotechnological solubilization of rock phosphate on media containing agro-industrial wastes. Appl. Microbiol. Biotechnol., 61, 435-440. Velazquez, E. Igual, J. M. Willens, A. Fernandez, M. P. Munoz, E. Mateos, P. F. Abril, A. Toro, N. Normand, P. Cervants, E. Gillis, M. and Martinez-Molina E. [2001]. Mesorhizobium chacoense sp. nov., a novel species that nodulates Prosopis alba in the Chaco Arido region [Argentina]. Int. J. Syst. Evol. Microbiol., 51, 1011–1021. Velazquez, E. Cervants, E. and Igual, J. M. et al. [1988]. Analysis of LMW RNA profiles of Frankia strains by staircase electrophoresis. Syst. Appl. Microbiol., 21, 539-545. Velazquez, E. Cruz-Sanchez, J. M. Mateos, P. F. and Martinez-Molina, E. [1998]. Analysis of stable low-molecular-weight RNA profiles of members of the family Rhizobiaceae. Appl. Environ. Microbiol, 64, 1555–1559. Wani, P. A. Khan, M. S. and Zaidi, A. [2007a]. Synergistic effects of the inoculation with nitrogen fixing and phosphate solubilizing rhizobacteria on the performance of field grown wheat. J. Plant. Nutr Soil Sci., 170, 283-287. Weisburg, W. G. Barns, S. M. and Lane, D. J. [1991]. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol., 173, 697-703. Whitelaw, M. A. [2000]. Growth promotion of plants inoculated with phosphate- solubilizing fungi. Adv Agron, 69, 99-151. Yamamoto, S. Kasai, H. Arnold, D. L. Jackson, R. W. Vivian, A. and Harayama, S. [2000]. Phylogeny of the genus Pseudomonas: intrageneric structure reconstructed from the nucleotide sequences of gyrB and rpoD genes. Microbiol., 146, 2385-2394. Yao, Q. Li, X. Feng, G. and Christie, P. [2001]. Mobilization of sparingly soluble inorganic phosphates by the external mycelium of an arbuscular mycorrhizal fungus. Plant Soil, 230, 279-285.

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Chapter 6

PRACTICAL USE OF PHOSPHATE SOLUBILIZING SOIL MICROORGANISMS Olga Mikanova∗ and Jaromir Kubat Research Institute of Crop Production, Drnovska 507, 161 06 Prague, Czech Republic

ABSTRACT

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From the sustainable agriculture point of view, at least a part of phosphorus necessary for plants needs to be supplied from other sources like, chemical phosphatic fertilizers. The use of chemical fertilizers due in part to the environmental hazards and its cost is generally discouraged. Use of soil microflora is one of the options to provide plants with sufficient phosphorus. The procedure adopted for identifying the Psolubilizing activity of soil microflora usually aims at determining P-solubilizing activity of the strains on solid or in liquid cultures [in vitro] followed by strain testing in association with plants [in vivo] in pot experiments or under field trials. The assessment of P-solubilizing activity of microbes in association with plants is generally decisive. Nitrogen fixing bacteria in general are well-known for their ability to fix atmospheric nitrogen and are commonly used worldwide for inoculation of leguminous plants. However, the ability to release phosphorus has also been found among certain nitrogen fixing genera like, Rhizobium, Bradyrhizobium and Sinorhizobium. In this chapter an attention is paid to highlight the role of nitrogen fixing bacteria as P-solubilizer besides other microbes, and their role in crop improvement which seems to be a very prospective concept for sustainable agriculture. Bacterial preparations, though despite all problems, are one of the options as to how some environmental and partly economic constraint faced by the farming communities can be resolved.

∗

Correspondence to:: [email protected]

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6.1. INTRODUCTION Phosphorus [P] is one of the principal macro-biogenic elements necessary for almost all the metabolic processes of plant growth and development. Among the most significant metabolic processes in plants on which phosphorus has important effects are [i] photosynthesis [ii] nitrogen fixation [iii] crop maturation [flowering and fruiting, including seed formation] [iv] root development [v] strength of straw in cereal crops and [vii] improvement of crop quality, especially of forages and of vegetables [Brady, 1990]. Although the total P content in soil is usually rather high, the most of it is in a wide variety of mineral and organic compounds that are not available to plants. That is why the P often becomes a limiting factor to plant growth. As low P status tends to limit the production of arable crops, the application of soluble P fertilizers such as, superphosphate has been widely used. However, much of the soluble P applied as fertilizer react with the soil constituents and is converted to forms less available to plants [Whitelaw et al., 1999]. Up to 90% of the P added to soil as mineral fertilizers is transformed to hardly soluble compounds [Goldstein, 1986] or washed away into fresh and ground waters thereby requiring purification of such polluted water [Shigaki et al., 2006]. In order to overcome this discrepancy, the P uptake efficiency of the the cultivated plants should be improved. One of the possibilities to reach this goal is the solubilization of hardly soluble phosphates by soil microorganisms. About 20–30% of the soil microorganisms are able to transform the hardly soluble phosphates into soluble form [Domey, 1987; Mikanová and Kubát, 1994]. The ability to release phosphorus from hardly soluble compounds was found also in bacteria of the genus Rhizobium, Bradyrhizobium and Sinorhizobium spp. [Dhingra et al., 1988; Mikanová et al., 1995]. Generally, soil microorganisms play an important role in an integrated soil fertility management and therefore, in a sustainable agriculture. The interest in sustainable agriculture has stimulated research on the rational use of plant growth-promoting rhizobacteria, among others, aiming at an introduction of microorganisms that may improve the P availability to plants or enhance their activity. This may be attained to a greater extent by inoculating seeds or soil with specific organisms possessing growth enhancing ability [biofertilizers]. The strategy adopted for improving plant productivity through the use of naturally occurring beneficial microbes however, should include: selection of good quality inoculants, awareness among end users [farmers] about inoculation technology, effective inoculant delivery system and formulation of the policy to exploit biofertilizers successfully [Wani and Lee, 1995]. In some cases, biofertilizers though, may not increase the crop yields dramatically as observed with mineral fertilizers application, yet a gradual and modest increase in the use of bacterial inoculants is likely to reduce the reliance on chemical fertilizers and could act as an alternative to environmentally hazardous chemical fertilizers. And hence, the use of such inexpensive microbial inoculants with greater promise for sustaining productivity while maintaining the fertility of soils should be promoted around the globe to overcome both environmental hazards and economic constraint associated with the use of chemical fertilizers [Bashan, 1998].

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6.2. PHOSPHORUS IN SOIL Phosphorus is present in soil both in inorganic and organic compounds. Natural sources of phosphorus include mineral phosphates [e.g. fluorapatite, chlorapatite, inorganic salts of phosphoric acid with calcium [calcium hydrogen phosphate, calcium phosphate], aluminum [variscite] or iron [strengite]]. Formation of salts of phosphoric acid depends on pH of the environment. Calcium salts predominate in alkaline soils while aluminum and ferric salts are abundantly found in acid soils. Organic phosphorus accounts for some 25-85% of the total content of soil P. As far as P availability to plants is concerned, mineral soil P may be divided into three categories- [i] P soluble in the soil solution [P available for plant uptake] [ii] labile P in the solid phase [complements of the soil solution] and [iii] stable P in the solid phase. Upon exhaustion of labile phosphorus, even stable P may be released; however, it is a very slow process [Hartikainen, 1991]. Only a small soil phosphorus stock contained in the soil solution serves as an immediate source of P for plants. When this pool is depleted, system balance is disturbed and some of the labile P is released into the soil solution. Mendoza et al., [1990] reported that phosphate concentration in the soil solution may continuously change due to phosphate sorption and consequent release from soil.

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6.3. MINERAL PHOSPHORUS FERTILIZATION The conventional approach to improve phosphorus nutrition for optimum crop yields is to apply chemical fertilizer containing soluble phosphate. Regular and high volumes of mineral fertilizers are used in order to achieve high yields. However, the applied phosphate is also subjected to a fixation processes, resulting in a large fraction of the fertilizer P becoming unavailable for plant use. Nevertheless, abundance of natural rock phosphates is rather limited and are extremely difficult and costly to excavate. From the environmental point of view, in the long run it will be more and more imperative to enhance efficiency of phosphorus uptake from the soil stock and phosphorus usage from mineral phosphorus fertilizers. One of such options to attain this objective is solubilization of hardly soluble phosphates, which in turn provide soluble form of P to plants.

6.4. PHOSPHORUS MOBILIZATION AND SOLUBILIZATI ON 6.4.1. MOBILITY OF PHOSPHORUS IN SOIL Phosphorus may move within the soil profile through soil organisms [plants, animals, microorganisms], soil water movement and in the direction of concentration gradient [diffusion]. The effect of higher plants on P mobility in soil is of great importance. In fact all labile P may be included in this process, while the extent and speed of movement depends on the amount of P taken by plant roots. Subsequently, P moves through plants to the aboveground part and returns back to soil through waste. It results in specific distribution of organic P forms with the highest concentration being usually in the soil surface layer and the root zone. The amount of P that moves within soil is determined by P concentration and the intensity of movement of soil solution. This type of movement of soil P is important for both

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ensuring P supply to roots and possible P wash-out into subsoil. In the case of low P concentration in the soil solution, P movement is negligible and loss caused by P leaching is minor. Activity of soil microorganisms contributes to a higher P concentration in the soil solution as well as a greater potential movement of P. Penetration is higher in organically fertilized soils than in soils fertilized with an equivalent amount of P in the inorganic form [Anderson, 1980]. Diffusion is another process in which transport of P from one system to another takes place by means of movement of molecules or ions. The particles stop moving when the system is in equilibrium. If there is a concentration gradient in the system, the system attempts a change in order to achieve a balanced status. This mechanism is partially applied for P movement in close proximity of the root zone [Eghball et al., 1990].

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6.4.2. Possibilities of Phosphorus Mobilization Appropriate crop rotation, particularly inclusion of leguminous plants in the cropping pattern, also facilitates P mobilization. As a result of more aggressive root exudates, most of these plants can make better use of P from soil compounds. Another advantage of including leguminous plants in agronomic practices is that their root system reaches considerable depth [particularly that of clover and alfalfa]. And hence, such plants are capable of taking up P from deeper horizons. It is also equally important to mention the role of symbiosis of leguminous plants with bacteria of the genus Rhizobium, which infect roots of leguminous plants and some of which have the ability to release P from hardly soluble compounds. Also, rich root residues that remain in soil after harvest is a source of organic P and nutrients supporting development of soil microflora. Another measure to mobilize P is to enrich soil with organic matter. Organic matter of plant and animal origin entering soil are a good source of energy and allow nutrient cycling in soil. Application of organic matter decreases P sorption and consequently increases P uptake by plants. Organic matter is also a substrate containing a large quantity of soil microorganisms, particularly fungi and bacteria, some of which show P-solubilizing activity. Phosphorus mobilization is also affected by root exudates. For example, Hoffland et al. [1989 a, b] quantified the amount of acid exudates released by oilseed rape to agar which either did or did not contain P. Oilseed rape plants grown with no source of P released acid exudates while plants grown in a medium containing P did not release acid exudates. These exudates contained some organic acids, such as citric and malic acids. Soil macrofauna also plays an important role in releasing and moving P in soil. For instance, soil invertebrates [e.g. earthworms] contribute to disintegration of organic residues and add P when mixed with other soil components. Earthworms have long been recognized by farmers as beneficial to soil constituting an important group of secondary decomposers. Crushing of the residues increases its surface and microorganisms can decompose organic substances more efficiently. As a bioinoculant, vermicompost increased N and P availability by enhancing biological nitrogen fixation and P-solubilization [Padmavathiamma et al., 2008]. In soils, the enzymes [e.g., acid or alkaline phosphatases] synthesized by majority of microorganisms break down organic P [mineralization] and make it available to plants. Approximately 2 to 5 % of the total amount of P in soil can be temporarily bound in microbial biomass and the rate of P uptake from a solution is twice as high in fungi and nine

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times as high in bacteria as the intake rate of P by plant roots. If the water soluble P is depleted in the soil solution, it is replaced, as a result of chemical balancing by P bound to soil particles.

6.4.3. Mechanisms of P-Solubilization The primary solubilization mechanism involves the use of organic acids released by Psolubilizing microorganisms resulting in a decrease of medium pH [Mikanova and Kubat, 1994, Taha et al., 1969; Sperber, 1958; Whitelaw et al., 1999]. The type of acids produced and their quantity depends on the microorganism involved in the solubilization process. Organic acids, such as lactic, citric, succinic, fumaric, malic, and oxalic acids, are known to take part in solubilization of hardly soluble P [Illmer and Schinner, 1992]. Of all these organic acids, citric acid has shown the highest P-solubilization [Gaur, 1990]. However, some P-solubilizing microorganisms produce low-molecular-weight organic acids, like, gluconic and keto-gluconic acids [Rodrígues and Fraga, 1999]. Other substances contributing to phosphate solubilization are humic and fulvic acids. In a study, Inskeep and Silvertooth [1988] reported that humic and fulvic acids inhibit precipitation of hydroxyapatite. Moreover, some authors however, believed that the P is solubilized by mechanism other than organic acid production [Gyaneshwar et al., 2002]. For example, Tao et al. [2008] found a significantly negative linear correlation between culture pH and P solubilized from inorganic P by P-mineralizing bacterial strains. The results suggested that P-solubilization and Pmineralization could coexist in the same bacterial strain.

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6.5. FACTORS AFFECTING MINERAL P-SOLUBILIZATION Optimum pH for solubilization of P depends on the type of insoluble P present in soil and various ionic forms of P present in the soil solution. It is H2PO4-that is the best plant-available form which occurs in the maximum amount when pH equals 3.5–5.5. The higher the pH, the greater is the share of HPO42- and PO43-. Hartikainen and Simojoki [1997] found that if H2SO4 was added to soil, it led to a significant release of available P in soils. Menon and Chien [1990] also observed that acidification of raw phosphates with H2SO4 has a favourable effect on maize [Zea mays] yield in comparison with the non-acidified phosphates. Nevertheless, this seemingly simple dependence is complicated by the presence or absence of other compounds or ions in relation to various pH levels. The solubility of P is greatly affected at low pH when Fe3+ and Al3+ are present while at an alkaline soil reaction, the presence of Ca2+ influences the P solubility. The low fertility of acid soils is mainly due to the transformation of soluble forms of P into form of poor solubility, particularly Fe-P and Al-P complexes [Pérez et al., 2007]. In the alkaline reaction, the presence of Ca2+ ions [even calcium phosphate], which is hardly available to plants, may be formed. Despite all these facts, Brady [1990] reported that the maximum solublization of P in soil occurs when the pH is between 6 and 7. The pH value also has an impact on microorganisms affecting solubilization. Bacteria prefer a neutral to slightly alkaline environment for maximum solubilization while fungi prefer a slightly acid to neutral environment. Data in the literature

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suggest that about 20–30% of soil microorganisms are able to transform the difficultly soluble phosphates to soluble forms. Soil microflora, particularly P-solubilizing bacteria and Psolubilizing fungi, play the most important role in releasing phosphorus from hardly soluble compounds in soil. However, instability of the P solubilizing activity of some strains after several cycles has been reported [Halder et al., 1990; Illmer and Schinner, 1992]. Another factor that strongly affect the amount of P released into soil is the type and origin of insoluble phosphate. For example, Kapoor [1995] reported that calcium phosphate, variscite and strengite can be solubilized faster than rock phosphates [RP]. In a follow up study, Xiao et al. [2008] assessed the impact of different P sources on content of soluble P and concluded that the the type and origin of a given RP strongly affected the solubility. Presence of soluble phosphates in soil also greatly influence the solubilization of the hardly soluble phosphates. In series of experiments, it has been found that colonization by VAM-fungi was inhibited by increasing phosphorus fertilization [Menge et al., 1978, Amijee et al., 1989, Thomson et al., 1986] which could possibly be due to a change in quality and quantity of root exudates and a decreased number of fungal entry points in roots [Braunberger et al. 1991]. Mikanová et al. [1997] in consequent studies also confirm that there are some bacteria whose P-solubilizing activity is inhibited by the presence of plant-available P while others are insensitive to such changes in soil. The inhibition of P-solubilizing activity in the presence of soluble P may account for a decreased efficiency of the inoculation of soil with Psolubilizing microorganisms, especially soils well supplied with P. In earlier study, we tried to find a correlation between P-solubilizing activity of 12 strains of Rhizobium spp. and Sinorhizobium spp. procured from the rhizobia culture collection of the RICP [Mikanova and Novakova, 2002] and soluble potassium dihydrogen phosphate in liquid culture medium- [g/l: glucose 10 asparagin 1, K2SO4 0.2, MgSO4.7 H2O 0.4, yeast autolysate 0.2]. A 30 ml of this medium was transferred into 250 ml flasks and 0.02 g Ca3 [PO4]2 was added to each flask. Except the control, the medium was then amended with different doses of the soluble dihydrogen potassium phosphate. The medium was filtered after incubation and the remaining tricalcium phosphate [TCP] was separated by filtration. Medium was poured onto the filter and rinsed with hot distilled water [in order to remove slime and soluble phosphates]. Filter paper with the remaining TCP was dried for 15 min. at 105o C and afterwards hydrolysed with 2N H2SO4 for 18 h. The solution was filtered again and two ml of the filtrate was adjusted to 100 ml with distilled water. Hydrolysed phosphate was determined spectrophotometrically by the method described by Murphy and Riley [1962] and the Psolubilizing activity was expressed as a difference between the TCP added and its remainder after the incubation. The results are shown in Table 1. All tested bacterial strains showed a high P-solubilizing activity when medium was devoid of soluble P. Interestingly, addition of water soluble potassium dihydrogen phosphate inhibited the P-solubilizing activity of the selected microorganisms. While this activity wasinhibited in majority of the tested strains in media amended with 15–20 mM phosphate/l, some of the tested strains [e.g., Rhizobium trifolii D558, D659 and D48l] demonstrated a high P-solubilizing activity even in medium containing 20 mM phosphate/l. More importantly, the P-solubilizing activity of the strain D558 was not completely inhibited even at a concentration of 25 mM phosphate while P-solubilizing activity of strain D600 was terminated at a concentration of l5 mM P/l.

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Table 1. Phosphate solubilizing activity of strains of Rhizobium leguminosarum, Rhizobium trifolii and Sinorhizobium meliloti grown in medium amended with or without different concentrations of soluble KH2PO4. Rhizobial strains

D558 D659 D481 D561 D663 D661 D600 D662 D562 D564 D163 D4 Control

Concentration of insoluble phosphate [mgP/l] at different rates of KH2PO4 [mM/l] 5 10 15 20 25 0 14.30 19.58 22.42 23.88 31.66 33.66 14.30 17.70 20.44 23.20 28.76 38.00 14.98 16.26 21.42 23.84 30.78 35.70 17.11 25.09 28.64 33.28 33.91 39.44 17.98 22.50 22.66 26.38 32.04 36.96 18.78 22.96 24.92 29.32 32.40 34.62 20.15 25.59 27.89 37.67 39.09 40.06 22.25 26.81 28.94 31.07 36.09 40.79 23.54 30.11 30.03 32.87 36.59 42.32 24.13 30.07 35,96 37.93 40.76 40.17 31.08 32.12 33.40 34.54 35.92 35.80 31.10 34.80 36.30 34.30 30.90 36.45 40.00 40.00 40.00 40.00 40.00 40.00

Strains D558, D659, D481, D663, D661 and D662 represent Rhizobium trifolii; D561, D600, D562 and D564 represent R. leguminosarum; D163 and D4 represent Mesorhizobium meliloti, respectively; Values are mean of three replicates.

6.6. PRACTICAL USE OF P-SOLUBILIZING SOIL MICROORGANISMS Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

6.6.1. P-Solubilizing Bacteria Among bacteria, the genera Azotobacter, Erwinia, Bacillus, Pseudomonas, Escherichia, Enterobacter, Burkholderia, Pantoea, Serratia, Ralstonia etc. isolated from soil or root rhizosphere of various plants have been reported as P-solubilizers [Rodríques and Fraga, 1999]. Phosphate-solubilizing microorganisms form a clear zone around their growth which indicates the P-solubilizing efficiency of a bacterial strain. The P-solubilizing activities of microbial strains are tested used insoluble P salts namely, tricalcium phosphate [Ca3[PO4]2 and iron phosphate [FePO4] [Pérez et al. 2007]. Moreover, the abundance of P-solubilizing bacteria in soil depends on the site of collection. For example, in a study, Yahya and AlAzawi [1989] isolated P-solubilizing bacteria from different soil samples and found different numbers of these bacteria. The order of bacteria density followed an order: vegetables > legumes > grass > crops > orchards. The ability to release phosphorus has also been found in the genera Rhizobium, Bradyrhizobium and Sinorhizobium [Halder et al., 1991; Halder and Chakrabartty, 1993; Abd-Alla, 1994; Mikanova and Kubat, 1999]; though, rhizobia are generally regarded as symbiotic partners of legumes and are mainly known for their nitrogen fixing ability and hence, are commonly used for inoculating leguminous plants.

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6.6.2. P-Solubilizing Fungi The ability of fungi to release P from phosphates has been known for many years. Venkateswarlu et al. [1984] tested different soil microorganisms for their P-solubilizing activity and found out that fungi are much more efficient in releasing P than bacteria. Increase of plant-available P on calcareous soil caused by the genera Penicillium and Aspergillus is reported by numerous authors [Salih et al. 1989; Asea et al. 1988; Whitelaw 2000; Mittal et al. 2008]. Another group of fungi capable of solubilizing P includes vesicular-arbuscular mycorrhizal [VAM] fungi: found in association with majority of farm crops. They improve the uptake of nutrients especially P from soil. The most commonly used strains belong to genera Glomus and Gigaspora [Khurana and Dudeja, 1995]. However, it is difficult to culture VAM fungi in vitro on a nutrient medium but can be propagated using plant roots. And hence, application of AM-fungi at large-scale in agricultural production systems is considerably more difficult.

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6.7. DETERMINATION OF P-SOLUBILIZING ACTIVITY OF MICROORGANISMS The P-solubilizing activity of soil microorganisms are determined by the formation of clearing zones on agar plates, quantitative assay of solubilized phosphate concentration in liquid cultures and assessment of P accumulated within inoculated plants grown either in pot experiments or in field environments. The assessment of microbes for P-solubilizing activity [PS] on agar plates is advantageous because this method is quick and allows a rapid screening of a large number of microorganisms. Effective strains are identified by the development of clearing zones around microbial growth when medium is supplemented with the hardly soluble phosphate such as, tricalcium phosphate [TCP]. The P-solubilizing activity is further assessed quantitatively using liquid cultures [Mikanová and Kubát, 1994; Mikanová and Kubát, 2006]. And finally, the impact of PSM on different plants in terms of P uptake by plants is tested in a greenhouse, in pots or small-plot or field experiments.

6.7.1. P-Solubilizing Activity in Liquid Cultures In our study, we assessed the PS activity of microorganisms isolated from the rhisosphere of cereals grown in pot and field trials at the Research Institute of Crop Production, Praha– Ruzyne, Czech Republic. These isolates were cultivated on Hirte-agar both in liquid cultures and on agar plates. The isolated bacteria showed a distinct P-solubilizing activity on agar plates and most of them maintained this activity even in shaken liquid culture. During one week incubation, about 96% bacterial isolates transformed hardly soluble calcium phosphate into soluble phosphate ions [Table 2]. The highest phosphorus concentration determined was 73.5 mg/l for the isolate No. 88, which solubilized 73.3% of TCP. A portion of the supplied phosphorus was incorporated into newly formed microbial biomass. The P-solubilizing activity of all the tested organisms was related to the drop in pH of the medium suggesting the production of organic acids by the bacterial isolates.

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Table 2. The amount of the P solubilized, change in pH, zones of clearing and colony forming units [cfu] of rhizospheric microbes

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Bacterial isolate No. 88 86 85 26 15 24 22 3 30 11 14 25 13 12 21 20 7 19 202 18 6 27 8

P solubilized [mg P/l] 73.5 63.0 63.0 62.9 47.9 45.9 43.4 40.5 37.1 36.9 36.8 32.5 21.0 16.3 11.8 11.7 11.6 10.9 6.5 6.4 5.6 3.7 0.0

pH 3.8 3.4 3.5 4.5 4.1 4.9 4.5 5.6 5.9 5.4 4.9 4.7 5.9 7.8 6.2 8.1 5.5 8.0 6.7 8.6 5.8 7.6 8.3

Zones [cm] 0.20 0.70 0.05 0.10 0.30 0.20 0.30 0.25 0.15 0.20 0.10 0.20 0.10 0.20 0.15 0.50 0.20 0.30 0.05 0.10 0.20 0.20 0.40

Log cfu/ml 6.00 6.30 5.48 9.00 9.00 6.85 10.00 6.00 7.00 6.48 7.30 6.00 7.48 8.48 10.00 8.78 6.30 9.00 8.30 7.78 7.48 7.60 7.48

Each value is a mean of three replicates.

Figure 1. The relation between phosphorus-concentration [mg P per l] and pH value of the medium influenced by Rhizobium leguminosarum.

Moreover, we also analyzed the strains of Rhizobium leguminosarum for its PS activity and about 76% of the tested strains displayed PS activity with concomitant drop in pH of the Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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liquid culture medium. The relationship between the amount soluble phosphate concentration and pH value of the medium is presented in Figure 1.

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6.8. INOCULATION EFFECTS The major objective of inoculating microbes is to increase soil fertility. Application of beneficial microbes in agricultural practices started 60 years ago. A group of bacteria, referred to as “plant growth-promoting rhizobacteria” [PGPR], is being used extensively as inoculants [biofertilizers] to facilitate plant growth around the world [Vessey, 2003]. In recent years, biofertilizers have emerged as an important component of the integrated plant nutrient supply system [IPNSS] and hold a great promise to improve crop yields through environmentally safe nutrient supplies to cultivated crops. Though, the application of microbial fertilizers somehow, has not achieved its anticipated targets [Wu et al., 2005] yet the use of biofertilizers has the following advantages, like, low price, easy application, preservation of soil and the environment, wide range of applications, utilization in places where fertilizers or chemical preparations are not allowed [water protection zones or natural preserves] and sustained increase in crop yields. In the Czech Republic, the application of biological preparations has had a long tradition. The preparation named rizobin [former nitrazon] containing nodular bacteria capable of fixing atmospheric nitrogen [rhizobia] in symbiosis with plants has been manufactured in the Czech Republic since 1949. Traditionally, the selection and testing of appropriate strains for the prepation of rizobin is based on measuring the nitrogenase activity. However, the selection and identification of P-solubilizing rhizobia in recent times has attracted greater attention of the scientists. And hence, both N2 fixation and P-solubilizing ability of rhizobial preparation [rizobin] are evaluated before they are applied either in pot or under field trials [Mikanova and Kubat, 1999]. In this regard, we assessed the P-solubilizing activity of seven nitrogenase producing strains each of Sinorhizobium fredii and Bradyrhizobium japonicum procured from Czech Rhizobia Collection [Table 3]. Four of the strains [D563, D504, D538 and D574] demonstrating high nitrogenase activity and greater P-solubilizing potential were used for the production of rizobin for soybean [Glycine max]. Soybean var. marple arrow was seeded in pots containing five kg of sifted soil. Before seeding, the pots were fertilized with hardly soluble Gafsa phosphate [@ 45 kgP/ha] and superphosphate [@ 45 kgP/ha]. After seeding, the seeds were either inoculated with the rizobin or a lab-prepared preparation from a mixture of rhizobial strains without P-solubilizing activity [D576, D344 and D216]. A control without inoculant was also included. At the maturity of soybean, the seed dry matter was determined [Table 4]. The results show that the yield of dry matter of soybean was affected by both inoculation and fertilizer application. The highest yield of seed dry matter was found in the variant inoculated with rizobin. While comparing the variants fertilized with Gafsa phosphate, the highest yields were recorded in the variant inoculated with the rizobin although no statistically significant differences were observed. This result indicates that upon inoculation with bacteria capable of releasing P from hardly soluble compounds it is even possible to effectively use less soluble fertilizers as reported by others [Wu et al. 2005; Chang and Yang 2009; Zaidi et al. 2003].

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Table 3. The amount of P solubilized and change in pH of the medium by Sinorhizobium fredii and Bradyrhizobium japonicum Strain D563 D504 D538 D574 D576 D344 D216

P solubilized [mg P/l] 64.7 58.3 56.4 43.7 1.8 0.0 0.0

pH 5.1 4.6 4.8 7.6 8.6 7.7 8.2

Strains D563, D574 and D576 represent Sinorhizobium fredii; D504, D538, D344 and D216 represent Bradyrhizobium japonicum; Values are mean of three replicates.

Table 4. The average grain yield of soybean in pot experiment Inoculation Rizobin [P-solubilizing rhizobial strains [D563, D504, D538, D574] Rhizobial strains without Psolubilizing activity [D576, D344, D216]

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Control [un- inoculated]

Fertilizer Gafsa Superfos. 0 Gafsa Superfos. 0 Gafsa Superfos 0

Seed dry matter [g/pot] 42.2bc 50.2d 48.7cd 33.6ab 44.8cd 33.2ab 25.6a 22.5a 23.9a

Mean values followed by same letters are significantly not different at P ≤ 0.05 by Tukey’s method; Strains D563, D574 and D576 represent Sinorhizobium fredii; D504, D538, D344 and D216 represent Bradyrhizobium japonicum.

CONCLUSION AND FUTURE PROSPECT A sufficient phosphorus supply for cultivated crops is one of the most important issues of agricultural system. Exploitation of P-solubilizing activity of microorganisms together with other inherent plant growth promoting activities seems a promising prospect. However, attention should be paid to identify new combinations of phosphate-solubilizing bacteria and other PGPR strains for optimum yields. In this context, genetic manipulation of phosphatesolubilizing bacteria offers a great potential to improve the P-solubilizing capabilities of microorganisms [Rodriguez and Fraga, 1999]. Composting of organic wastes with the rock phosphates could further be an alternative for using low-grade RP [Kapoor, 1995; Viveganandan, 2002; Biswas and Narayanasamy, 2006]. In this regard, Chang and Yang [2009] reported that thermo-tolerant phosphate-solubilizing bacteria released more soluble P and hence, could be used to prepare bioinoculant that may promote the growth under adverse environmental conditions. Additionally, in order to improve of the efficiency of inoculation, establishment and survival of the introduced microorganisms should be improved. A promising approach to enhance the adaptation of inocula may be the isolation of P-

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solubilizing organisms from a particular locality where it has to be applied. For instance, Hamdali et al. [2008] demonstrated that actinomycetal strains isolated from Moroccan phosphate mines had a better ability to solubilize RP than the reference strain suggesting that these strains had a specific adaptation to their ecological niche, rich in insoluble RP. In conclusion, bacterial inoculants, despite all problems, are one of the options which can help to resolve some contemporary environmental and partly economic problems facing agrarian community around the globe.

ACKNOWLEDGMENTS This work was supported by the Ministry of Agriculture of the Czech Republic – MZE0002700604.

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REFERENCES Abd-Alla, M.H.[1994]. Solubilization of rock phosphate by Rhizobium and Bradyrhizobium. Folia Microbiol. 39 : 53-56. Amijee, F., Tinker, P.D. and Stribley, D.P.[1989]. The development of endomycorrhizal systems VII. A detailed study of the effect of soil phosphorus on colonization. New Phytol. 111 : 435-446. Anderson, G.[1980]. Assesing organic phosphorus in soils. The role of phosphorus in agriculture. [F.E.Khasawneh,E.C.Sample,E.J.Kamprath, eds]. Madison, Wisconsin, American Society of Agronomy 411-413. Antoun, H.[2002]. Field and greenhouse trials performed with phosphate-solubilizing bacteria and fungi. First International Meeting on Microbial Phosphate Solubilization. Salamanca, Spain. pp. 29-31. Asea, P.E.A., Kucey, R.M.N. and Stewart, J.W.B.[1988]. Inorganic phosphate solubilization by two Penicillium species in solution culture and soil. Soil Biol. Biochem. 20 : 459-464. Bajpai, P.D., Rao, W.V.B.[1971]. Phosphate solubilizing bacteria part I. Soil Sci. Plant Nutrit. 17 : 41-43. Bashan, Y.[1998]. Inoculants of plant growth-promoting bacteria for use in agriculture. Biotechnol. Adv. 16 : 729-770. Barroso, C.B. and Nahas, E.[2005]. The status of soil phosphate fractions and the ability of fungi to dissolve hardly soluble phosphates. Applied Soil Ecology. 29[1] : 73-83. Biswas, D.R., Narayanasamy, G.[2006]. Rock phosphate enriched compost: an approach to improve low-grade Indian rock phosphate. Bioresour. Technol. 97 : 2243-2251. Brady, N.C.[1990]. The nature and properties of soil. Cornell University and United States agency for international development, pp. 351-368. Braunberger, P.G., Miller, M.H. and Peterson, R.L.[1991]. Effect of phosphorus nutrition on morphological characteristics of vesicular-arbuscular mycorrhizal colonization of maize. New Phytol. 119 : 107-113. Chang, C.H., Yang, S.S. [2009]. Thermo-tolerant phosphate-solubilizing microbes for multifunctional biofertilizer preparation. Bioresour. Technol. 100 : 1648-1658.

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141

Dhigra, K.K., Sekhom, H.S., Sandhu, P.S., Bhandari, S.C.[1988]. Phosphorus- Rhizobium interaction studies on biological nitrogen fixation and yield of lentil. J. Agric. Sci. 110 : 141-144. Domey, S.[1987]. Untersuchungen zur mikrobiellen Phosphatmobilisierung in der Rhizosphare von Winterweizen, Winterrogen und Luzerne. [Dizertation], Jena,-IPE. pp. 92. Eghball, B., Sander, D.H., Skopp, J.[1990]. Diffusion, adsorption and predicted longevity of banded phosphorus fertilizer in three soils. Soil Sci. Soc. Amer. J. 54 : 1161-1165. Gaur, A.C.[1990]. Phosphate solubilizing microorganisms as biofertilizers. Omega Sci. Publishers, New Delhi, 283 pp. Goldstein, A.H.[1986]. Bacterial solubilization of mineral phosphates: Historical perspective and future prospects. Am. J. Alternative Agric. 1 : 51-57. Gyaneshwar, P., Kumar, G.N., Parekh, L.J., Poole, P.S.[2002]. Role of soil microorganisms in improving P nutrition of plants. Plant Soil 245 : 83-93. Halder, A.K., Mishra, A.K., Bhattacharyya, P., Chakrabartty, P.K.[1990]. Solubilization of rock phosphate by Rhizobium and Bradyrhizobium. J. Gen. Appl. Microbiol. 36 : 81-92. Halder, A.K., Mishra, A.K. and Chakrabartty, P.K.[1991]. Solubilization of inorganic phosphates by Bradyrhizobium. Ind. J. Exp. Biol. 29 : 28-31. Halder, A.K., Chakrabartty, P.K.[1993]. Solubilization of inorganic phosphate by Rhizobium. Folia Microbiol. 38 : 325-330. Hamdali, H., Bouizgarne, B., Hafidi, M., Lebrihi, A., Virolle, M.J., Ouhdouch, Y.[2008]. Sceening for rock phosphate solubilizing Actinomycetes from Moroccan phosphate mines. Appl. Soil Ecol. 38 : 12-19. Hartikainen, H.[1991]. Potential mobility of accumulated phosphorus in soil as estimated by the indices of Q/I plots and by extractant. Soil. Sci. 152 : 204-209. Hartikainen, H., Simojoki, A.[1997]. Changes in solid- and solution-phase phosphorus in soil on acidification. Eur. J. Soil Sci. 48 : 493-498. Hoffland, E., Findenegg, G.R., Nelemans, J.A.[1989a]. Solubilization of rock phosphate by rape I. Plant Soil 113 : 155-160. Hoffland, E., Findenegg, G.R., Nelemans, J.A.[1989b]. Solubilization of rock phosphate by rape II. Plant Soil 113 : 161-165. Illmer, P., Schinner, F.[1992]. Solubilization of inorganic phosphates by microorganisms isolated from forest soil. Soil Biol. Biochem. 24 : 389-395. Inskeep, W.P., Silvertooth, J.C.[1988]. Inhibition of hydroxyapatite precipitation in the presence of fulvic, humic, and tannic acids. Soil Sci. Soc. Am. J. 52 : 941-946. Kapoor, K.K.[1995]. Phosphate mobilization through soil microorganisms. Proc. Seminar on National Resource Management, Hisar, 46-61. Khurana, A.L., Dudeja, S.S.[1995]. Role of vesicular-arbuscular mycorrhizae [VAM] in sustainable agriculture. Proc. Seminar on National Resource Management, Hisar, 37-45. Mendoza, R.E., Canduci, A., Aprile, C.[1990]. Phosphate release from fertilized soils and its effect on the changes of phosphate concentration in soil solution. Fertil. Res. 23 : 165172. Menge, J.A., Steirle, D., Bagyaraj, D.J., Johnson, F.L.V.and Leonard, R.T.[1978]. Phosphorus concentration in plant responsible for inhibition of mycorrhizal infection. New Phytol. 80 : 575-578.

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Olga Mikanova and Jaromir Kubat

Menon, R.G., Chien, S.H.[1990]. Phosphorus availability to maize from partially acidulated phosphate rocks compacted with triple superphosphate. Plant and Soil 127 : 123-128. Mikanova, O., Kubat, J.[1994]. Phosphorus solubilization from hardly soluble phosphates by soil microflora. Rostl. Výr. 40 : 833-840. Mikanova, O., Kubat, J., Vorisek, K., Randova, D.[1995]. The capacity of the strains Rhizobium leguminosarum to make phosphorus available. Rostl. Výr. 41 : 423-425. Mikanova, O., Kubat, J., Vorisek, K., Simon, T., Randova, D.[1997]. Influence of soluble phosphate on P-solubilizing activity of bacteria. Rostl. Výr. 43 : 421-424. Mikanová, O., Kubát, J.[1999]. The practical use of the P-solubilization activity of Rhizobium species strains. Rostl. Výr., 45 : 407-409. Mikanová, O., Kubát, J.[2006]. Phosphorus Solubilizing Microorganisms and their Role in Plant Growth Promotion. Microbial Biotechnology in Agriculture and Aquaculture, Volume II, pp. 556, Science Publishers, New Hampshire, USA, ISBN 1-57808-443-1, 111-145. Mikanova, O., Novakova, J.[2002]. Evaluation of the P-solubilizing activity of soil microorganisms and its sensitivity to soluble phosphate. Rostl. Výr. 48 : 397-400. Mittal, V., Singh, O., Nayyar, H., Kaur, J., Tewari, R.[2008]. Stimulatory effect of phosphatesolubilizing fungal strains [Aspergillus awamori and Penicillium citrinum] on the yield of chickpea [Cicer arietinum L. cv. GPF2]. Soil. Biol. Biochem. 40 : 718-727. Padmavathiamma, P.K., Li, L.Y., Kumari, U.R. [2008]. An experimental study of vermibiowaste composting for agricultural soil improvement. Biores. Technol. 99 : 1672–1681. Pérez, E., Sulbarán, M., Ball, M.M., Yarzábal, L.A.[2007]. Isolation and characterization of mineral phosphate-solubilizing bacteria naturally colonizing a limonitic crust in the south-eastern Venezuelan region. Soil. Biol. Biochem. 39 : 2905–2914. Rodrígues, H., Fraga, R.[1999]. Phosphate solubilizing bacteria and their role in plant growth promotion. Biol. Advanc. 17 : 319-339. Salih, H.M., Yahya, A.I., Abdul-Rahem, A.M. and Munam, B.H.[1989]. Availability of phosphorus in a calcareous soil treated with rock phosphate or superphosphate as affected by phosphate-dissolving fungi. Plant and Soil 12: 181-185. Shigaki, F., Sharpley, A.N., Prochnow, L.I.[ 2006]. Animal-based agriculture, phosphorus and management and water quality in Brazil: options for the future. Sci. Agric. 63 : 194209. Sperber, J.I.[1958]. Solution of apatite by soil microorganisms producing organic acids. Aust. J. Agric. Res. 9 : 782-787. Taha, S.M., Mahmoud, S.A.Z., Halim El-Damaty, A., Abd El-Hafez, A.M.[1969]. Activity of phosphate dissolving bacteria in egyptian soils. Plant Soil 31 : 149-160. Tao, G.C., Tian, S.J., Cai, M.Y., Xie, G.H.[2008]. Phosphate-solubilizing and –mineralizing abilities of bacteria isolated from soils. Pedosphere. 18[4] : 515-523. Thomson, B.D., Robson, A.D. and Abbot, L.K.[1986]. Effects of phosphorus on the formation of mycorrhizas by Gigaspora calospora and Glomus fasciculatum in relation to root carbohydrates. New Phytol. 103 : 751-765. Venkateswarlu, B., Rao, A.V. and Raina, P.[1984]. Evaluation of phosphorus solubilisation by microorganisms isolated from aridisols. J. Indian Soc. Soil. Sci. 32 : 273-277. Vessey, J.K.[2003]. Plant growth promoting rhizobacteria as biofertilizers. Plant Soil. 255 : 571-586.

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Viveganandan, G. and Jauhri, K.S.[2002]. Efficacy of rock phosphate based soil implant formulation of phosphabacteria in soybean. Indian J. Biotechnol. 1:180-187. Wani, S.P., Lee, K.K.[1995]. Role of microorganisms in sustainable agriculture. Proc. Seminar on National Resource Management. Hisar. pp. 62-88. Whitelaw, M.A., Harden, T.J., Helyar, K.R.[1999]. Phosphate solubilisation in solution culture by the soil fungus Penicillium radicum. Soil Biol. Biochem. 31 : 655-666. Whitelaw, M.A.[2000]. Growth promotion of plants inoculated with phosphate-solubilizing fungi. Adv. Agron. 69 : 99-151. Wu, S.C., Cao, Z.H., Li, Z.G., Cheung, K.C., Wong, M.H. [2005]. Effect of biofertilizer containing N-fixer, P and K solubilizers and AM fungi on maize growth: a greenhouse trial. Geoderma. 125 : 155-166. Xiao, C.Q., Chi, R.A., Huang, X.H., Zhang, W.X., Qiu, G.Z., Wang, D.Z.[2008]. Optimization for rock phosphate solubilization by phosphate-solubilizing fungi isolated from phosphate mines. Ecol. Eng. 33 : 187-193. Yahya, A.I. and Al-Azawi, S.K.[1989]. Occurrence of phosphate-solubilizing bacteria in some Iraqi soils. Plant and Soil. 117 : 135-141. Zaidi, A., Khan, M.S., Amil, M.[2003]. Interactive effect of rhizotrophic microorganisms on yield and nutrient uptake of chickpea [Cicer arietinum L.]. Europ. J. Agronomy. 19 : 1521.

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In: Phosphate Solubilising Microbes for Crop Improvement ISBN 978-1-60876-112-8 Editor: M. S. Khan and A. Zaidi © 2009 Nova Science Publishers, Inc.

Chapter 7

PHOSPHATE-SOLUBILIZATION BY PSYCHROPHILIC AND PSYCHROTOLERANT MICROORGANISMS: AN ASSET FOR SUSTAINABLE AGRICULTURE AT LOW TEMPERATURES Harshita Negi∗, Kuheli Das, Anil Kapri and Reeta Goel Department of Microbiology, College of Basic Sciences and Humanities, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar-263145, India

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ABSTRACT Low temperature habitats are colonized predominantly by psychrophiles and psychrotolerant microorganisms, which posses several adaptive characteristics to overcome such adverse conditions. These predominantly include production of antifreeze proteins and expression of various stress induced genes. However, for higher order organisms like plants, survival and sustenance under cold conditions needs to be backedup by additional factors like plant-growth promoting rhizo-microorganisms. In this direction, the development of efficient phosphate-solubilizers has been carried through mutagenic approaches. These mutant strains have shown higher P-solubilizing efficiency than their respective wild types, under both in-vitro and in-situ conditions. Pseudomonas fluorescens mutants [e.g., CRPF8 and CRM] have shown a significant increase in growth parameters of both leguminous and non-leguminous cultivars, in the presence of tricalcium phosphate as the sole ‘P’ source. These strains are not only competitive survivors at 100C, but are also good rhizosphere colonizers, which can be used as an alternative to phosphatic fertilizers for improving crop productivity under stressed environment. This chapter focuses on the exploitation of P-solubilizing potential of psychrophiles and psychrotolerant microorganisms.

∗

Correspondence to: E-mail: [email protected]

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7.1. INTRODUCTION Phosphorus [P] is second only to nitrogen [N] as the most limiting element needed by the plants. Phosphorus, classified as a macronutrient, is a component of certain enzymes and proteins, adenosine diphosphate [ADP], adenosine triphosphate [ATP], ribonucleic acids [RNA], deoxyribonucleic acids [DNA] and phytin. Consequently, plants cannot grow without a reliable supply of this nutrient. It is also involved in controlling key enzyme reactions and in the regulation of metabolic pathways [Theodorou and Plaxton, 1993; Srivastava, 2004]. The concentrations of soluble P in soil vary from 0.05 to 10 ppm. Moreover, a major portion [more than 80%] of the phosphatic fertilizers added to soil becomes immobile and unavailable for plant uptake because of adsorption, precipitation or conversion to insoluble, fixed organic form. It is generally fixed as tri-calcium phosphate, Ca3 [PO4]2 in alkaline soils and as ferric phosphate [FePO4] and aluminium phosphate [AlPO4] in acidic soils, which therefore needs to be solubilized, where phosphate-solubilizing microorganisms [PSMs] play an important role [Kucey et al., 1988]. Phosphate-solubilizing microorganisms include various bacteria [e.g., Bacillus megaterium, B. circulans, B. subtilis, Pseudomonas striata, P.fluorescence], fungi like, Aspergillus awamori, A. niger, Penicillium sp., Trichoderma harzianum, T. viride and yeasts like, Saccharomyces cerevisiae and Schwanniomyces occidentalis which are capable of solubilizing fixed phosphates in soil [Hedge, 1998]. The PSMs are more concentrated in the rhizospheric region of plants than in bulk soil [Gaur, 1990; Vesquez et al., 2000]. Many soil and rhizospheric microorganisms have this ability to release phosphate from sparingly soluble mineral phosphates found in soils and are important in facilitating soil phosphate to plants. However, their performance is directly influenced by environmental factors especially under stress conditions [Pal, 2001]. Temperature is one of the important factors that immediately affect the interior of the cell. Bacteria not only respond to elevated temperatures [heat shock], but also at downshifted temperatures [cold shock] by synthesizing a group of heat and cold shock proteins, respectively. These proteins are important for the survival of bacteria at higher or lower temperatures [Jones et al., 1987; Yura et al., 1993]. Temperature has profound effects on cellular process in all organisms. Due to their high surface to volume ratio, prokaryotes have to stringently cope with temperature changes and adapt to a given temperature regime, allowing them to be classified as thermophilic, mesophilic and psychrophilic microorganisms [Smirnova et al., 2001]. It has been reported that temperature has perplexing effects onto the P-solubilizing abilities of naturally occurring psychrotolerant strains of Pseudomonas fragi [Selvakumar et al., 2009].

7.2. PSYCHROPHILIC AND PSYCHROTOLERANT MICROORGANISMS The ability of bacteria to grow and reproduce at 00C was first described by Forster in 1887. A few years later, Schmidt-Nielsen [1902] for the first time used the term ‘psychrophilic’ to define this bacterial type. As only the minimum growth temperature was considered in this early definition, some confusion arose due to the lack of differentiation between ’cold-loving’ and ‘cold-tolerant’ adaptation types. However, with the isolation of

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true psychrophiles in the 1960s, Morita ended the confusion with a new definition. He coined the term ‘psychrophilic’ for those microorganisms whose cardinal growth temperatures [minimum, optimum, and maximum] are at or below 0, 15 and 20°C, respectively. The microorganisms which had their optima from 20 to 40oC, but could grow at 0oC were defined as psychrotolerants or psychrotrophs. Predominant microorganisms found in such habitats are Acinetobacter, Aeromonas, Alcaligens, Bacillus, Chromobacterium, Clostridium, Corynebacterium, Flavobacterium, Pseudomonas, Vibrio and some yeast [Herbert, 1986]. Psychrophilic organisms exhibit a wide range of adaptations like incorporation of specific lipid constituents into their cytoplasmic membranes to maintain fluidity and critical ability to transport substrates and nutrients under chilling temperatures [Allen et al., 2001]. Further, production of antifreeze proteins [AFPs] and accumulation of compounds that inhibit icecrystallization are other means of adaptation by bacteria [Mazur, 1966; Gilbert et al., 2004]. AFPs are structurally diverse group of protein with the ability to modify ice crystal structure [Urrutia et al., 1992] and inhibit re-crystallization of ice by adsorbing onto the surface of ice crystals via vander Waals interactions and/or hydrogen bonds [Devries, 1986; Ewart et al., 1999; Krembs and Engel, 2001]. During cold acclimation, many psychrophiles are known to accumulate antifreeze proteins [Griffith et al., 1995; Duman and Olsen, 1993; Griffith and Ewart et al., 1999; Khan and Goel, 2008]. The plant growth promoting rhizobacterium Pseudomonas putida GR12-2 was isolated from the rhizosphere of plants growing in the Canadian high arctic [Sun et al., 1995; Xu et al., 1998]. This bacterium was able to grow and promote root-elongation of spring and winter canola [Brassica napus L] at 50C, a temperature at which only a relatively small number of bacteria were able to proliferate and function. Moreover, the bacterium survived freezing temperature ranging from -200C to -500C and could secrete an antifreeze protein into the growth medium at 50C. Katiyar and Goel [2004] also reported the presence of antifreeze proteins in a cold tolerant mutant of P. fluorescence.

7.3. LIMITATION IN SUSTAINABLE AGRICULTURE AT LOW TEMPERATURE Phosphorus is by far the least mobile and available to plants in comparison to all other major nutrients in most soil conditions especially in arid soil. Although phosphorus is abundant in soils in both organic and inorganic forms, it is frequently a major or sometimes the prime limiting factor for plant growth. The bioavailability of soil inorganic phosphorus in the rhizosphere varies considerably with plant species, nutritional status of soil and ambient soil conditions. In India, the agricultural production has intensified over the past few decades; producers have become more dependent on agrochemicals for plant disease management. Furthermore, the growing costs of agro-chemicals, particularly in the less affluent regions of the world, and consumer demand for pesticide-free foods have prompted the search for viable biological alternatives. Moreover, the soil of Indian Himalayan region [IHR] is generally acidic in reaction, low in moisture and organic matter. These soil constraints often limit crop productivity at high altitude conditions that exhibit chilling temperatures. As phosphorus availability plays a very crucial role in crop production, water soluble phosphorus [WSP] applied in acid soils is rapidly fixed to unavailable forms. To circumvent phosphorus deficiency, PSMs could play an important role in supplying phosphate to plants

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in a more environmentally-pleasant and sustainable manner. However, the variability in the performance of PSM has dreadfully restricted the large-scale application of PSMs in sustainable agriculture. Despite the divergence in their performance, PSMs are widely and frequently applied in agronomic practices in order to optimize the productivity of crops while maintaining the health of soils [Khan et al., 2007].

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7.4. PHOSPHORUS AND SOIL FERTILITY Phosphorus is one of the essential plant nutrients constituting about 0.2% of the total plant biomass. Soils usually contain 1.32 to 15.5 x10-2 % ‘P’ and insoluble phosphate that is not directly available to plants and/or micro-organisms [Hayman 1975]. Several groups have described the relationship between total organic P content and other soil properties such as, organic matter content, N and pH. Rock phosphate [RP] is a good source of P for acid soils, but ineffective in neutral to alkaline soils [Awasthi et al., 1977]. Though, the average content of organic phosphorus in soils ranges from 5 to 50% of total P [Harrison, 1987], the forest soils have a higher organic P content than arable or cultivated soils. The availability of this nutrient for plants is limited by different chemical reactions especially in arid and semi-arid soils. Phosphorus plays a significant role in several physiological and biochemical plant activities like photosynthesis, transformation of sugar to starch, and transportation of the genetic traits. A large reservoir of organic phosphorus exists in forms, which are unavailable to plants. Precipitation or fixation to insoluble complex minerals is due to the union of phosphorus with elements such as iron and aluminium in acidic soils, and calcium in alkaline soils, denying the plant upto 75% of all soluble P [Goldstein, 1966; Kucey et al., 1989] thereby generating a 0.002-0.5% concentration of the mineral in the soil [Chabot et al., 1993]. This has forced many crop raisers to apply upto four times the required amount of phosphorous to plants. As an example, sugarcane [Saccharum officinarum] requires between 45 and 200 kg of phosphorous per hectare. This procedure generates an increase in the application of chemical fertilizers and therefore, a hike in the production cost. Nevertheless, the production and simultaneous application of bio-preparations especially the PSM could improve the availability of soluble phosphorus, which in turn, may lead to a substantial decline in the use of phosphate fertilizers and thus, have a positive effect on the environment besides the cost factor. Low organic matter coupled with low native soil P concentrations is a major constraint limiting crop productivity, particularly in case of soybean-wheat system on vertisols in the Indian semi-arid tropics. Phosphorus promotes N2 fixation in legume crops and is vital for photosynthesis, energy transfer and formation of sugars [Gaur, 1990]. Legumes need high amount of P in readily available form around their roots for rhizobia and the host plant. Only a small fraction of phosphate fertilizer is utilized by crops, while remaining portion of applied P gets fixed in the soil or rendered unavailable to crop plants. Continuous efforts are therefore, made to increase the efficiency of soil [with pH more than 7] by adding P-solubilizing microorganisms.

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7.5. PHOSPHATE-SOLUBILIZING ACTIVITIES AT LOW AND AMBIENT TEMPERATURES As described earlier, P is a macronutrient and essential for optimum plant growth. Plants require higher amount of P during the young stage both in autumn and spring in the zone of moderate climate. Therefore, P-solubilization both at moderate and low temperature is extremely important. For example, Gaind and Gaur [1991] reported thermotolerant phosphate-solubilizing micro-organisms able to solubilize P at 450C and assessed their impact on the performance of mung bean [Vigna radiata [L.] wilczek] cultivars. Seed inoculation of mung bean showed a better establishment of temperature tolerant strains as revealed by the rhizosphere population. The inoculation improved nodulation, the available P2O5 content of the alluvial soil, root and shoot biomass, straw and grain yield and phosphorus and nitrogen uptake of the crop. Different methods to solubilize phosphate for increasing its availability by biologically mediated processes such as mineralization and immobilization by PSMs have been reviewed earlier [Illmer and Schimmer, 1995]. High arctic and low temperature habitats require strains with greater adaptability to low temperature. As psychrophilic and psychrotolerant microorganisms widely occur in natural and artificial environments, such as in cold rooms and refrigerated transport systems, they take part in natural turnover of a variety of organic and inorganic compounds under cold conditions [Maresin and Schinner, 1999]. Although, reports indicating functionality of PGPR strains at low temperatures are available [Sun et al., 1995; Mishra and Goel, 1999; Saxena et al., 1999], studies on P solubilizing activity of microbes at low temperature [100C] are scarce. However, Das et al. [2003] documented the potentiality of fluorescent pseudomonas in plant growth promotion at low temperature conditions. They conducted comparative P solubilization activity of three mesophilic strains of Pseudomonas fluorescens and their cold tolerant mutants under in-vitro conditions, using NBRIP broth at 100C and 250C. The cold tolerant mutant of the Pseudomonas fluorescens strain GRS1 showed significantly high P solubilization both at 100C and 250C, followed by mutant of ATCC13525 at 250C and PRS9 at 100C [Table 1]. Table 1. Phosphate solubilizing potentials of cold tolerant mutants of P. fluorescens strains grown in Pikovskya’s medium supplemented with tri-calcium phosphate P. fluorescens strains a GRS1 CRPF1 CRPF2 CRPF3 CRPF4 CRM

Days of incubation at 100C 250C 4 3 7

7

6

4

6

7

6

7

4

4

P solubilized [μg/ml] at 100C 250C 1.65 14.95 3.81 3.81 [+2.6] [+2.3] 10.74 17.74 [+10.6] [+16.2] 1.77 9.99 [+1.6] [+8.4] 17.38 17.82 [+17.2] [+16.3] 19.40 23.99 [+19.3] [+22.5]

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Days of incubation at 0

0

10 C

25 C

3

6

7

6

4

6

4

3

a

CD at 5% ATCC13525

6

5

CRPF8

5

6

CRPF9

5

5

CD at 5% a PRS9 CRPF5 CRPF6 CRPF7

CD at 5%

P solubilized [μg/ml] at 0

10 C 1.89 31.00 1.90 [+9.7] 10.04 [+9.7] 5.890 [+2.790] 2.19 147.84 61.302 [-113.5] 60.124 [-114.7] 4.99

250C 4.91 184.60 13.23 [-5.5] 11.05 [-7.7] 3.80 [-15.0] 2.47 155.00 255.208 [+100.2] 131.812 [-23.2] 1.65

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Each value is a mean of three replicates; Values in parentheses indicate percent increase/decrease in P solubilized over wild type; a represents wild type.

There are several reports indicating that soil inoculation with phosphate-solubilizing bacteria [PSB] improves solubilization of fixed soil P and externally applied P, thereby enhancing crop yields. The availability coefficient ratio, relative agronomic efficiency and relative economic efficiency of RP have been also improved due to bacterization as compared to the RP alone [Dubey, 2001]. The continuous turn over of enriched phosphocompost containing aqueous slurry, RP and P-solubilizing micro-organisms increased dry matter accumulation in plants, seed yield and P uptake by soybean [Glycine max] relative to chemical fertilizer [Manna et al., 2001]. Further, Katiyar and Goel [2003] have documented P solubilization potential at 25oC and observed their in situ impact at 250C in green house. Later, they documented the siderophore hyper-producing [17 fold] mutant, of fluorescent Pseudomonads. The mutant [PRS9] promoted growth of mung bean var. PM-4 both at 25 and 100C [2004].

7.6. PSMS FOR SUSTAINABLE AGRICULTURE AT LOW TEMPERATURES Phosphate solubilizing microorganisms inhabit majority of soils, release P from insoluble sources by different mechanisms [Venkateswarlu et al., 1984; Kumar et al., 1999; Nautiyal et al., 2000] and hence, are considered among the most effective plant assistants to supply P at a favorable level. Microbes expressing highest P-solubilizing efficiency are selected and used to develop bio-fertilizers for their ultimate use to enhance plant growth by providing P in a readily absorbable form at ambient and low temperatures. Among the soil bacterial

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communities, Pseudomonas striata, Bacillus sircalmous and enterobacters are considered as the potential P- solubilizers. Application of these bio-inoculants enhance an abundant population of active and effective microorganisms to the root activity zone which thereby increases the ability to uptake more nutrients by plants. In particular, Pseudomonas fluorescens is considered as an important member of rhizosphere colonizing community. Phosphate solubilizing bacteria like Malic sp. and Socsinic sp. are able to affect the solubility of low dissolvable inorganic P compounds by secreting different organic acids. The Pseudomonas sp. has a considerable P uptake efficiency. Due to the ecotype diversity of this species and its tolerance under various environmental stresses, this bacterium is of special importance as a biological fertilizer. In order to harness the potential benefits of biofertilizers in commercial agriculture, the consistency of their performance must be improved at both ambient as well as low temperature [Katiyar and Goel, 2003]. In temperate regions, effectiveness of micro-organisms is reduced in degrading organic pollutants such as oils, lipids and xenobiotics such as, phenolic and non-phenolic aromatic compounds due to seasonal variations in temperature which makes the agricultural lands infertile. However, bioagumentation and inoculation of contaminated environment with specific cold adapted micro-organisms in mixed culture can help the biodegradation of recalcitrants [transformation or mineralization] due to high specificity at low temperature [Maresin and Schinner, 1999]. Further, phosphate solubilization and growth promotion by psychrotolerant bacteria namely, Serratia marcescens strain SRM, Pantoea dispersa strain 1A and Pseudomonas fragi CS11RH1, isolated from a high altitude garlic [Allium sativum] rhizosphere of the Indian Himalayan region has been reported [Selvakumar et al., in 2007, 2008 and 2009]. These isolates grew and solubilized P at temperatures ranging from 4 to 30°C. Besides solubilizing P they produced indole acetic acid [IAA] and hydrogen cyanide [HCN]. Seed bacterization with the isolate significantly increased the percent germination, rate of germination, plant biomass and nutrient uptake of wheat [Triticum aestivum] seedlings.

7.7. FIRST REPORTED PSYCHROTOLERANT MUTANTS FOR HIGHER ALTITUDES To enhance the growth of agriculturally important bacterial strains at low temperature, cold resistant mutants of Pseudomonas fluorescens were developed using N-methyl 1- N nitro N-nitrosoguanidine [MNNG] [Mishra and Goel, 1999]. Efforts to produce inoculants from Pseudomonas and to use it under low temperature conditions, is a potentially useful and environmental friendly way to help plant growth and development through the enhancement of this natural phenomenon. Cold resistant mutants of plant growth promoting bacterium Pseudomonas fluorescens GRS1 developed by MNNG treatment were able to grow and promote root and shoot elongation of wheat both at 250C and 100C, a temperature at which wild type was unable to proliferate and function. Cold tolerant mutants of P. fluorescens strains GRS1, PRS9 and ATCC13525 were screened for P-solubilizing activity under in vitro conditions and plant growth promoting potentials [Das, 2004]. Mung bean var. PM-2 was used as test crop for in situ studies as it is an important leguminous plant and responds better in terms of aerial growth. Under in vitro conditions, cold tolerant mutants CRPF8 and CRM were found as efficient plant growth promoters as compared to their wild type strains at 250C

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and 100C, respectively. Moreover, in situ studies of cold tolerant mutant CRPF8 at 280C with Phaseolus vulgaris var. PM-2 in sand culture with different P sources have shown a significant increase in root and shoot length along with biomass of the plants after 30 days of sowing with insoluble P source over negative control having no P source [Table 2]. Table 2. Effect of insoluble P [Ca3[PO4]2] and soluble P [KH2PO4] on growth parameters of mung bean [var. PM-2] plants bacterized with Pseudomonas fluorescens ATCC13525 and its cold tolerant mutant CRPF8 at 30 days after germination P source

Strains Control

Insoluble P

ATCC13525 CRPF8 Control

Soluble P

ATCC13525 CRPF8 Control ATCC13525

No P CRPF8

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CD at 5%

Root length [cm] 6.9±0.32 8.1±0.26 [+18.1] 7.7±0.44 [+12.5] 8.3±0.41 8.5±0.64 [+2.4] 8.9±0.31 [+6.9] 5.0±0.19 6.7±0.77 [+34.0] 7.3±0.25 [+45.5] 1.126

Growth parameters Shoot length Fresh weight [cm] [g/plant] 21.0±1.38 2.2±0.11 22.0±0.81 3.1±0.13 [+4.6] [+38.6] 22.7±0.65 3.6±0.26 [+8.5] [+64.1] 24.5±0.82 2.9±0.13 25.5±0.89 3.3±0.19 [+4.3] [+11.2] 28.1±0.29 4.0±0.04 [+14.9] [+35.0] 17.9±0.32 1.7±0.20 20.4±0.35 2.5±0.17 [+14.1] [+47.7] 22.6±1.02 2.8±0.14 [+25.9] [+60.5] 2.054 0.506

Dry weight [g/plant] 0.44±0.00 0.61±0.01 [+39.4] 0.72±0.00 [+64.0] 0.59±0.00 0.65±0.00 [+11.2] 0.79±0.00 [+35.1] 0.34±0.00 0.51±0.02 0.55±0.00 [+61.0] 0.201

Each value is a mean of 10 independent replicates; ± represents standard deviation; Values in parentheses indicates % increase over control.

Further, comparable results were obtained with reference to positive control having soluble P source. Inoculation with mutant strains CRPF8 also resulted in enhanced chlorophyll content of the plants over wild type and control [having non-bacterized seeds], when different sources of P as tri-calcium phosphate [1.715 μg/gm fresh weight], KH2PO4 [2.019 μg/gm fresh weight] and no P [1.639 μg/gm fresh weight] were used [Figure 1]. Moreover, at 100C, CRM [a cold tolerant mutant of Pseudomonas fluorescens, GRS1] showed better plant growth promotion when soil was treated with tri-calcium phosphate as a source of P over wild type mesophilic strain GRS1 and control [non-bacterized seeds]. In the presence of CRM, all the measured agronomical parameters were comparable to positive control [P in soluble form] but were greater than negative control [without P]. The CRM inoculation increased the shoot length, root length, fresh weight and dry weight by 0.33 fold, 1.13 fold, 67.47% and 6.09%, respectively, over control [Table 3]. The chlorophyll content was also significantly increased in the presence of CRM at 100C in comparison to GRS1 when different sources of P as TCP [1.3257 μg/gm fresh weight], KH2PO4 [1.4463 μg/gm fresh weight] and no P source [1.2435 μg/gm fresh wt.] were used [Figure 2].

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Figure 1. Effect of different P sources on chlorophyll content of mung bean [var. PM-2] treated with normal nutrient solution supplemented with insoluble P, Ca3[PO4]2, soluble P [KH2PO4] and no P using Pseudomonas fluorescence ATCC 13525 and its cold tolerant mutant CRPF8 at 280C. Lines above bars indicate standard errors of mean.

Figure 2. Effect of different P sources on chlorophyll content of mung bean [var. PM-2] treated with normal nutrient solution supplemented with insoluble P, Ca3[PO4]2, soluble P [KH2PO4] and no P with Pseudomonas fluorescence GRS1 and its cold tolerant mutant CRM at 100C. Lines above bars indicate standard errors of mean.

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Table 3. Effect of insoluble P [Ca3 [PO4]2] and soluble P [KH2PO4] on growth parameters of mung bean [var. PM-2] plants bacterized with Pseudomonas fluorescens GRS1 and its cold tolerant mutant CRM at 30 days after germination P source

Strains Control

Insoluble P

GRS1 CRM Control

Soluble P

GRS1 CRM Control

No ‘P’

GRS1 CRM

CD at 5%

Root length [cm] 5.2±0.12 9.4±0.13 [+81.1] 11.1±0.31 [+113.3] 7.3±0.31 10.0±0.32 [+37.9] 11.9±0.24 [+63.7] 5.0±0.07 8.6±0.14 [+70.0] 10.9±0.29 [117.3] 0.577

Growth parameters Shoot length Fresh weight [cm] [g/plant] 8.6±0.64 1.7± 1.65 10.6±0.36 2.04±0.05 [+23.3] [+22.9] 11.4±0.78 2.8±0.10 [+32.9] [+67.5] 8.7±0.52 1.8± 0.05 10.8±1.01 2.3±0.10 [+24.8] [+27.7] 11.5±0.75 2.8±0.11 [+32.87] [+57.1] 8.3±0.85 1.6±0.03 8.8±0.48 1.9±0.20 [+5.04] [+19.4] 11.3±0.69 2.6±0.03 [36.0] [+65.0] 1.823 0.272

Dry weight [g/plant] 0.34± 0.10 0.43±0.07 [+27.2] 0.56±0.00 [+65.1] 0.35±0.10 0.45±0.07 [+28.7] 0.56± 0.00 [+59.4] 0.32± 0.00 0.37±0.00 [+16.1] 0.51± 0.05 [+59.6] 0.109

Each value is a mean of 10 independent replicates; ± represents standard deviation; Values in parentheses indicates % increase over control.

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Table 4. Effect of Psudomonase fluorescens ATCC13525 and its cold tolerant mutant CRPF8 on P content and yields of Phaseolus vulgaris [var. PM-2] grown in soil treated with different concentrations of chemical fertilizers

Treatment

P content at harvest [mg/g plant ]

% increase and decrease in P content

Yield [q/ha]

% increase and decrease in yield

0% CF + ATCC13525 100% CF + ATCC13525 0% CF + CRPF8 100% CF + CRPF8 50% CF + CRPF8 35% CF + CRPF8

1.49 2.98 2.74 4.75 4.86 4.75

-50.16 -0.33 -8.36 +55.86 +62.54 +58.86

4.41 5.69 4.44 5.95 7.26 6.96

-11.8 +13.8 -11.2 +19.00 +45.2 +39.2

Each value is a mean of four replicates; CF indicates chemical fertilizer; bacterial load on each seed was 1.9×108 cells.

Furthermore, in a study, field trials were conducted to evaluate P economy in the presence of phosphatic fertilizers maintaining the concentration of bacterial P fertilizers [P.F] constant [1.9×108 cfu/seed] for each treatment and varying the dose of chemical fertilizer [C.F.] from 0, 100, 50 and 35%, respectively using Phaseolus vulgaris var. PM-2 as test plant [Das, 2004]. A significant increase in root length was found in plots where CPPF8 was used as a phosphatic fertilizer unlikely to the plots where ATCC13525 was used as a positive control. All the CRPF8 treated plots showed significant increase in root and shoot length as

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compared to control crop having 100% C.F. and 0% P.F. [Plate 1a]. Treatments having 0% C.F. and ATCC13525 or CRPF8 as P.F. showed minimum root lengths [Plate 1b and c]. Shoot length exhibited a significant increase in the treatments with C.F. in combination with the bioinoculants CRPF8 as in comparison to ATCC13525. Further, the influence was considerably higher with 50% of C.F. and decreased with increasing concentration [100%]. Plots treated with 50% C.F. and CRPF8 as P.F. showed maximum percentage increase in nodule number over control crop having 100% C.F. and 0% P.F [Plate 1d]. The P content in plants at harvest demonstrated a significant increase in the treatment having different concentrations of C.F. and CRPF8 as a P.F. [4.75 – 4.86 mg/g plant] in comparison to the treatments having ATCC13525 as the a phosphatic fertilizer. While comparing the percent increase and decrease in P content of the test plant grown under identical environmental conditions and within the same calendar year, a maximum increase in P content and yield of crop was found in the treatment having 50% C.F. and CRPF8 as P.F. followed by the treatments having CRPF8 as a P.F. with 100% C.F. and 35% C.F., respectively [Table 4].

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7.8. ECONOMY PERSPECTIVE OF BACTERIAL PHOSPHATIC FERTILIZER Economy of crop is calculated in terms of the cost benefit ratio and harvest index. A significant increase in net profit [NP] of mung bean [var. PM-2] was found in treatments having 50% CF and CRPF8 used as a bacterial P fertilizer [Rs. 5327= approx.105 US dollar], followed by treatment having 35% CF and CRPF8 [Rs. 4880= approx. 96 US dollar] in comparison to crop grown under identical physiological conditions in the same calendar year [NP=Rs. 1498] [Table 5]. Cost Benefit Percentage also showed a significant increase in the treatment having 50% CF and CRPF8 in comparison to normal crop. Further, harvest index percent was also maximum for the former treatment. It was concluded from this study that cold tolerant mutant CRPF8 is a good phosphatic fertilizer in ambient temperature as compared to its wild type strain ATCC 13525. Table 5. Effect of Ps. fluorescens ATCC13525 and its cold tolerant mutant CRPF8 on cost economy of mung bean [var. PM-2] crop grown with different percentage of chemical fertilizers

Treatment

Production cost [Rs]

Return [Rs.]

Net Profit [Rs.]

0% CF+ATCC13525 100% CF+ ATCC13525 0% CF+ CRPF8 100% CF +CRPF8 50% CF +CRPF8 35% CF+ CRPF8 Control

8685.00 9702.00 8685.00 9702.00 9193.00 9040.00 9702.00

8820.00 11380.0 8880.00 11900.00 14520.00 13920.00 11200.00

135 1678 195 2198 5327 4880 1498

Cost Benefit percent 1.55 17.29 2.24 22.65 57.95 53.98 15.44

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Harvest Index [%] 3.068 3.574 2.936 3.661 4.714 4.490

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Moreover, both worked as good phosphatic fertilizers in combination with chemical fertilizer, when compared to a control crop having no phosphatic fertilizers and 100% chemical fertilizer [Table 5]. An increase in available P to plants through the inoculation of phosphate-solubilizing micro-organisms has been reported several times in pot experiments and under field conditions [Chabot et al., 1996]. As the nutrient availability of soil increases with the use of phosphatic fertilizers which in turn help the nutrient uptake by plants. Hence, in regard to the cold agro-ecosystems, it is suggested that a cold tolerant mutant strain CRPF8 could be developed as a phosphatic fertilizer which in combination with 50% chemical fertilizer may reduce the chemical inputs and hence, the cost of crop production, besides protecting the environment from hazards of chemical fertilizer.

Plate 1. Morphological and symbiotic differences in Phaseolus vulgaris var. PM-2 grown in soil treated with different concentrations of chemical fertilizer [C.F.] wherein bacterial phosphatic fertilizer [P.F.] was constant for all treatments.

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7.9. FUTURE PROSPECTS Psychrophilic P-solubilizers like, Pseudomonas fluorescence, P. putida, P. striata, Acinetobacter, Bacillus sircalmous, etc. has immense potential for increasing soil fertility. Production of crops like, wheat, rice, millets, maize, barley, potato and legumes, which can grow at higher altitude, can be increased using these strains. This would also bring a dramatic decrease in the usage of chemical fertilizers, if not completely and ultimately the cost of production could be lowered for marginal farmers. Further, it would prevent acidification of soil and lead to a sustainable crop-production, whereby the soil-fertility is not at stake. Moreover, PSMs boast of additional plant growth promoting properties like, biocontrol action against plant pathogens and production of siderophores, IAA, phytohormones, HCN, ACC deaminase, etc., which could indirectly improve crop-production.

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CONCLUSION Plants require the greater concentration of P at the young stage, therefore, the Psolubilization by microbes and consequent uptake of P by plants growing at moderate and low soil temperature becomes extremely important. The development of physiologically efficient cold-tolerant strains, especially Pseudomonas fluorescence through mutagenesis [GRS1 and PRS1] has paved way for resultant mutants with greater adaptability and rhizocompetence, which co-incidentally harbor tremendous P-solubilization potential. This could be an important leap towards commercialization of bacterial strains as phosphatic fertilizers to improve crop-productivity at lower temperatures [100C]. These rhizosphere competent microorganisms brings significant increase in plant growth and symbiosis in the presence of insoluble tricalcium phosphate and rock phosphate as the sole P-source in soil through organic acid production, formation of oxo-acids from sugars, ion-chelation and exchange reactions in the growth environment. The application of these bio-inoculants can be considered as an appropriate substitute for chemical fertilizers in sustainable agricultural systems because psychrophiles are not only confined to permanently cold environments but also live and develop in temperate habitats. Moreover, the ability of cold tolerant P solubilizers to function efficiently in combination with chemical fertilizers may be exploited further to improve crop productivity vis-à-vis reduce fertilizer application in cold regions.

REFERENCES Allen, D., Huston, A.L., Wells, L.E. and Deming, J. W. [2001]. Biotechnological use of psychrophiles. In G. Bitton, [Ed.], Encyclopedia of environmental microbiology [pp. 117]. New York, John Wiley and Sons. Awasthi, P.K., Luthra, K.L. and Jaggi, J.N. [1977]. Use of Indian rock phosphates for direct application as phosphorus fertilizers. Fertilizers News, 12, 44. Chabot, R., Antoun, H. and Cescas, M.C. [1996]. Growth promotion of maize and lettuce by phosphate solubilizing Rhizobium leguminosarum biovar phaseoli. Plant and Soil, 184, 311-321.

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Harshita Negi, Kuheli Das, Anil Kapri et al.

Chabot, R.A., Hani and Cescas, M.M. [1993]. Stimulation do la croissance deimais et de la latitude romaine par des microorganisms dissolvant de phosphore inorganique. Can. J. Microbiol., 39, 941-947. Das, K. [2004]. P solubilization potential of plant growth promoting Pseudomonas mutants at low and ambient temperature. P.hD. Thesis. Pantnagar, India. G.B.P.U. of Ag. and Tech. Das, K., Katiyar, V. and Goel, R. [2003]. P solubilization potential of plant growth promoting Pseudomonas mutants at low temperature. Microbiol. Res., 158, 359-362. DeVries, A.L. [1986]. Antifreeze glycopeptides and peptides: Interactions with ice and water. In L. Packer [Ed.], Methods in Enzymology [vol. 127, pp. 293-303]. New York, Academic Press. Dubey, S.K. [2001]. Biomass and nitrogen accumulation pattern in soybean [Glycine max] as influenced by phosphate solubilizing bacteria in vertisols. Indian Journal of Plant Physiolog., 6, 4. Duman, J.G. and Olsen, T.M. [1993]. Thermal hysteresis protein activity in bacteria, fungi and phylogenetically diverse plants. Cryobiology, 30, 322–328. Ewart, K.V., Lin, Q. and Hew, C.L. [1999]. Structure, function and evolution of antifreeze proteins. Cellular and Molecular Life Sciences, 55, 271-283. Forster, J. [1887]. Ueber einige Eigenschaften leuchtender Bakterien.Zentralbl. Bakteriol. Parasitienkd, 2, 337-340. Gaind, S. and Gaur, A.C. [1991]. Thermotolernat phosphate solubilizing microorganisms and their interaction with mung bean. Plant and Soil, 133, 141-149. Gaur, A.C. [1990]. Phosphate solubilizing microorganisms as Biofertilizers. Omega Scientific Publisher. New Delhi, India, p.176. Gilbert, J. A., Hill, P.J., Dodd, C.E. and Laybourn-Parry, J. [2004]. Demonstration of antifreeze protein activity in Antarctic lake bacteria. Microbiology, 150, 171-180. Goldstein, A.H. [1966]. Bacterial solubilization of mineral phosphates: historical perspective and future prospects. Am. J. Alt. Agric, 20, 51-57. Griffith, M. and Ewart, K.V. [1995]. Antifreeze proteins and their potential uses in frozen foods. Biotechnology Advances, 13, 375-402. Griffith, M., Ala, P., Yang, D.S., Hon, W. C. and Moffatt, B.A. [1992]. Plant Physiology, 100, 593-597. Harrison, A.F. [1987]. Soil organic phosphorus. A Review of World Literature. C.A.B. International, Wallingford, U K, pp.257. Hayman, D. S. [1975]. Phosphorus cycling by soil microorganisms and plant roots. In N. Walker, [Ed.], Soil Microbiology[ pp. 67-92]. London, Butterworths. Hedge, N.G. [1998]. Organic farming: A boon for our farmers. Integrated Rural Development for Sustainable Livelihood. BAIF Publication. Herbert, R. [1986]. Psychrophilic organisms. In R. Herbert, and G. Codd [Eds.] Microbes in Extreme Environments [17, 1-23]. London, Academic Press. Illmer, P. and Schinner, F. [1995]. Solubilization of hardly-soluble AlPO4 with P-solubilizing microorganisms. Soil Biol. Biochem., 24, 389-395. Illmer, P. and Schinner, F. [1995]. Solubilization of inorganic calcium phosphates— Solubilization mechanisms. Soil Biol. Biochem., 27, 257-263. Jones, P.G., Van Bogelen, R.A. and Neidhardt, F.C. [1987]. Induction of proteins in response to low temperature in E. coli. J. Bacteriol. 169, 2092- 2095.

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159

Katiyar V. and Goel, R. [2004]. Siderophore mediated plant growth promotion at low temperature by mutant of fluorescent pseudomonads. Plant Growth Regulation. 42, 239244. Katiyar, V. and Goel, R. [2003]. Solubilization of inorganic phosphate and plant growth promotion by cold tolerant mutants of Pseudomonad fluorescens. Microbiol. Res., 158, 163-168. Katiyar, V. and Goel, R. [2004]. Improved plant growth from seed bacterization of a siderophore overproducing cold resistant mutant of fluorescent pseudomonad. J. Microbiol and Biotechnol., 14, 653-657. Khan, M. and Goel R. [2008]. Principles, applications and future aspects of cold-adapted PGPR. In Iqbal Ahmad [Ed.] Pichtel, Hayat. Plant Bacteria Interactions [195-209]. Germany, WILY-VCH Weinhim. Khan, M.S., Zaidi, A. and Wani, P.A. [2007]. Role of phosphate-solubilizing microorganisms in sustainable agriculture-A review. Agron. Sustain. Dev. 27, 29-43. Krembs, C. and Engel, A. [2001]. Abundance and variability of microorganisms and transparent exopolymer particles across the ice water interface of melting first-year sea ice in the Laptev Sea [Arctic]. Marine Biology, 13, 173-185. Kucey, M. N., Janzen, H. H. and Legett, M. E., [1989]. Microbially mediated increases in plant available phosphorus. Adv. Agron., 42, 198-228. Kucey, R.M.N. [1988]. Effect of Penicillium bilaji on the solubility and uptake of P and micronutrients from soil by wheat. Can. J. Soil Sci., 68, 261-270. Kumar, V., Behl, R.K. and Nurula, N. [1999]. Establishment of phosphate solubilizing strains of Azotobacter chroococcum in rhizosphere and their effect on wheat cultivars under green house conditions. Microbiol. Res., 156, 87-93. Manna, M.C., Ghosh, P.K., Ghosh, B.N. and Singh, K.N. [2001]. Comparative effectiveness of phosphate enriched compost and single superphosphate on yield, uptake of nutrients and soil quality under soybean wheat rotation. J. of Agric. Sci., 137, 45-54. Maresin, R. and Schinner, F. [1999]. Biotechnological application of cold adapted organisms. Springer-Verlag. Berlin, Germany. Mazur, P. [1966]. Physical and chemical basis of injury in single-celled micro-organisms subjected to freezing and thawing. In H.T. Merman [Ed.], Cryobiology [pp. 214-315]. New York, Academic Press. Mishra, M. and Goel, R. [1999]. Development of a cold resistant mutant of growth promoting Pseudomonas fluorescens and its functional characterization. J. Biotechnol., 75, 71-75. Morita, R.Y. [1975]. Psychrophilic bacteria. Bacteriol. Rev., 39, 146-167. Nautiyal, C.S., Bhadauria, S., Kumar, P., Lal, H., Mondal, R. and Verma, D. [2000]. Stress induced phosphate solubilization in bacteria isolated from alkaline soils. FEMS Microbiol. Lett., 182, 291-296. Pal, K.K., Tilak, K.V.B.R., Saxena, A.K., Dey, R. and Singh, C.S. [2001]. Antifungal characteristics of a fluorescent Pseudomonas strain involved in the biological control of Rhizoctonia solani. Microbiol. Res., 155, 233-242. Saxena, A. and Goel, R. [1999]. Growth promotory activities of standard and antarctic Pseudomonads. Physiol. Mol. Biol. Plants, 57, 181-184. Schmidt-Nielsen, S. [1902]. Ueber einige psychrophile Mikroorganismen und ihr Vorkommen. Zentr. Bakteriol. Parasitenkd Infektionsk. Hyg. Abt. II., 9, 145-147.

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Selvakumar, G., Kundu, S., Joshi P., Nazim S., Gupta, A. D., Mishra, P. K. and Gupta, H. S. [2008]. Characterization of a cold-tolerant plant growth-promoting bacterium Pantoea dispersa 1A isolated from a sub-alpine soil in the North Western Indian Himalayas. World J Microbiol Biotechnol., 24, 955–960. Selvakumar, G., Mohan, M., Kundu S., Gupta, A.D., Joshi, P., Nazim, S. and Gupta, H.S. [2007]. Cold tolerance and plant growth promotion potential of Serratia marcescens strain SRM [MTCC 8708] isolated from flowers of summer squash [Cucurbita pepo]. Letters in Applied Microbiology, ISSN 0266-8254. Selvakumar, G., Joshi, P., Nazim, S., Mishra, P.K., Bisht, J.K. and Gupta, H.S. [2009]. Phosphate solubilization and growth promotion by Pseudomonas fragi CS11RH1 [MTCC 8984], a psychrotolerant bacterium isolated from a high altitude Himalayan rhizosphere. Biologia, 64, 239-245. Smirnova, A., Li. H. Weingar, H., Aufhammer, S., Burse, A., Fiuis, K., Schenk, A. and Ullrich, M. [2001]. Thermoregulated expression of virulence factors in plant-associated bacteria. Arch. Microbiol., 178, 393-399. Srivastava, S., Yadav, K.S. and Kundu, B.S. [2004]. Prospects of using phosphate solubilizing Pseudomonas as biofungicides. Indian. J. Microbiol., 44, 91-94. Sun, X., Griffith, M., Pasternak, J.J. and Glick, B.R. [1995]. Low temperature growth, freezing survival and production of antifreeze protein by the plant growth promoting rhizobacterium Pseudomonas putida GR12-2. Can. J. Microbiol., 41, 776-784. Theodorou, M.E. and Plaxton, W.C. [1993]. Metabolic adaptations of plant respiration to nutritional phosphate deprivation. Plant Physiol., 101, 339-347. Urrutia, M., Duman, J.G. and Knight, C.A. [1992]. Plant thermal hysteresis proteins. Biochemica et Biophysica Acta, 1121, 199-206. Venkateswarlu, B., Rao, A.V., Raina, P. and Ahmad, N. [1984]. Evaluation of phosphorus solubilization by microorganisms isolated from aridisols. J. Indian Sc. Soil Sci., 32, 273277. Vesquez, P., Holiguin, G., Puente, M.E., Lopez C. A. and Bashan, Y. [2000]. Phosphate solubilizing microorganisms associated with the rhizosphere of mangroves in semi and coastal lagoon. Biol. Fertil. Soils, 30, 460-468. Xu, H., Griffith, M., Pattern, C.L. and Glick, B.R. [1998]. Isolation and characterization of an antifreeze protein with ice nucleation activity from the plant growth promoting rhizobacterium Pseudomonas putida GR12-2. Can. J. Microbiol., 44, 64-73. Yura, T., Nagai, H., and Mori, H. [1993]. Regulation of the heat-shock response in bacteria. Ann. Rev. Microbiol., 47, 321-350.

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Chapter 8

BENEFICIAL MICROBES IN SUSTAINABLE TROPICAL CROP PRODUCTION Zulkifli Haji Shamsuddin∗1, O. Radziah1 and Halimi Mohd. Saud2 1

Department of Land Management Department of Agriculture Technology Faculty of Agriculture, Universiti Putra Malaysia, Serdang, Selangor, Malaysia. 2

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Sustainability of a biological species in a specific environment depends largely on its ability to survive and be competitive to the other species. The review includes discussions on the mutual association between the associative/symbiotic rhizobacteria and the plant systems along with the respective organisms for a sustainable agro-ecosystem. The influence of the highly weathered soils of the humid tropics which often limit plant nutrient availability can be alleviated with a better understanding of these plantrhizobacteria associations. Emphasis is also given to the nitrogen fixing bacteria, phosphate and potassium- solubilizing bacteria, and the beneficial fungi as the major biofertilizers with significant effects on tropical plant growth. These biofertilizers often exhibit bio-enhancing impacts due to the production of plant growth regulators. An effective biofertilizer production strategy for the rhizobacteria and fungi are discussed together with the synergistic mode of applying them for sustainable crop production. The plant-microbe interactions possess unique features to overcome abiotic and biotic stresses to ensure their competitiveness under the adverse, acidic and humid tropical environment. The unique and versatile plant-rhizobacteria associations and the bioenhancing effects of plant growth regulators clearly demonstrate the beneficial partnerships potentially available in the natural ecosystem. To complement these, improvements in biofertilizer production and application are achieved through exploitation of the inexpensive and abundant organic residues.

∗

Correspondence to: [email protected]

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8.1. INTRODUCTION The humid tropics are blessed with plentiful rainfall [>2500 mm/year] and sunshine essential for luxurious plant growth. This has resulted in lush and dense vegetation which encourage the perpetuation of diverse species of flora and fauna and the survival of many higher animal species. Sustainability of a biological species in a specific environment depends largely on its ability to survive and outcompete the other species. It also hinges on the ability of plant species to associate facultatively or symbiotically with other organisms for mutual benefit. The mutual association between the root-associated and colonizing bacteria [rhizobacteria] and the plant systems [legumes and non-legumes] is vital not only for those organisms but also for the sustainability of the agro- ecosystem. The less commonly appreciated phenomenon which occurs during the plant-rhizobacteria association is the contribution towards nutrient cycling. Nitrogen is generally considered the most demanded plant nutrient. Thus, an effective nitrogen cycling is vital especially because biological nitrogen [N2] fixation [BNF] by N2-fixing bacteria, independently or in association with legumes or non-legumes, is the sole biological mechanism whereby gaseous nitrogen is recycled into the biological system again. Effective plant-rhizobacteria associations also improve the organic matter status and consequently soil fertility. Soils of the humid tropics continuously undergo the weathering process resulting in highly problematic low pH [ malonate/malate > tartrate > lactate > gluconate > acetate > formate [Ryan et al. 2001]. This result serves to confirm the ability of the strains tested in mobilizing P from insoluble sources, in particular those producing altogether citrate, malate and tartarate. Henri et al. [2008] isolated three P. fluorescens strains [CB501, CD511 and CE509] from acidic soils of Cameroon, having the ability to solubilize the three phosphate types [Ca3[PO4]2, AlPO4·H2O or FePO4·2H2O]. The results showed that calcium phosphate [Ca-P] solubilization resulted from the combined effects of pH decrease and carboxylic acids synthesis. At pH 4, it was solubilized by most of the organic acids. However, the synthesis of carboxylic acids was the main mechanism involved in the process of aluminium phosphate [Al-P] and Fe-P solubilization. Both were mobilized at pH 4 by citrate, malate, tartrate, and on a much lower level by gluconate and trans-aconitate. In few other cases, the degree of solubilization was not necessarily correlated with acidity or with the decline in pH [Krishanaraj, 1987; Asea et al., 1988]. Solubilization of Ca-P has even been reported to occur even in the absence of organic acid [Illmer and Schinner, 1992]. An HPLC analysis of the culture suspension of Pseudomonas did not detect any organic acid even though the bacterium solubilized unavailable forms of P [Illmer and Schinner, 1995]. In each of these cases, acidification of the medium resulted and was postulated that H+ excretion originating from NH4 assimilation contributed to acidification [Parks et al., 1990]. Krishanaraj [1996] derived MPS- mutants from Pseudomonas and compared with their wildtype with respect to the Pi release in the TCP broth, drop in pH and identification of organic acid released in the medium. It was found that a highly coordinated reaction caused the dissolution of insoluble P. In the event of P stress, glucose is utilized and gets converted to organic acids that provide H+ and get co-transported into the external mileu with H2PO4- or HPO4-2. These reactions are hypothesized to involve the membrane enzymes and organic acid transporters.

9.5.2. Production of Inorganic Acids The solubilization of inorganic P in some cases is attributed to the production and release of inorganic acids [Richardson, 2001; Reyes et al., 2001]. In the special case of ammonium and sulfur oxidizing chemoautrophs, nitric acid and sulfuric acids are produced [Dugan and Lundgren, 1965]. The inorganic acids convert Ca3[PO4]2 to di- and monobasic phosphates with the net result of an enhanced availability of the element to plants. Nitric or sulphuric acids produced during the oxidation of nitrogenous materials or inorganic compounds of sulfur react with RP and thereby increase the soluble P. The oxidation of elemental sulfur is a simple and effective means of providing utilizable phosphates. For example, a mixture may

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be prepared with soil or manure, elemental sulfur and RP. As the sulfur is oxidized to sulfuric acid by Thiobacillus, there is a parallel increase in acidity and net release of soluble P. Nitrification of ammonium salts also leads to a slight but significant liberation of soluble P from RP composts. However, biological sulfur or ammonium oxidation has never been adopted on a commercial scale because of the availability of cheaper and more efficient means of preparing fertilizers. Gaur [1990] observed solubilization of Mussourie rock phosphate [MRP] in soil amended with ammonium sulphate. The available P increased greatly in soil inoculated with PSM and the increase in solubilization was more with fungal inoculation followed by bacteria and yeast. Application of 1% farmyard manure further improved P solubilization. The structural complexicity and particle size of P, and the quantity of organic acid secreted by microbes was also reported to affect P solubilization.

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9.5.3. Other Mechanisms of Phosphate Solubilization Although phosphate solubilization commonly requires acid production, other mechanisms may account for ferric phosphate mobilization. In flooded soil, the iron available as insoluble ferric phosphates may be reduced leading to the formation of soluble iron with concomitant release of P into solution. Such increases in the availability of P on flooding may explain why rice cultivated under water has a lower requirement for fertilizer P than the same crop grown in dry land agriculture. Phosphorus may also be made available for plant uptake by certain bacteria that librates H2S. Fermentative microorganisms produce H2S from sulphur-containing aminoacids or anaerobic sulphate reducing bacteria like, Desulphovibrio and Desulfatomaculum causes reduction of sulphate to H2S when the redox potential is low. Hydrogen sulfide reacts with ferric phosphate to yield ferrous sulfide and librates the phosphate. Humic and fulvic acids are the other chelating substances produced during the decomposition of organic materials. Mishra et al. [1982] reported that 5% solution of humic acid in alkali could solubilize 362 µg P per gram of RP. The action of humic and fulvic acid is due to the presence of hydroxyl, phenolic and carboxyl groups [Banger et al., 1985]. Respiratory H2CO3 production by plants and soil organisms has been found as an alternate mechanism of mineral phosphate solubilization [Juriank et al., 1986]. The CO2 produced in the rhizosphere due to decomposition of organic matter by microbes has also been reported to be involved in increased P availability to plants. The reaction may be with CO2 directly or due to formation of carbonic acid which react with Ca3[PO4]2 forming CaHPO4 or Ca[H2PO4]2 and CaCO3.

9.6. ORGANIC PHOSPHATE SOLUBILIZATION The chief source of organic P compounds entering the soil is the vast quantity of vegetation that undergoes decay. Agricultural crops commonly contain 0.05 to 0.50% P in their tissues and this element is found in several compounds or groups of substances in plants i.e., phytin, phospholipids, nucleic acids, phosphorylated sugars, coenzymes and related compounds. Phosphorus may also be present as inorganic orthophosphate, especially in vacuoles and internal buffers. The phosphorus in phytin, phospholipids and nucleic acids is

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found as phosphate. Phytin is the calcium magnesium salt of phytic acid. The nucleic acids RNA and DNA consist of a number of purine and pyrimidine bases, pentose sugar and phosphate. In bacterial cell, the bulk of P is in RNA, usually accounting for one-third to somewhat more than one-half of all the P. DNA contributes from 2 to 10% of the total P content. The acid-soluble fraction of bacterial protoplasm contains ortho-and metaphosphate, sugar phosphates, many of the coenzymes and adenosine phosphates. In this process of organic phosphate solubilization, microorganisms convert the organic P to inorganic forms [Deubel et al., 2000]. Thus, the bound element in the plant residue material and in soil organic matter is made available to succeeding populations of plants by the action of bacteria, fungi and actinomycetes. The mineralization and immobilization of this element are related to the analogous reactions of nitrogen. As a rule, phosphate release is most rapid under conditions favoring ammonification [nitrogen mineralization]. Thus, a highly significant correlation is observed between the rates of N and P conversion to inorganic forms. The nitrogen mineralized being from 8-15 times the amount of phosphate made available. There is also a correlation between C [CO2 release] and P mineralization [a ratio of 100 to 300:1]. The results showed that the ratio of C:N:P mineralized microbiologically at the equilibrium condition is similar to the ratios of three elements in humus. Phosphorus can be released from organic compounds in soil by three groups of enzymes[i] nonspecific phosphatases, which perform dephosphorylation of phosphor-ester or phosphor-anhydride bonds in organic matter [ii] phytases, which specifically cause P release from phytic acid and [iii] phosphonatases and C-P lyases enzymes that perform C-P cleavage in organophosphonates. The main activity apparently corresponds to the work of acid phosphatases and phytases because of the predominant presence of their substrates in soil. Availability of organic phosphate compounds for plant nutrition could be a limitation in some soils resulting from precipitation with soil particle ions. Therefore, the capability of enzymes to perform the desired function in the rhizosphere is a crucial aspect for their effectiveness in plant nutrition.

9.6.1. Nonspecific Acid Phosphatases A single phosphatase enzyme may catalyze the cleavage of ethyl phosphate, glycerophosphate and phenyl phosphate. On the other hand, diesters may require different enzymes for their breakdown. Phosphatases acting on phospholipids and hydrolyzing nucleic acids have diesters as their substrates. The phosphatase enzyme catalyzing hydrolysis of the monoesters often has distinct optima in pH for maximum activity i.e., active at low pH ranges are acid phosphatases whereas the enzymes active at high pH ranges are termed as alkaline phosphatases. Bacterial nonspecific acid phosphatases [phosphohydrolases] [NSAPs] are formed by three molecular families, which have been designated as molecular class A, B and C [Thallar et al., 1995a]. From their cellular location, these enzymes seem to function as organic phosphoester scavengers, releasing inorganic phosphates from nucleotides and sugar phosphates and thus providing the cell with essential nutrients [Beacham, 1980; Wanner, 1996]. Several acid phosphatase genes from Gram-negative bacteria have been isolated and characterized [Rossolini et al., 1998]. These cloned genes encoding acid phosphatase

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represent an important source of material for genetic transfer to PGPR strains. For example, the acpA gene isolated from Francisella tularensis expresses an acid phosphatase with optimum action at pH 6, with a wide range of substrate specificity [Reilly et al., 1996]. Also, genes encoding nonspecific acid phosphatases class A [PhoC] and class B [NapA] isolated from Morganella morganii are very promising, since the biophysical and functional properties of the encoded enzymes were extensively studied [Thallar et al., 1994; Thallar et al., 1995b]. Besides, they are P-irrepressible enzymes showing broad substrate action and high activity around pH 6 and 300C. These enzymes have also potential biotechnological applications. Macaskie et al. [1997] reported on the successful use of class A NSAPs as tools for environmental bioremediation of uranium-bearing waste water and on heavy metal biomineralization, particularly nickel [Bonthrone et al., 1996; Baskanova and Macaskie, 1997]. Moreover, the transfer and expression of these genes encoding for NSAPs into plant growth promoting rhizobacteria could result in bacterial strains with improved phosphate solubilizing activity using recombinant DNA technology. Roderiguez et al. [2000a] isolated a gene from Burkholderia cepacia that facilitates phosphatase activity. This gene codes for an outer membrane protein that enhances synthesis in the absence of soluble phosphates in the medium and could be involved in P transport to the cell. Roderiguez et al. [2006] constructed a plasmid for the stable chromosomal insertion of the phoC phosphatase gene from Morganella morganii using the delivery system developed by Lorenzo et al. [1990]. This plasmid was transferred to Azospirillum spp. and the strains with increased phosphatase activity were obtained. Two nonspecific periplasmic acid phosphatase genes [napD and napE] were cloned from Rhizobium meliloti [Deng et al., 1998; 2001]. The napA phosphatase gene from the soil bacterium Morganella morganii was transferred to Burkholderia cepacia IS-16, a strain used as biofertilizer, using the broad host range vector PRK293 [Fraga et al., 2001]. An increase in extracellular phosphatase activity of the recombinant strain was achieved.

9.6.2. Phytases Phytate is the primary source of inositol in its basic form and the major stored form of phosphate in plant seeds and pollen. Monogastric animals are incapable of using the P bound in the phytate because their gastrointestinal tracts have low levels of phytase activity. Thus, nearly all the dietary phytate phosphorus ingested by these species is excreted, resulting in P pollution in areas of intensive animal production. Supplemental microbial phytase in cornsoybean meal diets for swine and poultry effectively improves phytate phosphorus utilization by these animals and reduces their fecal P excretion by up to 50% [Lei et al., 1993]. Therefore, phytases have emerged as very attractive enzymes for industrial and environmental applications. Most phytases belong to high molecular weight acid phosphatases. The phytase enzyme librates phosphate from phytic acid or its calcium-magnesium salt, phytin with the accumulation of inositol. Some species make intracellular phytase while others excrete extracellular phytase enzymes. Moreover, some phytases are reasonably specific and act chiefly on inositol phosphates whereas nonspecific phosphatases remove phosphorus from dissimilar organic compounds. Phytase activity is widespread and about 30-50% of the bacterial isolates from soil synthesizes this enzyme. Its activity in nature is enhanced by addition of carbonaceous materials that increase the size of community. Species of

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Aspergillus, Rhizopus, Cunninghamella, Arthrobacter, Streptomyces, Pseudomonas and Bacillus have been found to synthesize the enzyme. Phytate is the major component of organic forms of P in soil [Richardson, 1994]. The ability of plants to obtain P directly from phytate is very limited. However, the growth and P nutrition of Arabidopsis plants supplied with phytate was improved significantly when they were genetically transformed with the phytase gene [phyA] from Aspergillus niger [Richardson et al., 2001a]. This resulted in improved P nutrition such that the growth and P content of the plant was equivalent to control plants supplied with inorganic P. The enhanced utilization of inositol phosphate by plants in the presence of soil microbes has also been reported [Richardson et al., 2001b]. Therefore, developing agriculture inoculants with high phytase production would be of great interest for improving plant nutrition and reducing P pollution in soil. Thermally stable phytase gene [phy] from Bacillus sp. DS11 [Kim et al., 1998d] and from B. subtilis VTT E-68013 [Kerovuo et al., 1998] have been cloned. Han et al. [1999] reported that 1.4 kb DNA fragment containing the coding region of the phyA gene from Aspergillus niger was expressed in Saccharomyces cerevisiae. The recombinant active extracellular phytase from S. cerevisiae effectively hydrolyzed phytate phosphorus from corn or soybean meal in vitro. Acid phosphatase phytase genes from E. coli [appA and appA2 genes] have also been isolated and characterized [Roderiguez et al., 1999; Golovan et al., 2000]. The bi-functionality of these enzymes makes them attractive for solubilization of organic P in soil. Also, neutral phytases have great potential for genetic improvement of plant growth promoting rhizobacteria. Neutral phytase genes have been cloned from B. subtilis and B. licheniformis [Tye et al., 2002]. For example, a phyA gene was cloned from the FZB45 strain of B. amyloliquefaciens, having plant growth promoting activity [Idriss et al., 2002]. It showed the highest extracellular phytase activity and the diluted culture filtrates of these strains stimulated growth of maize seedlings under limited P in the presence of phytate. Culture filtrates obtained from a phytase negative mutant strain, whose phyA gene was disrupted, did not stimulate plant growth. In addition, growth of maize seedlings was enhanced in the presence of purified phytase and the absence of culture filtrate. These experiments provided strong evidence that phytase activity can be important for stimulating plant growth under limited P in soil and support the potential of using phytase genes to improve or transfer the P-solubilizing trait to PGPR strains used as agricultural inoculants.

9.7. ISOLATION OF MINERAL PHOSPHATE SOLUBILIZING [MPS] GENES The repression of mineral phosphate solubilizing activity observed in presence of increasing levels of inorganic P in the medium indicated the physiological regulation of the system. Goldstein [1986] reported the complete inhibition of MPS activity by Erwinia herbicola by addition of 20 mM Pi in the medium. Similarly, it was found that externally added K2HPO4 inhibited the MPS activity of Pseudomonas Psd 201 [Krishanaraj, 1996]. The phosphate stress induction of MPS activity and repression of MPS activity by externally added Pi indicated the physiologically regulated gene expression of MPS activity in bacteria. Based on these observations, Goldstein [1986] proposed the existence of mps genes in

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Erwinia herbicola. Several genes were induced under P starvation in E. coli and constituted the Pho regulon. Recently, the transcriptional control of Pho regulon has been extensively studied in E. coli [Makino et al., 2007], Bacillus subtilis [Huelett et al., 2007] and Saccharomyces cerevisiae [Ogawa et al., 2007]. Using shotgun-cloning experiments, Goldstein and Liu [1987] cloned gene[s] involved in mineral phosphate-solubilization from Gram-negative bacteria Erwinia herbicola and showed that GDH mediated dissimilatory bypass system, involving direct oxidation of glucose to gluconic acid in the periplasmic space was responsible for the mineral phosphate solubilization in Erwinia herbicola. Expression of this mps gene allowed production of GA in E. coli HB101 and conferred the ability to solubilize hydroxyapatite [MPS+ phenotype]. MPSE. coli can synthesize GDH, but not PQQ, thus it did not produce GA. On screening a cosmid pHC 76 library from Erwinia herbicola, they found that a 55 kb insert DNA was able to transform E. coli. Transposon mutagenesis of the cosmid construct pMCG 898 carrying a 4.5 kb insert showed that the essential gene was localized in a 1.8 kb region. Based on sequence comparison and minicell analysis, Liu et al. [1992] deciphered that the gene codes for an enzyme pyrrolquinoline quinone [PQQ], a cofactor for the enzyme glucose dehydrogenase [GDH]. The quinoprotein GDH controls a unique step in direct oxidation of glucose [Duine et al., 1979]. The cloned 1.8 kb locus encoded protein was found similar to the gene III product of a pqq synthesis gene complex from Acinetobacter calcoaceticus and to pqqE of Klebsiella pneumoniae [Liu et al., 1992]. The authors suggested that the E. herbicola DNA fragment functions as a PQQ synthase gene and that probably, some E. coli strains contain some cryptic PQQ synthase genes that could be complemented by this single open reading frame [ORF]. Coincidentally, nucleotide sequence analysis of a 7 kb fragment from Rhanella aquatilis genomic DNA that induced hydroxyapatite solubilization in E. coli, showed two complete ORFs and a partial ORF. One of the cloned proteins showed similarity to pqqE of E. herbicola, K. pneumoniae and A. calcoaceticus [Kim et al., 1998b] while the partial ORF is similar to the pqqC of Klebsiella pneumoniae. These genes complemented the cryptic pqq genes in E. coli, thus allowing GA production. Another type of gene [gabY] involved in GA production and MPS was cloned from Pseudomonas cepacia [Babu-khan et al., 1995]. The deduced amino acid sequence showed no homology with previously cloned direct oxidation pathway [GA synthesis] genes, but was similar to histidine permease membrane-bound components. In the presence of gabY, GA is produced only if E. coli strain expresses a functional glucose dehydrogenase [gcd] gene. It was speculated that this ORF could be related to the synthesis of PQQ by an alternative pathway, or the synthesis of a gcd cofactor different from PQQ [Babu-khan et al., 1995]. In addition, a DNA fragment from Serratia marcescens induced quinoprotein glucose-mediated gluconic acid production in E. coli, but showed no homology to pqq or gcd genes [Krishanaraj and Goldstein, 2001]. They suggested that this gene acted by regulating GA production under cell-signal effects. Other isolated gene JM 109 [pKKY] involved in the MPS phenotype was obtained from genomic DNA fragment of Enterobacter agglomerans using cosmid [pHC 79] genomic library [Kim et al., 1997]. The complementation of this gene in E. coli JM109 showed the MPS activity, although the pH of the medium was not altered. These results indicate that acid production is an important way, but not the only mechanism, of P solubilization by bacteria [Illmer and Schinner, 1995]. All these findings demonstrate the complexity of MPS in different bacterial strains, but at the same time, offer a basis for better understanding of phosphate solubilization process.

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9.7.1. Manipulation of MPS Genes for PGPR Improvement Expression of the mps genes from Ranella aquatilis in E. coli supported a much higher GA production and hydroxyapatite dissolution in comparison with the donor strain [Kim et al., 1998b]. The authors suggested that different genetic regulation of the mps genes might occur in both species. MPS mutants of Pseudomonas spp. showed pleiotropic effects, with apparent involvement of regulatory mps loci in some of them [Krishanaraj et al., 1999]. Two distinct classes of mutants namely, nonsolubilizers [MPS-] and delayed expression types [MPSd] were obtained through nitrosoguanidine and Tn5 mutagenesis of Pseudomonas strain Psd 201. These mutants also showed different phenotypic classes with respect to metabolic and cell surface properties. The nature of pleiotropies shown by these mutants indicated that these mutational lesions might have occurred in some of the regulatory mps loci since the level of expression of zone and time of solubilization got affected in some mutants [Krishanaraj et al., 1999]. Gene bank of the MPS+ wild type Pseudomonas sp Psd 201 was mobilized from E. coli into MPS- derivative strain Pseudomonas Psd 207. Two clones were isolated which could restore MPS+ phenotype to Psd 207 and had an insert of the size of 11.8 kb that might contain one or more mps loci. Expression of the mps genes in a different host could be influenced by the genetic background of the recipient strain, the copy number of the plasmids present and metabolic interactions. Thus, genetic transfer of any isolated gene involved in MPS to induce or improve phosphate-dissolving capacity in PGPB strains, is an interesting approach. An attempt to improve MPS in PGPR strains, using a PQQ synthase gene from E. herbicola was carried out [Rodriguez et al., 2000b]. This gene was subcloned in a broad-host range vector pKT230. The recombinant plasmid was expressed in E. coil and transferred to PGPR strains of Burkholderia cepacia and Pseudomonas aeruginosa, using tri-parental conjugation. Several of the exconjugants that were recovered in the selection medium showed a larger clearing halo in medium with tricalcium phosphate as the sole P source. This indicates the heterologous expression of this gene in the recombinant strains, gave rise to improved MPS ability in these PGPRs. A bacterial citrate synthase gene was reported to increase exudation of organic acids and P availability to the plant when expressed in tobacco roots [Lopez-Bucio et al., 2000]. Citrate overproducing plants yielded more leaf and fruit biomass when grown under P-limiting conditions and required less P-fertilizer to achieve optimal growth. This shows the putative role of organic acid synthesis genes in P uptake in plants.

9.8. AGRONOMIC SIGNIFICANCE OF MINERAL PHOSPHATE SOLUBILIZING BACTERIA Phosphatic biofertilizers were first prepared in USSR using Bacillus megaterium var. phosphaticum as P-solubilizing bacteria and the product was named as ‘phosphobacterin’. It was extensively used in collective farming for seed and soil inoculation to cover an area of 14 million hectares annually and reported to give 5-10% increase in crop yields. Inoculation experiments conducted with phosphobacterin and other PSM for various crops like oat, wheat, potatoes, groundnut, peas, soybean, tomatoes and tobacco showed an average 10-15% increase in yields in about 30% of the experiments conducted [Kundu and Gaur, 1980a;

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Agasimani et al., 1994; Dubey, 1997]. The variations under field conditions are expected due to the effect of various environmental conditions and survival of the inoculant strains in the soil. The first evidence to show that inoculation of seedling with P solubilizing bacteria increased the P-uptake and yield of oat was by Gerretson [1948]. Subsequently, improved plant growth responses and increased Pi uptake on addition of RP has been reported [Banik and Dey, 1983b; Rachewad et al., 1992; Bagyaraj et al., 2000]. The agronomic influence of some commonly used bacterial species is listed in Table II. Inoculation of these bacteria along with RP resulted in increased availability of Pi for plant utilization [Hebbara and Suseeladevi, 1990; Jisha and Alagawadi, 1996]. It was observed that inoculation of mineral phosphate solubilizing bacteria [MPSB] along with application of 17.5 kg P ha-1 as MRP resulted in increased dry matter in chickpea and was as effective as single super phosphate [Prabhakar and Saraf, 1990]. With the current emphasis on organic farming, the use of MPSB as inoculants gains more importance. Kundu and Gaur [1980b] observed increased plant growth and P-uptake in wheat on inoculation with B. polymyxa and P. striata under pot house conditions. They also observed positive effect on inoculation with a mixture of P. striata and A. awamorii in rice crop [Kundu and Gaur, 1984]. Datta et al. [1982] reported increased yield of rice in soils inoculated with Bacillus firmis in presence of RP under field conditions. Increase in dry matter production and P-uptake from 10-27% and 15-34%, respectively was observed by inoculation of Penicillium bilaji in chernozemic soil with low P availability in wheat crop [Kucey, 1987; 1988].

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Table II. Inoculation effect of mineral phosphate solubilizing bacteria Bacteria Pseudomonas striata and Bacillus polymyxa Pseudomonas putida

Crop Wheat

Conditions Greenhouse

Canola

Greenhouse

Pseudomonas striata

Rice

Greenhouse

Bacillus subtilis, B. circulans and Aspergillus niger Pseudomonas striata

Mungbean

Field

Groundnut

Field

Pseudomonas sp.

Chickpea

Greenhouse

Pseudomonas striata

Soybean

Field

Enterobacter cloacae, Burkholderia cepacia and Serratia marcescens Azospirillum lipoferum and Bacillus megaterium Pseudomonas fluorescens

Bamboo

Greenhouse

Wheat

Greenhouse

Maize

Greenhouse

Response Increased P uptake and yield Increased P uptake and yield Increased P uptake and yield Increased nodulation and grain yield

Reference Kundu and Gaur, [1980b] Lifschitz et al., [1987] Monod et al., [1989] Gaind and Gaur, [1991]

High pod yield and P uptake Increased P uptake and dry matter Increased yield and P content Increased dry matter

Agasimani et al., [1994] Krishnaraj, [1996] Dubey, [1997]

Increased shoot P and shoot weight Increased grain yield and P content

El Komy, [2005]

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Maheshkumar, [1997]

Henri et al., [2008]

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The addition of RP [low P solubility] had little effect while mono-ammonium phosphate [commercial fertilizer with high soluble P content] resulted in the highest yields and P uptake. The addition of P. bilaji to these P sources did not increase P availability but increased release of P from soil [Kucey, 1987]. Rachewad et al. [1992] reported that addition of PSB along with RP resulted in increased P uptake by sunflower under field conditions. Jisha and Algawadi [1996] suggested that PSM could be used in combination with RP to make P available in the soil. Seed yield, total dry matter yield and P content in different plant parts at various stages increased with Pseudomonas striata alone or in conjunction with superphosphate and RP. Seed treatment with P. striata further enhanced the yield and P content of soybean [Dubey, 1997]. de Freitas et al. [1997] observed that inoculation with PSB significantly increased the number and weight of pods and seed yield of canola [Brassica napus] but did not affect the Puptake. Saraf et al. [1997] showed that PSB inoculation, increased seed yield [10.3q ha-1] of chickpea as compared to control [8.8 q ha-1]. Increased grain yield [13-69%] and uptake of N and P was reported in chickpea by inoculation of PSB along with phosphatic fertilizers. The grain and straw yield of chickpea was found to increase with increasing level of P [0-60 kg P2O5 ha-1] which were further improved by inoculation of PSB [Sarawgi et al., 1999; 2000]. Significantly higher yield [19.5 q ha-1] was observed in soybean on PSB inoculation and on addition of SSP [26.4 kg P ha-1] as compared to control [16.3q ha-1] by Dubey [2001]. Sharma [2003] observed that addition of RP with PSB increased grain yield [0.9 to 1.8 t ha-1], Nuptake [18 to 38 kg ha-1], P-uptake [2.7 to 6.6 kg ha-1] and K- uptake [16 to 41 kg ha-1] in rice-wheat cropping system. Plant growth-promoting fluorescent pseudomonad isolate PGPR1, which produced siderophore and indole acetic acid, and solubilized TCP under in vitro conditions, significantly enhanced the pod yield [23-26%, respectively], haulm yield and nodule dry weight over the control during three years and also suppressed the soil-borne fungal diseases like, collar rot of peanut caused by A. niger in field trials [Dey et al., 2004]. Henri et al. [2008] conducted a greenhouse trial in Zea mays by inoculation of three Pseudomonas fluorescens strains [CB501, CD511 and CE509], having the ability to solubilize the three phosphorus types. Inoculation of P. fluorescens strains showed positive effects on the growth, grain yield and P uptake. The results revealed that strain CB501 was the best plant growth promotor with a global effect of +37%, followed by strain CE509 [+21.2%] and strain CD511 [+16.7%]. Thus, inoculation with phosphate-solubilizing P. fluorescens strains made more soluble P available to the growing maize plants and could have stimulated microbial growth and activity.

9.8.1. Interactions of P-Solubilizing Bacteria with Other Beneficial Microbes Plant growth promoting rhizobacteria have been found to stimulate plant growth by facilitating the uptake of minerals into the plant roots [Lifshitz et al., 1987]. Several experiments conducted in legume and non-legume crops by coinoculation of PSM with diazotrophs have shown synergistic effects with regard to increase in population of both bacteria and significant increase in crop yields in comparison to single inoculation. However, field inoculation trials have been rather inconsistent [Kucey et al., 1989]. The synergistic effect was observed after co-inoculation of nitrogen-fixing bacteria with PSM. Ocampo et al.

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[1975] studied interactions between Azotobacter and phosphobacteria in the rhizosphere of lavender plants [Lavendula spica L.] and it was observed that there was always more counts of Azotobacter and phosphobacteria in the rhizosphere of coinoculated plants in comparison to singly inoculated plants. Plant growth was maximum when seedlings were inoculated with both microorganisms. Similarly, the inoculation of phosphate-solubilizing bacteria either alone or in combination with A. chroococcum enhanced the yield and nutrient uptake of cotton and wheat in field trials [Kundu and Gaur, 1980c; 1982]. Increased phosphorus availability by P. putida to common bean plants on coinoculation with Rhizobium phaseoli has been found to increase nodulation of common bean [Grimes and Mount, 1984]. The seed inoculation with thermo-tolerant PSM [viz. Bacillus subtilis, B. circulans and Aspergillus niger] improved nodulation, available P2O5 content of soil, root and shoot biomass, straw and grain yield, P and N uptake by mungbean [Gaind and Gaur, 1991]. High pod yield and P uptake in groundnut due to inoculation of Pseudomonas striata were also recorded [Agasimani et al., 1994]. Soybean seeds inoculated with Bradyrhizobium japonicum grown in a plot with inoculation of PSB showed significantly higher noculation and yield [Chandra et al., 1995]. Increased nodulation, yield attributes, seed index and seed yield have also been reported due to combined inoculation of P. striata and B. japonicum [Dubey, 1997; Kumrawat et al., 1997]. A significant increase in nitrogenase activity, growth and grain yield of pea was found due to dual inoculation of Rhizobium leguminosarum and PSB [Srivastava et al., 1998]. El Sayed [1999] observed that coinoculation of Rhizobium leguminosarum and P- solubilizing Pseudomonas striata significantly increased the dry matter content, grain yield, N and P uptake of lentil over the uninoculated control. Sonboir and Sarawgi [2000] reported increased nutrients uptake [N, P and K], grain yield and pods plant-1 with increasing level of P in chickpea that was further enhanced by inoculation of PSB. Balyan et al. [2002] observed more nodulation, yield, and N-uptake when PSB was coinoculated with Rhizobium in mungbean. A positive response of PSB and Rhizobium along with P was observed in black gram by Tanwar et al. [2002]. Jain and Singh [2003] found that Rhizobium, PSB and potassium [50 kg/ha] increased P and N uptake by chickpea. Inoculation of PSB along with Azospirillum increased the grain and straw yield of barley by 6.1 and 9.2% as compared to control [Yadav et al., 2004]. El Komy [2005] observed that combined inoculation of Azospirillum lipoferum and Bacillus megaterium increased the shoot weight, total nitrogen yield and shoot P in wheat in relation to the control. In some cases, plant growth promoting rhizobacteria selected for a particular trait also showed other beneficial activities. For example, bacteria selected for phytohormone production also showed P solubilization or biological control activity. Thus, there is no clear separation of growth promotion in plants and biological control induced by bacterial inoculants [Lugtenberg et al., 1991; Goel et al., 2002]. Bano and Musarrat [2004] isolated Pseudomonas sp. NJ-101 from agricultural soil that exhibited efficient degradation of the insecticide carbofuran. The ability to produce HCN and siderophore stipulated its role in biological control and caused inhibition of Fusarium sp. Concurrent production of indole acetic acid [IAA] and solubilization of inorganic P revealed its plant growth promotion potential and its significance in management of the agro-environmental and phytopathological problems. Similarly, growth promotion and yield enhancement of peanut [Arachis hypogaea L.] was studied by application of PGPR [Dey et al., 2004]. Nine different isolates of PGPR were selected based on ACC-deaminase activity from the peanut rhizosphere. These isolates were identified as Pseudomonas species. Four of these isolates, viz. PGPR1, PGPR2, PGPR4

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and PGPR7, produced siderophore and IAA. In addition, Pseudomonas fluorescens PGPR1 also possessed the properties like TCP solubilization and ammonification, and inhibited A. niger under in vitro conditions, the causative agent of collar rot disease in peanut. Mycorrhizal associations are best known to improve plant growth in nutritionally deficient soils. The main effect of mycorrhizal infection on plant growth is attributed to the stimulation of P uptake by fungal hyphae [Gianinazzi-Pearson, 1996; Harrison, 2005]. Synergistic interaction between PSM and vesicular arbuscular mycorrhizal [VAM] fungi has been found and the positive responses were associated with low concentration of active calcium in soils. Ghosh and Poi [1990] reported improved nodulation, plant growth, P uptake and PSM population due to combined inoculation with Bacillus polymyxa and Glomus fasciculatum in soybean, groundnut, mungbean and lentil. Tilak et al. [1995] reported that dual inoculation with Pseudomonas striata and VAM fungi [G. fasciculatum and G. mosseae] significantly increased the bean yield, root biomass and total P uptake by soybean plant over uninoculated control in alluvial sandy soils. The P-solubilizing bacteria behaved as mycorrhiza helper bacteria [MHB] because they promoted root colonization when associated with mycorrhizal fungi [Garbaye, 1994]. Toro et al. [1997] reported that combined inoculation of G. intraradices and Bacillus subtilis significantly increased plant biomass, N, and P accumulation in onion plant tissues. The inoculated rhizobacteria may have released Pi from the added RP and at least 75% of the P in dually inoculated plants was derived from the added RP. Kim et al. [1998c] observed a significantly higher soluble P concentration in tomato with the inoculation of PSB and AM-fungi. Thus, these mycorrhizosphere interactions between bacterial and fungal plant association contributed to biogeochemical P cycling and promoted a sustainable nutrient supply to plants.

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CONCLUSION Soil microorganisms play a pivotal role in various biogeochemical cycles and are responsible for the cycling of nutrients in the plant utilizable form [Wall and Virginia, 1999]. Phosphate-solubilizing microorganisms and other beneficial rhizobacteria cause the release of nutrients in plant utilizable form and exert beneficial effects on plant growth [Glick, 1995]. Thus, microbes influence above ground ecosystems by contributing to plant nutrition, plant health, soil structure and soil fertility. Therefore, microorganisms offer an environment friendly sustainable system and help in maintaining soil nutrient status. Torsvik et al. [1990] estimated 4000 different bacterial genomic units per gram of soil based on DNA-DNA reassociation and reported that soil microflora is of great importance because it has both beneficial and detrimental influences in the plant rhizosphere. A healthy rhizosphere population can also help plants to deal with biotic and abiotic stresses [pathogens, drought and soil contamination]. Recently, different methods and techniques have been developed to characterize and conserve various agriculturally important microbial communities from different environments for their optimal utilization in agriculture [Kirk et al., 2004; Naik et al., 2008]. The knowledge generated on biodiversity and genetic manipulation of Psolubilizing bacteria will be useful to design strategies for use of these bacterial strains as inoculants in sustainable and organic agriculture. However, the selection of P- solubilizing

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microorganisms as possible inoculation tool for P-deficient soils should focus on the integral interpretation of laboratory assays, greenhouse experiments as well as field trials. The complex interactions between the PSB, other PGPR, the plant and the environment are responsible for the variability observed in solubilization of bound phosphates, Pi uptake and plant growth promotion. The inconsistency in performance of these PGPR strains is a major constraint to the wide spread use of PSB in commercial agriculture. Genetic manipulation of PGPRs has the potential to construct significantly better strains with improved P solubilization efficacy [Rodriguez et al., 2006]. Future strategies are required to clone genes from PSM involved in solubilization of insoluble P and to transfer those genes into the bacterial strains having good colonization potential along with other beneficial characteristics such as, nitrogen fixation. Further, the efficacy of phosphate-solubilizing bacteria can be improved by developing the better cultural practices and delivery systems that favor their establishment in the rhizosphere. The applications of mixture of PGPRs with different beneficial activities including P solubilization ability, may be a more ecologically sound approach because it may result in better colonization and better adaptation to the environmental changes occurring throughout the growing season. In near future, the biotechnological approaches used in manipulation of bacterial traits will improve the efficiency of P solubilization in bacteria and their inoculation as phosphatic biofertilizer may enhance plant growth and crop productivity in soil.

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REFERENCES Abd-Alla, M. H. [1994]. Use of organic phosphorus by Rhizobium leguminosarum biovar viceae phosphatases. Biology and Fertility of Soils 18, 216-218. Agasimani, C. A., Mudalagiriyappa, M. V. and Sreenivasa, M. H. [1994]. Response of groundnut to phosphate solubilizing microorganisms. Groundnut News 6, 5-7. Agnihotri, V. P. [1970]. Solubilization of insoluble phosphates by some soil fungi isolated from nursery seed beds. Canadian Journal of Microbiology 16, 877-880. Asea, P. E. A., Kucey, R. W. N. and Stewart, J. W. B. [1988]. Inorganic phosphate solubilization by two Penicillium species in solution culture and soil. Soil Biology and Biochemistry 20, 459-464. Babu-khan, S., Yeo, C., Martin, W. L., Duron, M. R., Rogers, R. and Goldstein, A. [1995]. Cloning of a mineral phosphate solubilizing gene from Pseudomonas cepacia. Applied and Environonmental Microbiology 61, 972-978. Bagyaraj, D. J., Krishanaraj, P. U. and Khanuja, S. P. S. [2000]. Mineral phosphate solubilization: Agronomic implications, mechanism and molecular genetics. Proceedings of Indian National Science Academy B66, 69-82. Balyan, S. K., Chandra, R. and Pareek, G. R. [2002]. Enhancing nodulation in Vigna mungo by applying higher quantity of Rhizobium in planting furrows and PSB. Legume Research 25, 160-164. Banger, K. C., Yadav, K. S. and Mishra, M. M. [1985]. Transformation of rock phosphate during composting and the effect of humic acid. Plant and Soil 85, 259-266.

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Banik, S. and Dey, B. K. [1981]. Solubilization of inorganic phosphate and production of organic acids by microorganisms isolated in sucrose-tricalcium phosphate agar plates. Zentralbl Microbiology 136, 478-486. Banik, S. and Dey, B. K. [1983a]. Alluvial soil microorganisms capable of utilizing insoluble aluminium phosphates as a sole source of phosphorus. Zentralbl Microbiology 138, 437442. Banik, S. and Dey, B. K. [1983b]. Available phosphate content of an alluvial soil as influenced by inoculation of some isolated phosphate solubilizing microorganisms. Plant and Soil 69, 353-364. Banik, S. and Dey, B. K. [1983c]. Phosphate solubilizing potentiality of microorganisms capable of utilizing aluminium phosphate as a sole phosphate source. Zentralbl Microbiology 138, 17-23. Bano, N. and Musarrat, J. [2004]. Characterization of a novel carbofuran degrading Pseudomonas sp. with collateral Biocontrol and plant growth promoting potential. FEMS Microbiology Letters 231, 13-17. Bardiya, M. C. and Gaur, A. C. [1972]. Rock phosphate dissolution by bacteria. Indian Journal of Microbiology 12, 269-271. Barea, J. M., Navarro, E. and Montoya, E. [1976]. Production of plant growth regulators by rhizosphere phosphate solubilizing bacteria. Journal of Applied Bacteriology 40, 129134. Bashan, Y., Moreno, M. and Troyo, E. [2000]. Growth promotion of the sea-water irrigated oil seed halophyte Salicornia bigelovii inoculated with mangrove rhizosphere bacteria and halotolerant Azospirillum spp. Biology and Fertility of Soils 32, 265-272. Baskanova, G. and Macaskie, L. E. [1997]. Microbially-enhanced chemisorption of nickel into biologically-synthesized hydrogen uranyl phosphate: a novel system for the removal and recovery of metals from aqueous solutions. Biotechnology and Bioengineering 54, 319-329. Beacham, I. R. [1980]. Periplasmic enzymes in Gram-negative bacteria. International Journal of Biochemistry 10, 877-883. Bieleski, R. L. [1973]. Phosphate pools, phosphate transport and phosphate availability. Annual Review of Plant Physiology 24, 225-252. Bonthrone, K. M., Baskanova, G., Lin, F. and Macaskie, L. E. [1996]. Bioaccumulation of nickel by intercalation into polycrystalline hydrogen uranyl phosphate deposited via an enzymatic mechanism. Natural Biotechnology 14, 635-638. Carrillo, A. E., Li, C. Y. and Bashan, Y. [2002]. Increased acidification in the rhizosphere of cactus seedlings induced by Azospirillum brasilense. Naturewissenschaften 89, 428-432. Cattelan, A. J., Hartel, P. G. and Furhmann, F. F. [1999]. Screening of plant growth promoting rhizobacteria to promote early soybean growth. Soil Science Society of America Journal 63, 1670-1680. Chandra, K., Mukherjee, P. K., Karmakar, J. B. and Sharma, B. K. [1995]. Effect of phosphate solubilizing bacteria on rhizobial symbiosis in soybean at rainfed conditions of Manipur. Environment and Ecology 13, 436-438. Chen, Y. P., Rekha, P.D., Arun, A. B., Shen, F. D., Lai, W. A. and Young, C. C. [2006]. Phosphate solubilizing bacteria from subtropical soil and their tri-calcium phosphate solubilizing abilities. Applied Soil Ecology 34, 33-41.

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Molecular Genetics of Phosphate Solubilization in Rhizosphere Bacteria…

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Datta, M., Banik, S. and Gupta, R. K. [1982] Studies on efficacy of a phytohormone producing PSB Bacillus firmus in augmenting paddy yield in acid soils of Nagaland. Plant and Soil 69, 365-373. de Freitas, J. R., Banerjee, M. R. and Germida, J. J. [1997]. Phosphate solubilizing rhizobacteria enhance the growth and yield but not phosphorus uptake of canola [Brassica napus L.]. Biology and Fertility of Soils 27, 358-364. Deng, S., Elkins J. G., Da, L. H., Botero, L. M. and McDermontt, T. R. [2001]. Cloning and characterization of second acid phosphatase from Sinorhizobium meliloti strain 104A14. Archives of Microbiology 176, 255-263. Deng, S., Summers, M. L., Kahn, M. L. and Mc Dermontt, T.R. [1998]. Cloning and characterization of a Rhizobium meliloti nonspecific acid phosphatase. Archives of Microbiology 170, 18-26. Deubel, A. and Merbach, W. [2005]. Influence of microorganisms on phosphorus bioavailability in soils. In: F. Buscot and A. Varma [Eds.], Microorganisms in Soils: Roles in Genesis and Functions. [pp. 177-191]. Berlin Heidelberg: Springer-Verlag. Deubel, A., Gransee, A. and Merbach, W. [2000]. Transformation of organic rhizodepositions by rhizosphere bacteria and its influence on the availability of tertiary calcium phosphate. Journal of Plant Nutrition and Soil Science 163, 387-392. Dey, R., Pal, K. K., Bhatt, D. M. and Chauhan, S. M. [2004]. Growth promotion and yield enhancement of peanut [Arachis hypogaea L.] by application of plant growth-promoting rhizobacteria. Microbiological Research 159, 371-394. Dubey, S. K. [1996]. Combined effect of Bradyrhizobium japonicum and phosphate solubilizing Pseudomonas striata on nodulation, yield attributes and yield of rainfed soybean [Glycine max] under different sources of phosphorus in vertisols. Indian Journal of Agricultural Sciences 66, 28-32. Dubey, S. K. [1997]. Coinoculation of phosphate solubilizing bacteria with Bradyrhizobium japonicum to increase phosphate availability to rainfed soybean on vertisol. Journal of Indian Society of Soil Science 45, 506-509. Dubey, S. K. [2001]. Associative effect of nitrogen fixing and phosphate solubilizing bacteria in rainfed soybean [Glycine max] grown in vertisols. Indian Journal of Agricultural Sciences 71, 476-479. Dugan, P. and Lundgren, D. G. [1965]. Energy supply for the chemoautotroph Ferrobacillus ferrooxidans. Journal of Bacteriology 89, 825- 834. Duine, J.A., Frank, J. and van Zeeland, J. K. [1979]. Glucose dehydrogenase from Acinetobacter calcoaceticus: a quino protein. FEBS Letters 108, 443-446. Dye, C. [1995]. Effect of citrate and tartrate on phosphate absorption by amorphous ferric hydroxide. Fertility Research 40, 129-134. El Komy, H. M. A. [2005]. Coimmobilization of Azospirillum lipoferum and Bacillus megaterium for successful phosphorus and nitrogen nutrition of wheat plants. Food Technology and Biotechnology 43, 19-27. El Sayed, S. A. M. [1999]. Influence of Rhizobium and phosphate solubilizing bacteria on nutrient and yield of lentil in New Valley [Egypt]. Egyptian Journal of Soil Science 39, 175-186. Fankem, H., Nwaga, D., Deubel, A., Dieng, L., Merbach, W. and Etoa, F. X. [2006]. Occurrence and functioning of phosphate solubilizing microorganisms from oil palm tree

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

220

S. S. Sindhu, M. K. Verma and Suman Mor

[Elaeis guineensis] rhizosphere in Cameroon. African Journal of Biotechnology 5, 24502460. Fernandez, L. A., Zalba, P., Gomez, M. A. and Sagardoy, M. A. [2007]. Phosphatesolubilization activity of bacterial strains in soil and their effect on soybean growth under greenhouse conditions. Biology and Fertility of Soils 43, 805-809. Fraga, R., Rodriguez, H. and Gonzalez, T. [2001]. Transfer of the gene encoding the NapA acid phosphatase from Morganella marganii to a Burkholderia cepacia strain. Acta Biotechnologica 21, 359-369. Gadagi, R. S. and Tongmin, S. A. [2002]. New isolation method for microorganisms solubilizing iron and aluminium phosphates using dyes. Soil Science and Plant Nutrition 48, 615-618. Gaind, S. and Gaur, A. C. [1991]. Thermotolerant phosphate solubilizing microorganisms and their interaction with mungbean. Plant and Soil 133, 141-149. Gaind, S. and Gaur, A. C. [1990]. Influence of temperature on the efficacy of phosphorus solubilizing microorganisms. Indian Journal of Microbiology 30, 305-310. Galar, M. L. and Bolardi, J. L. [1995]. Evidence for a membrane bound pyrrolquinoline quinine- linked glucose dehydrogenase in Acetobacter diazotrophicus. Applied and Environmental Microbiology 43, 713-716. Garbaye, J. [1994]. Helper bacteria: a new dimension to the mycorrhizal symbiosis. New Phytology 128, 197-210. Gaur, A. C. [1990]. Phosphate Solubilizing Microorganisms as Biofertilizer. pp. 176. New Delhi: Omega Scientific Publications. Gaur, A. C., Madan, M. and Ostwal, K. P. [1973]. Solubilization of phosphatic compounds by native microflora of rock phosphates. Indian Journal of Experimental Biology 11, 427429. Geelhoed, J. S., van Riemsdijk, W. H. and Findenegg, G. R. [1999]. Simulation of the effect of citrate exudation from roots on the plant availability of phosphate adsorbed on goethite. European Journal of Soil Science 50, 379-390. Gerretson, F. C. [1948]. The influence of microorganisms on the phosphate intake by the plants. Plant and Soil 1, 51-81. Ghosh, G. and Poi, S. C. [1990]. Response of Rhizobium, phosphate solubilizing bacteria and mycorrhizal organisms in legume crops. Environment and Ecology 16, 607-610. Gianinazzi-Pearson, V. [1996]. Plant cell responses to arbuscular mycorrhizal fungi: getting to the roots of the symbiosis. The Plant Cell 8, 1871-1883. Glick, B. R. [1995]. The enhancement of plant growth by free-living bacteria. Canadian Journal of Microbiology 32, 145-148. Goel, A. K., Sindhu, S. S. and Dadarwal, K. R. [2002]. Stimulation of nodulation and plant growth of chickpea [Cicer arietinum L.] by Pseudomonas spp. antagonistic to fungal pathogens. Biology and Fertility of Soils 36, 391-396. Goldstein, A. H. [1986]. Bacterial solubilization of microbial phosphates: Historical perspective and future prospects. American Journal of Alternative Agriculture 1, 51-57. Goldstein, A. H. [1995]. Recent progress in understanding the molecular genetics and biochemistry of calcium phosphate solubilization by Gram-negative bacteria. Biology of Agriculture and Horticulture 12, 185-193. Goldstein, A. H. [1996]. Involvement of the quinoprotein glucose dehydrogenase in the solubilization of exogenous phosphates by Gram-negative bacteria. In: Toriani-Gorini,

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Molecular Genetics of Phosphate Solubilization in Rhizosphere Bacteria…

221

A., Yagil, E. and Silver, S. [Eds.], Phosphate in microorganisms: Cellular and Molecular Biology. [pp. 197-203]. Washington, DC: ASM Press. Goldstein, A. H. [2007]. Future trends in research on microbial phosphate solubilization: one hundred years of insolubility. In: Velazquez, E. and Roderguez-Barrueco, C. [Eds.]. Proceedings of First International Meeting on Microbial Phosphate Solubilization. [pp. 91-96]. Berlin: Springer-Verlag. Goldstein, A. H. and Liu, S. T. [1987]. Molecular cloning and regulation of a mineral phosphate solubilizing gene from Erwinia herbicola. Biotechnology 5, 72-74. Goldstein, A. H., Rogers, R. D. and Mead, G. [1993]. Separating phosphate from ores via bioprocessing. Biotechnology 11, 1250-1254. Golovan, S., Wang, G., Zhang, J. and Forsberg, C. W. [2000]. Characterization and overproduction of the Escherichia coli appA encoded bifunctional enzyme that exhibits both phytase and acid phosphatase activities. Canadian Journal of Microbiology 46, 5971. Grimes, H. D. and Mount, M. S. [1984]. Influence of Pseudomonas putida on nodulation of Phaseolus vulgaris. Soil Biology and Biochemistry 16, 27-30. Gupta, R., Singal, R., Shankar, A., Kuhad, R. C. and Saxena, R. K. [1994]. Modified plate assay for screening of phosphate solubilizing microorganisms. Journal of General and Applied Microbiology 40, 255-260. Gyaneshwar, P., Naresh, K. G., Parekh, L. J. and Poole, P. S. [2002]. Role of soil microorganisms in improving P nutrition of plants. Plant and Soil 245, 83-93. Halder, A. K., Mishra, A. K. and Chakrabarty, P. K. [1991]. Solubilizing of inorganic phosphates by Bradyrhizobium. Indian Journal of Experimental Biology 29, 28-31. Halvorson, H. O., Kenyan, A. and Korenberg, H. L. [1990]. Utilization of calcium phosphates for microbial growth at alkaline pH. Soil Biology and Biochemistry 22, 887-890. Han, Y., Wilson, D. B. and Lei, X. G. [1999]. Expression of an Aspergillus niger phytase gene [phyA] in Saccharomyces cerevisiae. Applied and Environmental Microbiology 65, 1915-1918. Harrison, M. J. [2005]. Signaling in the arbuscular mycorrhizal symbiosis. Annual Review of Microbiology 59, 19-42. Hasan, R. [1994]. Phosphorus fertility status of soils in India. Phosphorus research in India. In: Dev, G. [Ed.], Proc. of Group Discussion, Indian Agricultural Research Institute, New Delhi. [pp. 8-12]. Gurgaon: Principal Publications. Hebbara, M. and Suseeladevi, L. [1990]. Effect of phosphorus solubilizing bacteria [PSB] on phosphorus availability to groundnut from rock phosphate. Current Research 19, 56-57. Henri, F., Laurette, N. N., Annette, D., John Q., Wolfgang, M, François-Xavier, E and Dieudonné, N. [2008] Solubilization of inorganic phosphates and plant growth promotion by strains of Pseudomonas fluorescens isolated from acidic soils of Cameroon. African Journal of Microbiology Research 2, 171-178. Holford, I. C. R. [1997]. Soil phosphorus: its measurement and its uptake by plants. Australian Journal of Soil Research 35, 227-239. Huelett, F. M., Sun, G. and Liu, W. [2007]. The Pho regulon of Bacillus subtilis is regulated by sequential action of two genetic switches. In: Toriani-Gorini, A., Yagil, E. and Silver, S. [Eds.], Phosphate in microorganisms: Cellular and Molecular Biology. [pp. 50-54]. Washington, DC: ASM Press.

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222

S. S. Sindhu, M. K. Verma and Suman Mor

Idriss, E. E., Makarewicz, O., Farouk, A., Rosner, K., Greiner, R., Bochow, H., Richter, T. and Borris, R. [2002]. Extracellular phytase activity of Bacillus amyloliquefaciens FZB45 contributes to its plant growth promoting effects. Microbiology 148, 2097-2109. Igual, J. M., Valverde, A., Ceravantes, E. and Velazquez, E. [2001]. Phosphate solubilizing bacteria as inoculants for agriculture: use of updated molecular techniques in their study. Agronomie 21, 561-568. Illmer, P. and Schinner, F. [1992]. Solubilization of inorganic phosphates by microorganisms isolated from forest soils. Soil Biology and Biochemistry 24, 389-395. Illmer, P. and Schinner, F. [1995]. Solubilization of inorganic calcium phosphates: solubilization mechanisms. Soil Biology and Biochemistry 27, 257-263. Illmer, P., Babato, A. and Schinner, F. [1995]. Solubilization of hardly soluble aluminium phosphate with phosphate solubilizing microorganisms. Soil Biology and Biochemistry 27, 265-270. Jain, L. K. and Singh, P. [2003]. Growth and nutrient uptake of chickpea [Cicer arietinum L.] as influenced by biofertilizers and phosphorus nutrition. Crop Research 25, 410-413. Jisha, M. S. and Alagawadi, A. R. [1996]. Nutrient uptake and yield of sorghum [Sorghum bicolor L. Moench] inoculated with phosphate solubilizing bacteria and cellulolytic fungus in cotton stalk amended vertisol. Microbiological Research 151, 1-5. Jisha, M. S. [1997]. Optimization of factors for efficient solubilization of mineral phosphates. Ph. D. thesis, PG School, IARI, New Delhi. Juriank, J. J., Dudley, L. M., Allen, M. F. and Knight, W. G. [1986]. The role of calcium oxalate in the availability of phosphorus in soils of semiarid regions: thermodynamic study. Soil Science 142, 255-261. Katznelson, H. and Bose, B. [1959]. Metabolic activity and phosphate dissolving activity of bacterial isolates from wheat roots, rhizosphere and non-rhizosphere soil. Canadian Journal of Microbiology 5, 79-85. Kerovuo, J., Lauracus, M., Nurminen, P., Kalkinen, N. and Apajalahti, J. [1998]. Isolation, characterization, molecular gene cloning and sequencing of a novel phytase from Bacillus subtilis. Applied and Environmental Microbiology 64, 2079-2085. Khan, M. S., Zaidi, A. and Wani, P. A. [2007]. Role of phosphate solubilizing microorganisms in sustainable agriculture-A review. Agronomy for Sustainable Development 27, 29-43. Kim, K. Y., Jordan, D. and Krishnan, H. B. [1998b]. Expression of genes from Rahnella aquatilis that are necessary for mineral phosphate solubilization in Escherichia coli. FEMS Microbiology Letters 159, 121-127. Kim, K. Y., Jordan, D. and McDonald, G. A. [1998c]. Effect of phosphate solubilizing bacteria and vesicular-arbuscular mycorrhizae on tomato growth and soil microbial activity. Biology and Fertility of Soils 26, 79-87. Kim, K. Y., McDonald, G. A. and Jordan, D. [1997]. Solubilization of hydroxyapatite by Enterobacter agglomerans and cloned Escherichia coli in culture medium. Biology and Fertility of Soils 24, 347-352. Kim, K.Y., Jordan, D. and McDonald, G.A. [1998a]. Enterobacter agglomerans, phosphate solubilizing bacteria and microbial diversity in soil: Effect of carbon sources. Soil Biology and Biochemistry 30, 995-1003.

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Molecular Genetics of Phosphate Solubilization in Rhizosphere Bacteria…

223

Kim, Y. O., Lee, J. K, Kim, H. K., Yu, J. H. and Oh, T. K. [1998d]. Cloning of thermostable phytase gene [phy] from Bacillus sp. DS11 and its overexpression in Escherichia coli. FEMS Microbiology Letters 162, 185-191. Kirk, J. L., Beaudette, L. A., Hart, M., Moutoglis, P., Klironomus, J. N., Lee, H. and Trevors, J. T. [2004]. Methods of studying soil microbial diversity. Journal of Microbiological Methods 58, 169-188. Kole, S. C. and Hazra, J. N. [1998]. Occurrence and acidity of tricalcium phosphate and rock phosphate solubilizing microorganisms in mechanical compost plants of Calcutta and an alluvial soil of West Bengal. Environment and Ecology 16, 344-349. Krishanaraj, P. U. and Goldstein, A. H. [2001]. Cloning of a Serratia marcescens DNA fragment that induces quino-protein glucose dehydrogenase-mediated gluconic acid production in Escherichia coli in the presence of Serratia marcescens. FEMS Microbiology Letters 205, 215-220. Krishanaraj, P. U. [1987]. Studies on beneficial microorganisms in crop plants. M. Sc. thesis, UAS, Bangalore. Krishanaraj, P. U. [1996]. Genetic characterization of mineral phosphate solubilization in Pseudomonas sp. Ph. D. thesis, IARI, New Delhi. Krishanaraj, P. U., Sadasivam, K. V. and Khanuja, P. S. [1999]. Mineral phosphate solubilization defective mutants of Pseudomonas sp. express pleiotropic phenotypes. Current Science 76, 1032-1034. Kucey, R. M. N. [1987]. Increased phosphorus uptake by wheat and field beans inoculated with a phosphorus solubilizing Penicillium bilaji strain and vesicular arbuscular mycorrhizal fungi. Applied and Environmental Microbiology 53, 2699-2703. Kucey, R. M. N. [1988]. Effect of Penicillium bilaji on the solubility and uptake of P and micronutrients from soil by wheat. Canadian Journal of Soil Science 68, 261-270. Kucey, R. M. N., Janzen, H. H. and Legett, M. E. [1989]. Microbially mediated increases in plant-available phosphorus. Advances in Agronomy 42, 198-228. Kumrawat, B., Dighe, J. M., Sharma, R. A. and Katti, G. V. [1997]. Response of soybean to biofertilizer in black clay soils. Crop Research 14, 209-214. Kundu, B. S. and Gaur, A. C. [1980a]. Effects of phosphobacteria on yield and phosphate uptake by potato crop. Current Science 48, 159. Kundu, B.S. and Gaur, A.C. [1980b]. Establishment of nitrogen and phosphate solubilizing bacteria in rhizosphere and their effect on yield and nutrient uptake of wheat crop. Plant and Soil 57, 223-230. Kundu, B. S. and Gaur, A. C. [1980c]. Effect of nitrogen fixing and phosphate solubilizing microorganisms as single and composite inoculants on cotton. Indian Journal of Microbiology 20, 225-229. Kundu, B. S. and Gaur, A. C. [1982]. Yield increase of wheat after inoculation with A. chroococcum and phosphobacteria. Current Science 51, 291-293. Kundu, B. S. and Gaur, A. C. [1984]. Rice response to inoculation with nitrogen fixing bacteria and PSM. Plant and Soil 78, 227-234. Larsen, S. [1967]. Soil phosphorus. Advances in Agronomy 19, 151-210. Lei, X. G., Ku, P. K., Miller, E. R., Yokoyama, M. T. and Ullrey, D. E. [1993]. Supplementing corn-soybean meal diets with microbial phytase maximizes phytate P utilization by winling pigs. Journal of Animal Sciences 71, 3368-3375.

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224

S. S. Sindhu, M. K. Verma and Suman Mor

Lifschitz, R. Kloepper, J. W., Kozlowski, M., Simonson, C., Carlson, J., Tipling, E. M. and Zaleska, T. [1987]. Growth promotion of canola [rape seed] seedlings by a strain of Pseudomonas putida under gnotobiotic conditions. Canadian Journal of Microbiology 33, 390-395. Liu, S. T., Lee, L. Y., Jai, C. Y., Hung, C. H., Chang, Y. S., Wolfram, J. H., Rogers, B. and Goldstein, A. H. [1992]. Cloning of an Erwinia herbicola gene necessary for gluconic acid production and enhanced mineral phosphate solubilization in E. coli HB101: Nucleotide sequence and probable involvement in biosynthesis of the co-enzyme pyrrolquinoline quinine. Journal of Bacteriology 174, 5814-5819. Lopez-Bucio, J., de la Vega, O. M., Guevara-Garcia, A. and Herreera-Estrella, I. [2000]. Enhanced phosphate uptake in transgenic tobacco plants that overproduce citrate. Natural Biotechnology 18, 450-453. Lorenzo, V., Herrero, M., Jakubjik, U. and Timmis, K. N. [1990]. Mini-Tn5 transposon derivatives for transposon mutagenesis, promoter probing and chromosomal insertion of cloned DNA in Gram-negative eubacteria. Journal of Bacteriology 172, 6568-6572. Lugtenberg, B. J. J., de Weger, L. A. and Bennett, J. W. [1991]. Microbial stimulation of plant growth and protection from disease. Current Opinion in Microbiology 2, 457-464. Macaskie, L. E., Young, P., Doyle, T. C., Roig, M. G., Diaz, M. and Monzano, T. [1997]. Bioremediation of uranium-bearing waste water: biochemical and chemical factors affecting bioprocess application. Biotechnology and Bioengineering 53, 100-109. Maheshkumar, K. S. [1997]. Studies on microbial diversity and their activity in soil under bamboo plantations. M. Sc. thesis, UAS, Dharwad. Maheshkumar, K. S.; Krishanraj, P. U. and Alagawadi, A. R. [1999]. Mineral phosphate solubilizing activity of Acetobacter diazotrophicus: a bacterium associated with sugarcane. Current Science 76, 874-875. Makino, K., Amemura, M., Kim, S. K., Nakata, A. and Shinagawa, H. [2007]. Mechanism of transcriptional activation of the phosphate regulon in Eschericia coli. In: Toriani-Gorini, A., Yagil, E. and Silver, S. [Eds.], Phosphate in microorganisms: Cellular and Molecular Biology. [pp. 5-12]. Washington, DC: ASM Press. Mehta, S. and Nautiyal, S. C. [2001]. An efficient method for qualitative screening of phosphate-solubilizing bacteria. Current Microbiology 43, 51-56. Mishra, M. M., Kapoor, K. K. and Yadav, K. S. [1982]. Effect of compost enriched with Mussorie rock phosphate on crop yield. Indian Journal of Agricultural Sciences 52, 674678. Monod, S. P. I., Gupta, D. N. and Chavan, A. S. [1989]. Enhancement of phosphate availability and phosphorus uptake in rice by phosphate solubilizing culture. Journal of Maharastra Agricultural University 14, 178-181. Mortensen, J. L. [1963]. Complexing of metals by soil organic matter. Soil Science Society of America Proceedings 27, 179-186. Naik, P. R., Raman, G., Narayanan, K. B. and Sakthivel, N. [2008]. Assessment of genetic and functional diversity of phosphate solubilizing fluorescent pseudomonads isolated from rhizospheric soil. BMC Microbiology 8, 230-243. Nautiyal, C. S. [1990]. An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiology Letters 170, 265-270.

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Molecular Genetics of Phosphate Solubilization in Rhizosphere Bacteria…

225

Ocampo, J. A., Barea, J. M. and Montoyar, E. [1975]. Interference between Azotobacter and phosphobacteria and their establishment in the rhizosphere as affected by soil fertility. Canadian Journal of Microbiology 21, 1160-1165. Ogawa, N., Hayashi, N., Salto, H., Noguchi, K., Yamashita, Y. and Oshima, Y. [2007]. Regulatory circuit for phosphatase genes in Saccharomyces cerevisiae: specific cis-acting sites in Pho promoters for binding the Pho4p. In: Toriani-Gorini, A., Yagil, E. and Silver, S. [Eds.], Phosphate in microorganisms: Cellular and Molecular Biology. [pp. 56-62]. Washington, DC: ASM Press. Ostwal, K. P. and Bhide, V. P. [1972]. Solubilization of tricalcium phosphate by soil Pseudomonas. Indian Journal of Experimental Biology 10, 153-154. Otani, T., Ae, N. and Tanaka, H. [1996]. Phosphorus [P] uptake mechanisms of crops grown in soils with low P status: II. Significance of organic acids in root exudates of pigeonpea. Journal of Soil Science and Plant Nutrition 42, 553-560. Parks, E. J., Olson, G. J., Brickman, P. E. and Baldi, F. [1990]. Characterization of high performance liquid chromatography [HPLC] of the solubilization of phosphorus in iron ore by a fungus. Indian Journal of Microbiology 5, 183-190. Prabhakar, M. and Saraf, C. S. [1990]. Dry matter accumulation and distribution in chickpea as influenced by genotype, P source and irrigation level. Indian Journal of Agricultural Sciences 60, 204-206. Puente, M. E., Bashan, Y., Li, C. Y. and Lebsky, V. K. [2004]. Microbial populations and activities in the rhizoplane of rock-weathering desert plants. Root colonization and weathering of igneous rocks. Plant Biology 6, 629-642. Rachewad, S. N., Raut, R. S., Malewar, G. U. and Ganure, C. K. [1992]. Effects of phosphate solubilizing biofertilizer on biomass production and uptake of phosphorus by sunflower. J. Maharastra Agric. Univ. 17, 480-481. Rajarathinam, K., Balamurugan, T., Kulasekarapandian, R., Veersami, S. and Jayabalan, M. [1995]. Isolation and screening of phosphate solubilizers from soil of Kamarajar district [Tamil Nadu]. Journal of Exotoxicology and Environmental Monitoring 5, 155-157. Rashid, M., Khalil, S., Ayub, N., Alam, S. and Latif, M. [2004]. Organic acids production and phosphate solubilization by phosphate solubilizing microorganisms [PSM] under in vitro conditions. Pakistan Journal of Biological Sciences 7, 187-196. Reilly, T. J., Baron, G. S., Nano, F. and Kuhlenschmidt, M. S. [1996]. Characterization and sequence of a respiratory burst-inhibiting acid phosphatase from Francisella tularensis. Journal of Biological Chemistry 271, 10973-10983. Reyes, I., Baziramakenga, R., Bernier, L. and Antoun, H. [2001]. Solubilization of phosphate rocks and minerals by a wild-type strain and two UV- induced mutants of Penicillium rugulosum. Soil Biology and Biochemistry 33, 1741-1747. Reyes, I., Bernier, L., Simard, R. R., Tanguay, P. and Antoun, H. [1999]. Characteristics of phosphate solubilization by an isolate of a tropical Penicillium rugulosum and two UVinduced mutants. FEMS Microbiology and Ecology 28, 291-295. Richardson, A. E. [2001]. Prospects for using soil microorganisms to improve the acquisition of phosphorus by plants. Australian Journal of Plant Physiology 28, 897-906. Richardson, A. E. [1994]. Soil microorganisms and phosphorus availability. In: Pankhurst, C. E., Doube, B. M. and Gupta, V. V. S. R. [Eds.], Soil Biota: Management in Sustainable Farming Systems. [pp. 50-62]. Victoria, Australia: CSIRO.

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226

S. S. Sindhu, M. K. Verma and Suman Mor

Richardson, A. E., Hadobas, P. A. and Hayes, J. E. [2001a]. Extracellular secretion of Aspergillus phytase from Arabidopsis roots enables plants to obtain phosphorus from phytate. Plant Journal 25, 641-649. Richardson, A. E., Hadobas, P. A., Hayes, J. E., O’Hara, C. P. and Simpson, R. J. [2001b]. Utilization of phosphorus by pasteur plants supplied with meso-inositol hexaphosphate is enhanced by the presence of soil microorganisms. Plant and Soil 229, 47-56. Rodriguez, E., Han, Y. and Lei, X. G. [1999]. Cloning, sequencing and expression of an Escherichia coli acid phosphatase/phytase gene [appA2] isolated from pig colon. Biochemical and Biophysical Research Communications 257, 117-123. Rodriguez, H. and Fraga, R. [1999]. Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnological Advances 17, 319-339. Rodriguez, H., Fraga, R., Gonzalez, T. and Bashan, Y. [2006]. Genetics of phosphate solubilization and its potential applications for improving plant growth promoting bacteria. Plant and Soil 287, 15-21. Rodriguez, H., Gonzalez, T. and Selman, G. [2000b]. Expression of a mineral phosphate solubilizing gene from Erwinia herbicola in two rhizobacterial strains. Journal of Biotechnology 84, 155-161. Rodriguez, H., Gonzalez, T., Goire, I. and Bashan, Y. [2004]. Gluconic acid production and phosphate solubilization by the plant growth promoting bacterium Azospirillum spp. Naturwissenschaften 91, 552-555. Rodriguez, H., Rossolini, G. M., Gonzalez, T., Jiping, L. and Glick, B. R. [2000a]. Isolation of a gene from Burkholderia cepacia IS-16 encoding a protein that facilitates phosphatase activity. Current Microbiology 40, 362-366. Rossolini, G. M., Schippa, S., Riccio, M. L., Berlutti, F., Macaskie, L. E. and Thallar M. C. [1998]. Bacterial non-specific acid phosphatases: physiology, evolution and use as tools in microbial biotechnology. Cellular and Molecular Life Sciences 54, 833-850. Roychaudhary, P. and Kaushik, B. D. [1989]. Solubilization of Mussoorie rock phosphate by cyanobacteria. Current Science 58, 569-570. Ryan, P. R., Delhaise, E. and Jones, D. L. [2001]. Function and mechanism of organic anion exudation from plant roots. Annual Review of Plant Physiology and Plant Molecular Biology 52, 527-560. Santhi, V. [1998]. Mechanism of mineral phosphate solubilization and growth promotion by diverse bacteria. M. Sc. Thesis, University of Agricultural Sciences, Dharwad. Saraf, C. S., Shivkumar, B. G. and Patil, R. R. [1997]. Effect of phosphorus, sulphur and seed inoculation on performance of chickpea [Cicer arietinum]. Indian Journal of Agronomy 42, 323-328. Sarawgi, S. K., Tiwari, P. K. and Tripathi, R. S. [2000]. Growth, nodulation and yield of chickpea as influenced by phosphorus, bacterial culture and micronutrients under rain-fed conditions. Madras Agriculture Journal 86, 181-185. Sarawgi, S. K., Tiwari, P. K. and Tripathi, R. S. [1999]. Uptake and balance sheet of nitrogen and phosphorus in gram [Cicer arietinum] as influenced by phosphorus biofertilizers and micronutrients under rain-fed conditions. Indian Journal of Agronomy 44, 768-772. Sharma, S. N. [2003]. Effect of phosphate solubilizing bacteria on the efficacy of Mussoorie rock phosphate in rice [Oryza sativa] - wheat [Triticum aestivum] cropping system. Indian Journal of Agricultural Sciences 73, 478-481.

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Molecular Genetics of Phosphate Solubilization in Rhizosphere Bacteria…

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Sheshardri, S., Kumaraswamy, R., Lakshminarasimhan, C. and Ignacimuthu, S. [2000]. Solubilization of inorganic phosphate by Azospirillum halopraeferans. Current Science 79, 565-567. Singh, C. P., Mishra, M. M. and Yadav, K. S. [1980]. Solubilization of insoluble phosphates by thermophilic fungi. Annals of Microbiology [Inst. Pasteur] 131, 289-296. Singh, H. P., Pareek, R. P. and Singh, P. A. [1984]. Solubilization of rock phosphate by phosphate solubilizers in broth. Current Science 53, 1212-1213. Singh, S. and Kapoor, K. K. [1994]. Solubilization of insoluble phosphate by microorganisms isolated from different sources. Environment and Ecology 12, 51-55. Sonboir, H. L. and Sarawgi, S. K. [2000]. Nutrients uptake, growth and yield of chickpea as influenced by phosphorus, Rhizobium and phosphate solubilizing bacteria. Madras Agriculture Journal 87, 149-151. Sperber, J. I. [1958]. Solution of apatite by soil microorganisms producing organic acids. Australian Journal of Agricultural Research 9, 778-781. Srivastava, T. K., Ahalwat, L. P. S. and Panwar, J. D. S. [1998]. Effect of phosphorus, molybdenum and biofertilizers on productivity of pea. Indian Journal of Plant Physiology 3, 237-239. Stalstorm, V.A. [1903]. Beitrag, Zur Kenntrusder einwinsking sterilizer and in garung befindlieher strife any dil Loslishkerd der phosphorus are destrical cum phosphorus. Zentralbl Bakteriol Abt 11, 724-732. Surange, S. and Kumar, N. [1993]. Phosphate solubilization under varying pH by Rhizobium from tree legumes. Indian Journal of Experimental Biology 31, 427-429. Taha, S. M., Mahamood, S. A. Z., Halim, E. I., Damaty, A. and Hafez, A. M. [1969]. Activity of phosphate dissolving bacteria in Egyptian soils. Plant and Soil 31, 149-160. Tanwar, S. P. S., Sharma, G. L and Chahar, M. S. [2002]. Effect of phosphorus and biofertilizers on growth and productivity of black gram. Annual Agricultural Research 23, 491-493. Thakkar, J., Narsian, V. and Patel, H. H. [1993]. Inorganic phosphate solubilization by certain soil bacteria: solubilization of natural rock phosphates and pure insoluble inorganic phosphate by Aspergillus awamorii. Indian Journal of Experimental Biology 31, 743747. Thallar, M. C., Berlutti, F., Schippa, S., Lombardi, G. and Rossolini, G. M. [1994]. Characterization and sequence of PhoC, the principal phosphatase-irrepressible acid phosphatase of Morganella morganii. Microbiology 140, 1341-1350. Thallar, M. C., Berlutti, F., Schippa, S., Lori, P. Passariello, C. and Rossolini, G. M. [1995a]. Heterogenous patterns of acid phosphatases containing low molecular mass polypeptides in members of the family Enterobacteriaceae. International Journal of Systematic Bacteriology 4, 255-261. Thallar, M. C., Lombardi, G., Berlutti, F., Schippa, S. and Rossolini, G.M. [1995b]. Cloning and characterization of the NapA acid phosphatase/phosphotransferase of Morganella morganii. Identification of a new family of bacterial acid phosphatase encoding genes. Microbiology 140, 147-151. Tilak, K. V. B. R., Saxena, A. K. and Sadasivan, K. V. [1995]. Synergistic effects of phosphate solubilizing bacterium Pseudomonas striata and arbuscular mycorrhizae on soybean. In: Sujan Singh and Adholya, A. [Eds.], Mycorrhizae: Biofertilizers for the Future. [pp. 224-226]. New Delhi: Tata Energy Research Institute.

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228

S. S. Sindhu, M. K. Verma and Suman Mor

Tomar, M. [2005]. Biodiversity of mineral phosphate solubilizing bacteria from chickpea, mustard and wheat rhizosphere. Ph. D. thesis submitted to CCS HAU, Hisar. Toro, M., Azcon, R. and Barea, J. M. [1997]. Improvement of arbuscular mycorrhiza development by inoculation of soil with phosphate solubilizing rhizobacteria to improve rock phosphate bioavailability [32P] and nutrient cycling. Applied and Environmental Microbiology 63, 4408-4412. Torsvik, V., Salte, K., Soerheim, R. and Goksoyr, J. [1990]. Comparison of phenotypic diversity and DNA heterogeneity in a population of soil bacteria. Applied and Environmental Microbiology 56, 776-781. Tye, A. J., Siu, F. K., Leung, T. Y. and Lim, B. L. [2002]. Molecular cloning and the biochemical characterization of two novel phytases from Bacillus subtilis 168 and Bacillus licheniformis. Applied and Environmental Microbiology 59, 190-197. van Schie, B. J., Hellingwerf, K. E., Vandijkan, J. P., Elferink, M. G. L., Van Diji, J. M., Kuenen, J. G. and Konigns, N. [1985]. Energy transduction by electron transfer via a pyrroquinoline quinine dependent glucose dehydrogenase in Escherichia coli, Pseudomonas aeruginosa and Acinetobacter calcoaceticum [var. Lowoffi]. Journal of Bacteriology 163, 493-499. Varsha-Narsian and Patel, H. H. [1995]. Inorganic phosphate solubilization by some yeast. Indian Journal of Microbiology 35, 127-132. Varsha-Narsian, Thakkar, J. and Patel, H. H. [1994]. Isolation and screening of phosphate solubilizing fungi. Indian Journal of Microbiology 34, 113-118. Venkateswarlu, B., Rao, A. V. and Raina, P. [1984]. Evaluation of phosphorus solubilization by microorganisms isolated from arid soils. Journal of Indian Society of Soil Science 32, 273-277. Wall, D. H. and Virginia, R. A. [1999]. Control of soil biodiversity- in sight from extreme environments. Applied Soil Ecology 13, 137-150. Wanner, B. L. [1996]. Phosphorus assimilation and control of the phosphate regulon. In: Niedhardt, F. C., Curtis, R. III, Ingraham, J. L., Lin, E. C., Low, K. B., Magasanik, B., Reznikoff, W. S., Riley, M., Schaechter, M. and Umbarger, H. E. [Eds.], Escherichia coli and Salmonella: Cellular and Molecular Biology. [2nd Edition, pp. 1357-1381]. Washington, DC: IASM Press. Yadav, K. S. and Dadarwal, K. R. [1997]. Phosphate solubilization and mobilization through soil microorganisms. In: Dadarwal, K. R. [Ed.], Biotechnological approaches in soil microorganisms for sustainable crop production. [pp. 293-308]. Jodhpur, India: Scientific Publishers. Yadav, B. N., Singh, D. and Singh, S. M. [2004]. Performance of barley [Hordeum vulgare L.] varieties under varying fertilizer levels and microbial inoculation. Agricultural Science Digest 24, 148-150.

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Chapter 10

STRATEGIES FOR DEVELOPMENT OF MICROPHOS AND MECHANISMS OF PHOSPHATE-SOLUBILIZATION Almas Zaidi∗, Md. Saghir Khan, Mohd. Oves and Munees Ahemad Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh-202002; U.P., India

ABSTRACT

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Although, phosphorus is abundant in soils in both organic and inorganic forms, it is frequently a major or even the prime limiting factor for plant growth. The bioavailability of soil inorganic P in the rhizosphere varies considerably with plant species, nutritional status of soil and ambient soil conditions. To overcome the P deficiency, phosphatesolubilizing microorganisms [PSM] could play an important role in supplying phosphate to plants in a more environment friendly and sustainable manner. The solubilization of phosphatic compounds by PSM is though, common under in vitro conditions; the performance of PSM in situ has been contradictory. The variations in the performance has thus, greatly hampered the large-scale application of PSM [s]. The challenge is therefore, how to make use of such biological resources to maintain soil health while increasing the crop productivity by providing P to plants through the application of PSM. Moreover, unlike, animal and plant ecologists which can quantify and identify different species through [relatively] easily identifiable traits, it is extremely difficult to do this with microbial communities, because microbes vary greatly in their numbers and their associated activities. The assessment of the composition and/or function of soil microbial communities including PSM thus, present a number of challenges, due to which less than 1% of bacterial species and an unknown percentage of fungi have been recovered so far. The present chapter focuses on the screening, development and mode of inoculants application and mechanisms of P solubilization by microbes.

∗

Correspondence to: [email protected]

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10.1. INTRODUCTION Phosphorus [P] plays a pivotal role in several physiological and biochemical plant activities, like, photosynthesis, transformation of sugar to starch, and transfer of the genetic traits. Moreover, the benefits of providing the plants with sufficient P are that it creates deeper and more abundant roots [Sharma 2002]. However, of the total soil P, only 1 to 5% is in plant available form [Molla and Chaudhury 1984]. In tropical soils including sub Saharan African soils, P deficiency is considered to be one of the main biophysical constraint to crop productivity and consequently to food security [Chien and Menon 1995; Sanchez et al. 1997; Khan et al. 2007]. On the other hand, in regions where soils are high in total P, including some temperate soils, deficiencies may also occur if soluble forms of P are not replenished following plant uptake of P from soil solution [Arcand and Schneider 2006]. For example, organically managed soils have been found to become deficient in plant available P over the long-term without external P inputs [Entz et al. 2001; Oehl et al. 2002]. The use of conventional environmentally unfriendly chemical phosphatic fertilizers is usually limited in developing countries due in part to the cost involved. Therefore, the focus has been shifted towards the use of naturally abundant microbial communities possessing exceptional qualities of phosphate solubilization under natural ecosystems. Of the heterogeneous microbial populations inhabiting rhizosphere, certain microorganisms capable of solubilizing insoluble P and collectively termed phosphate-solubilizing microorganisms [PSM] are of great practical agronomic interest, as they can be used as inoculants for crop improvement [Igual et al. 2001; Konietzny and Greiner 2004; Zaidi and Khan 2007; Wani et al. 2007a]. A large number of rhizospheric communities possessing PS potentials are known [Chen et al. 2006; Wani et al. 2007b; Ramachandran et al. 2007; Henri et al. 2008; Hameeda et al. 2008; Patel et al. 2008].]. And hence, the application of P-solubilizing microorganisms as inocula or the management of their populations in soil has become an alternative to improve P availability for plants [Silva Filho and Vidor 2000]. In addition to the P-solubilizing action, PSM also increases population of active and effective microorganisms in the root activity zone which increases plant ability to uptake more nutrient. The production of such fertilizers, however, requires the selection of beneficial soil microorganisms which possess the highest efficiency to facilitate plant growth by providing P nutrients in a readily absorbable form. The principal mechanism for mineral phosphate solubilization [mps] is the production of organic acids, while enzymes [e.g., phosphatases] play a major role in the mineralization of organic P in soil. The dissolution of insoluble P in soil however, is influenced greatly by changes in pH values, root exudates, variation in soil types, and plant genotypes. Several phosphatase-encoding genes have been cloned and characterized and a few genes involved in mps have been isolated. Therefore, genetic manipulation of PSM to improve their ability to facilitate plant growth may include cloning genes involved in both mineral and organic P solubilization, followed by their expression in selected rhizobacterial strains. Chromosomal insertion of these genes under appropriate promoters is an interesting approach [Rodríguez and Fraga 1999]. Inoculation by microphos [microbial inoculants with P-solubilizing potential] has been shown to improve P uptake by many plants [Rodriguez et al. 2006; Khan et al. 2009]. For example, the effect of seed inoculation by phosphate-solubilizing microorganisms and different levels of phosphorus chemical fertilizer on yield and yield components of barley

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[Hordeum vulgare] was studied in experimental farm of college of agronomy and animal sciences, University of Tehran, during 2006-2007 growing seasons using three P levels [0, 30 and 60 kg/ha] and three levels of PS bacteria of 0 [control], Pseudomonas putida [accessions number 9 and 41] along with two levels of mycorrhiza [with and without mycorrhiza]. Sole application of bacteria [accession 9] produced the maximum biological yield, while the application of same bacteria along with mycorrhiza further improved the production of seed yield. Seed inoculation with sole bacteria positively affected the number of the seeds per kernel. Application of mycorrhiza along with bacteria significantly increased leaf chlorophyll content. Phosphate-solubilizing bacteria [accession 41] in absence of any chemical phosphorus fertilizer had an appropriate performance and could increase biomass production to an acceptable level suggesting that it could serve as an alternative to chemical phosphorous fertilizer in agricultural systems [Mehrvarz et al. 2008]. Increased growth, yield and P uptake by other plants following microphos inoculation is reported [Saxena and Sharma, 2003; Ponmurugan and Gopi, 2006]. Strains from the genera Pseudomonas, Bacillus and Rhizobium are among the most powerful phosphate solubilizers [Khan et al. 2007].

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10.2. DEVELOPMENT OF PHOSPHATE-SOLUBILIZING MICROBIAL INOCULANTS Phosphate-solubilizing activities of the microbial populations have attracted their use as biofertilizer and hence, are receiving more attention by agronomists than microbiologists [Khan et al. 2007; Khan et al. 2009]. Therefore, the organisms showing greater solubilization [both qualitatively and quantitatively] of insoluble P under in vitro conditions are selected for bulk production; for their ultimate transmission to the farmers. The production of viable microbial preparations possessing P-solubilizing activity [microphos] involves three critical stages- [i] screening and selection and in vitro evaluation of P-solubilizing potentials of the microbial strains [ii] selection of carriers, mixing of inocula with selected carriers and proper development of microbial inoculants and [iii] testing of the quality of inoculants in terms of persistence of P- solubilizing activity, viable microbial load/g of carrier and proper distribution to the farmers.

10.2.1. Isolation, Screening and Selection of Phosphate-Solubilizing Microbes Microorganisms play a fundamental role in the biogeochemical cycling of elements including P in natural ecosystems. Of these microbes, PSM [s] are ubiquitous but vary in density, pigment formation [Figure 1] and mineral PS [mps] ability from soil to soil or from one production system to another and are influenced greatly by nutritional status of soils and environmental factors. However, the concentration of ore, temperature, and C and N sources and stressed environment greatly influences the PS potentials of these organisms under in vitro conditions [Zaidi 1999; Johri et al. 1999]. Phosphate-solubilizing microorganisms are generally isolated from conventional non-rhizosphere and rhizosphere soils, rhizoplane, phyllosphere, rock phosphate deposit area soil and marine environment, and polluted soils using serial plate dilution method or by enrichment culture technique.

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Figure 1. Pigment producing phosphate-solubilizing bacterial strains tentatively identified as Bacillus isolated from cauliflower rhizosphere [Plate A], unidentified PSB [Plate B] isolated from coconut rhizosphere and Serratia [Plate C].

Figure 2. Solubilization of tri-calcium phosphate on Pikovskaya plates by species of [A] Pseudomonas [B] Serratia [C] Bacillus and [D] Apergillus.

Gerretsen [1948] initially demonstrated that microbial activity in the rhizosphere could dissolve sparingly soluble inorganic P and increase plant growth. Subsequently, Pikovskaya [Pikovskaya 1948] suggested a medium [g/l] glucose 10; Ca3 [PO4] 2 5; [NH4]2 SO4 0.5; NaCl 0.2; MgSO4.7H2O 0.1; KCl 0.1; yeast extract 0.5; MnSO4 and FeSO4 trace; pH 7] for the isolation and screening of PSM. Recently, a few other methods, like, bromophenol blue dye method [Gupta et.al. 1994] and National Botanical Research Institute P [NBRIP] medium [Nautiyal, 1999] for the isolation and selection of P-solubilizing microbes have been suggested. After proper incubation of inoculated solid plates that contain insoluble P [e.g., tricalcium phosphate] for five to seven days [bacteria] and three to five days [fungi and actinomycetes] at 28 ± 2 0C, the P-solubilizing microbes are detected by the formation of clear halo around their colonies [Figure 2 and Figure 3]. Since P-solubilizing organisms

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exhibit many fold variations in P dissolving activity and instability with regard to their Psolubilizing activity [Illmer and Schinner 1992], they are repeatedly sub-cultured to test the persistence of P-solubilizing potential. Once the efficient PSM are selected, they are tested for their ability to solubilize insoluble P under liquid culture medium. Finally, the efficient Psolubilizing organisms are selected and used for the development of inoculants whose performance is tested under pot/field environments against various crops of economic importance. A flow chart briefly outlining the strategies for enumeration, qualitative assay of PS activity and selection of potential PS bacteria is presented in Figure 4.

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Figure 3. Solubilization of insoluble P by fungal strains on Pikovskaya [Plate A] and modified Pikovskaya plate [Plate B] amended with bromophenol blue.

Figure 4. Strategies adopted for isolation and screening of P solubilizing bacteria.

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10.2.2. Quantitative Assay of Phosphate

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The microbial strains expressing phosphate-solubilizing activity during screening process are further enriched by inoculating into Pikovskaya medium, incubated at 28 ± 2 0C for seven days and then observed on solid plates for halo formation. The colony forming a clear halo around their growth indicates solubilization of insoluble P by microbes. The solubilizing efficiency of such microbes are calculated by determining the solubilization index [SI] of the phosphate- solubilizing organism as- SI= TCD- CD/ TCD: Where TCD= total colony diameter + zone size and CD represent colony diameter. The colony forming clear halo around microbial growth indicating P solubilization are counted and further used to determine the relative P solubilizing efficiency [RPSE] in liquid Pikovskaya medium. For the quantitative measurement of P, 100 ml of Pikovskaya broth containing tricalcium phosphate is inoculated with one millilitre of 108 cells/ml of each culture. The flasks are then incubated for 10-15 days [bacteria] and about seven days [fungi] with shaking at 120 rpm at 28 ± 2 0C. A 20 ml culture broth from flask is removed and centrifuged for 30 min. and the amount of water soluble P released into the supernatant is estimated by the chlorostannousreduced molybdophosphoric acid blue method [King, 1932; Jackson, 1967]. To 10 ml of supernatant, 10 ml chloromolybdic acid and 5 drops of chlorostannous acid is added and volume is adjusted to 50 ml with distilled water. The blue colour developed is read at 600 nm. Amount of phosphate solubilized is calculated using the calibration curve of KH2PO4. The change in pH following solubilization is also recorded. The microbial cultures showing greater solubilization on both solid and liquid medium and maintaining the PS activity after several subcultures are the criteria for choosing the efficient PS strains for further studies.

10.2.3. Development of Inoculants Various carrier materials used for microphos production includes, soil, cow dung cake powder, peat and farmyard manure [FYM]. After the microphos is produced in bulk, they are packed in polythene bags and are stored for about three months at 25 ± 2 0C. Similarly, the two cultures of the same groups or different groups [one or two fungi/AM fungi together or one PSM and other PGPR] can be mixed together in order to produce a mixed/co- inoculant. However, before the two organisms, identical or different, are used, their compatibility towards each other and the persistence of P-solubilizing activity under in vitro conditions must be ascertained. If the two organisms show any kind of antagonisms under laboratory conditions, they should not be used together for developing a mixed or co-culture of the microphos. Approaches used in the production and application of phosphate-solubilizing microbes are shown in Figure 5.

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Figure 5. Approaches used for production and application of microbial phosphatic inoculants (Adapted from Khan et al. 2009].

10.3. MODE OF APPLICATION OF PHOSPHATE- SOL UBILIZING MICROORGANISMS Phosphate-solubilizing microorganisms can be used for all crops including paddy, millets, oilseeds, pulses and vegetables. Methods recommended for application include [i] seed treatment [ii] seedling dipping and [iii] soil application. Inoculation of seeds by microbial inoculants including microphos has traditionally been the most common and widely used method of microphos application [Khan et al. 2007]. In the bio-priming process, the healthy and rightly chosen seeds are dipped into the microbial broth for about 2h and are then mixed manually or mechanically with the carrier based microphos. After proper mixing, the coated seeds are allowed to dry under shade and then such seeds are applied under pot or field environment. This method allows the microbial inoculants to stick very firmly onto the seed surface ensuring that the seed has the sufficient microbial load [108 cells/seed]. In seed treatment method, 10 kg medium size seeds such as groundnut, wheat, cotton, maize etc., may be treated with 200 g of inoculant, whereas 100 g per acre inoculant is enough for treatment

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of small size seeds. This method, however, has certain limitations, like [i] the coated [inoculated] seeds may come in direct contact with any seed applied chemicals, which in turn, may adversely affect the survivability of the inoculated strains and [ii] the coated inocula may move away from rooting zones and hence, could be exposed to agro-chemicals after planting. However, the use of certain sticker solution [e.g., gum arabic] has been found to improve the adherence of the inocula onto the seeds. Seedling dipping is useful where the transplantation of seedlings is required. It is ideal for vegetable crops. Roots of seedlings are dipped in inoculant suspension well prepared in water in the ratio of 1:10 are kept immersed for about 5 min. Seedlings are then removed from the suspension and transplanted as early as possible. Suspension of one kg microphos in 10 to 15 liter of water is sufficient for treating seedlings for one acre. Another method of microphos introduction has been the soil application method where microbial inoculants [in liquid forms] are directly applied to soils by pouring the active and viable microbial cultures around the root regions of the plants. Alternatively, three to five kg of microphos could be mixed with farm yard manure and then resulting mixture is broadcasted at the time of last ploughing. This method allows a rapid and greater colonization of P-solubilizing organisms per unit area. In addition, the direct contact of inocula with chemically treated seeds is minimized. This method also offers advantages like [i] it is quick compared to seed inoculation technique which requires mixing of seeds with inoculants [ii] inoculants can withstand low moisture conditions better than carrier based inoculants and [iii] less expensive compared to other inoculation methods. Thus, in accordance with these considerations, two approaches can be applied for microphos applications- [i] the mono culture approach [MCA] where Psolubilizing microorganisms can be used alone, and [ii] the co-culture or multiple culture approach [CCA], where microphos prepared from two or more identical or different microbial strains can be mixed together and then applied under natural field/pot house conditions.

10.4. MECHANISMS OF P-SOLUBILIZATION The solubilization of P compounds by naturally-abundant P-solubilizing microbes is common under in vitro conditions [Asea et al. 1988; Turan 2006; Souchi et al. 2006a; Gupta et al. 2007]. Indeed, soil microorganisms are effective in releasing P from inorganic P through solubilization and from organic pools of total soil P by mineralization [Bishop et al. 1994]. The microbial biomass in soil also contains a significant quantity of immobilized P that is potentially available to plants [Brookes et al. 1984; Oberson et al. 2001]. The primary mechanisms attributed to the solubilization of insoluble P include organic acid [OA] production [Cunningham and Kuiack 1992] and H+ excretion [Illmer and Schinner, 1995]. The OA theory is well recognized and most widely accepted mechanism of P solubilization by PSM. The ability of OA to solubilize difficultly available/fixed form of P involves acidification, chelation and exchange reactions [Omar 1998]. Organic acids released by microbes in surrounding environment contribute to the lowering of solution pH [Cunningham and Kuiack, 1992; Maliha et al. 2004; Pradhan and Sukla, 2005] as they dissociate in a pH dependent equilibrium, in to their respective anion [s] or cation [s] as: Ca10F2 [PO4]6+12H+→10Ca2+H2PO4+2F-. The H+ ions favor solubilization of rock P and consequently release more P into solution [Goldstein, 1994; Welch et al. 2002].

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Microbes in general, export OA as anions which are actively transported across the plasma membrane. For example, the transfer of OA by phosphate-solubilizing fungi occurs by an H+symport transport system, causing acidification of the external solution [Netik et al. 1997]. Organic acids produced by P-solubilizing microorganisms can be detected by high performance liquid chromatography and enzymatic methods [Parks et al. 1990; Whitelaw 2000]. A list of OA released by the phosphate-solubilizing microorganisms can be found in Table 1. Though, OA are known to reduce pH values, yet they can solubilize rock P through chelation reaction. This is based on the facts that no correlation has also been found between pH and the amount of P solubilized [Subba Rao 1982; Asea et al. 1988], a mystery unexplained that why OA could not be detected from some PS microorganisms [Illmer and Schinner 1992; Chen et al. 2006]. It was later on suggested that the production of chelating substances could play an active role in solubilization of insoluble P [Illmer and Schinner 1995]. Thus, the chelating ability of the organic acids is also important, as it has been shown that the addition of 0.05M EDTA to the medium has the same solubilizing effect as inoculation with Penicillium bilaii [Kucey 1988]. In other study, Altomare et al. [1999] investigated the capability of the plant-growth promoting and biocontrol fungus T. harzianum T-22 to solubilize in vitro insoluble minerals including rock phosphate. Organic acids were not detected in the culture filtrates and hence, the authors concluded that acidification was probably not the major mechanism of solubilization as the pH never fell below five. The fungal-solubilizing activity was attributed both to chelation and to reduction processes, which could also play a role in the biocontrol of plant pathogens. The formation of complexes between a chelator and cations such as, Ca+ and Al+ depends on the number and kind of functional groups involved as well as the specific cations. Now, it has been reported that acids with greater number of carboxyl groups are generally more effective at solubilizing insoluble P [Kpomblekou-A and Tabatabai, 1994; Xu et al. 2004]. For instance, Ca+ has shown to form complexes more readily with tricarboxylic acids such as, citric acid, compared to dicarboxylic acids, such as malic and tartaric acid [Whitelaw, 2000]. However, conflicting reports on the ability of acids in dissolving P are available. For example, Sagoe et al. [1998b] found that dicarboxylic acids [e.g., oxalic and tartaric] were superior than tricarboxylic acid [citric] in terms of their solubilizing ability which could probably be due to the fact that oxalic and tartaric acids form poorly soluble precipitate with Ca+, and in turn effectively lower the solution saturation point [Sagoe et al. 1998b]. Recently, Yi et al. [2008] used four bacterial strains of Enterobacter sp. [EnHy-401], Arthrobacter sp. [ArHy-505], Azotobacter sp. [AzHy-510] and Enterobacter sp.[EnHy-402], possessing the ability to solubilize tri-calcium phosphate, in order to understand the mechanism of P-solubilization. These PS bacteria produced a significant amount of exopolysaccharide [EPS] and demonstrated a stronger ability for P-solubilization. Of these, the strain EnHy-401 with the highest EPS and OA production had a stronger capacity for P solubilization than the others. Further studies demonstrated that addition of EPS into medium could increase the amount of P solubilized by OA, but failed to release P from TCP alone. The synergistic effects of EPS and OA on TCP solubilization varied with the origin and the concentration of EPS in medium. Exo-polysachharides produced by EnHy-401 was most effective in promoting P release at an optimal concentration in medium. The increase in Psolubilization by EPS was attributed mainly to the participation of EPS which led to a change in the homeostasis of P-solubilization, pushing it towards P dissolved by holding free P in the medium, consequently resulting in greater P released from insoluble P. It was, therefore,

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suggested that EPS with the ability of P-holding may be a novel important factor in the microbial dissolution of TCP except for OA. A general mechanism of P solubilization by PS organism is briefly presented in Figure 6. Table 1. Organic acids involved in solubilization of insoluble P synthesized by phosphate solubilizing microbes Microorganisms Burkholderia cepacea DA23 Aspergillus niger Burkholderia, Serratia, Ralstonia and Pantoea Aspergillus flavus, A. candidus, A. niger, A. terreus, A. wentii, Fusarium oxysporum, Penicillium sp., Trichoderma isridae, Ttrichoderma sp.

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A. flavus, A. candidus Bacillus, Rhodococcus, Arthrobacter, Serratia and one Chryseobacterium, Delftia, Gordonia, Phyllobacterium, Arthrobacter ureafaciens, Phyllobacterium myrsinacearum, Rhodococcus erythropolis and Delftia sp. Penicillium oxalicum Burkholderia glathei [MB 14] Aspergillus flavus, A. niger, P. canescens Penicillium rugulosum Bacillus amyloliquefaciens, B. licheniformis, B. atrophaeus, Penibacillus macerans, Vibrio proteolyticus, xanthobacter agilis, Enterobacter aerogenes, E. taylorae, E. asburiae, Kluyvera cryocrescens, Pseudomonas aerogenes, Chryseomonas luteola A. niger Penicicllium variabile Pseudomonas cepacia Penicillium rugulosum Penicillium radicum Penicillium variabile A. niger A. awamori, A. foetidus, A. terricola, A. amstelodemi, A. Tamari, Bacillus polymyxa, B. licheniformis, Bacillus spp. A. japonicus, A. foetidus

Organic Acids produced Gluconic

References Song et al. [2008]

Citric, oxalic, formic, maleic

Kumari et al. [2008]

Gluconic Elizabeth et al. [2007] lactic, maleic, malic, acetic, tartaric, citric, fumaric, gluconic glutaric citric acid, gluconic acid, lactic, succinic, propionic

malic, gluconic, oxalic gluconate and acetate oxalic, citric, gluconic, succinic citric, gluconic lactic, itaconic, isovaleric, isobutyric, acetic

Akintokun et al. [2007] Akintokun et al. [2007] Chen et al. 2006

Shin et al. [2006] Kim et al. [2005] Maliha et al. [2004] Reyes et al. [2001] Vazquez et al. [2000]

Succinic gluconic gluconic , 2-ketgluconic Gluconic Gluconic Gluconic citric, oxalic, gluconic oxalic, citric

Vazquez et al. [2000] Fenice et al. [2000] Bar-Yosef et al. [1999] Reyes et al. 1999a Whitelaw et al. [1999] Vassilev et al. [1996a] Illmer et al. [1995] Gupta et al. [1994] Singal et al. [1994]

Penicillium bilaji

oxalic, citric gluconic succinic, tartaric citric, oxalic

A. niger, P. simplicissimum

citric

Cunningham and Kuiack [1992] Burgstaller et al. [1992]

Modified from Khan et al. [2009].

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Figure 6. Mechanisms of P solubilization by phosphate-solubilizing microbes [Adapted from Khan et al. 2009].

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10.4.1. Enzymatic Dissolution of Phosphates by Phosphate-Solubilizing Microbes Availability of organic P compounds for plant nutrition could be a limitation in some soils resulting from precipitation with soil particle ions. Such P can be released from organic compounds in soil by enzymes-[i] nonspecific phosphatases, which dephosphorylate phospho-ester or phosphoanhydride bonds of organic matter [ii] phytases, which specifically cause release of P from phytic acid and [iii] phosphonatases and C–P lyases that cleave the C–P of organophosphonates. The efficiency of microbial phosphatases in organic P depletion in the rhizosphere and P uptake by plants has been reported [Rodriguez et al. 2006]. Phosphatases, are released exterior to the cell and are involved in dissolution and mineralization of organic P compounds. Among phosphatases, acid phosphatase [To-O et al. 2000], a widely distributed enzyme, is commonly found in fungi [To-O et al. 1997; Omar and Abd-Alla 2000] and activate the release of inorganic P from P esters [Nozawa et al. 1998]. Acid phosphatase has been detected in vacuoles and vesicles [Ruch and Motta 1987] and other intracellular and extracellular [Ruch and Motta 1987] organs of fungi. For instance, the enzyme was found in the vacuoles of un-germinated conidia of Colletotrichum graminicola, and also during germination [Schadeck et al. 1998a; Schadeck et al. 1998b]. Under in vitro conditions, phosphatase activity is influenced by incubation time and increases with colony development. Moreover, it is influenced by the concentration of nutrients added to medium and an increase in its activity with increasing glucose level is reported [Goud et al. 2008]. Similarly, Aspergillus, Emmericella and Penicillium, isolated from arid and semi-arid regions of India hydrolyzed phytin and glycerophosphate [Yadav and Tarafdar, 2003]. The

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extracellular [E] phosphatases released by fungi were less than their intracellular [I] counterpart and the E:I ratio of fungi ranged from 0.39 to 0.86 for acid phosphatase and 0.29 to 0.41 for alkaline phosphatases. Fungal efficiency of hydrolyzing organic P compounds varied from 2.12–4.85 μg min−1 g−1 for glycerophosphate to 0.92–2.10 μg min−1 g−1 for phytin. The trend of efficiency was: Aspergillus sp.>Emmericella sp.>Penicillium sp. Similar phosphatase production is also reported for A. caespitosus and Mucor rouxii [Guimarães et al. 2006]. Another attractive application of P dissolving enzymes is the solubilization of soil organic P through phytate degradation mediated by enzyme phytase. Phytate is a major component of organic P in soil. Though, the ability of plants to obtain P directly from phytate is very limited, yet the growth and P nutrition of Arabidopsis plants supplied with phytate was improved significantly when they were genetically transformed with the phytase gene [phyA] derived from Aspergillus niger [Richardson et al. 2001a]. This lead to increase in P nutrition to such an extent that the growth and P content of the plant was equivalent to control plants supplied with inorganic P. Similar increase in utilization of inositol P by plants in the presence of microbial communities including P-solubilizing fungus [Aspergillus niger] capable of producing phytase is reported [Richardson et al. 2001b; Vassilev et al. 2007] [for more details see chapter 5]. Therefore, developing fungal inoculants with high phosphatase and phytase activity would be of great practical interest for augmenting plant nutrition and reducing P pollution in soil.

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CONCLUSION In modern agronomic practices, the replenishment of soil P most commonly involves the excessive application of chemical phosphatic fertilizers, an approach that poses severe threat to soil health and consequently the plant development. The use of natural resources like, phosphate-solubilizing microorganisms opens up a new horizon for better plant productivity besides protecting the agro-ecosystems from hazards of agrochemicals. However, the viability and sustainability of this technology largely depends on the development and distribution of good quality microphos to farming communities. The commercialization of microphos is however, a challenging task which requires a full-scale, cost-effective manufacturing, packaging and quality control systems. Moreover, it should be easy-to-use and shelf-stable formulations are needed to be developed. Extensive field research to confirm efficacy and comprehensive data on compatibility of PSM with seed-applied agro-chemicals are required. Thus, the search for identifying new microbes possessing P solubilizing activity continues to be an on-going process with the improved production methods and formulations, new applications, and continuing market research to monitor changing farmer needs.

REFERENCES Akintokun, AK; Akande, GA; Akintokun, PO; Popoola, TOS; Babalola, AO. Solubilization of insoluble phosphate by organic acid producing fungi isolated from Nigerian soil. International J. Soil Sci, [2007], 2, 301-307.

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Altomare, C; Norvell, WA; Bjorkman, T; Harman, GE. Solubilization of phosphates and micronutrients by the plant growth promoting and biocontrol fungus Trichoderma harzianum rifai 1295-22. Appl. Environ. Microbiol [1999], 65, 2926–2933. Arcand, MM; Schneider, KD. Plant-and microbial based mechanisms to improve the agronomic effectiveness of phosphate rock: a review. Ann. Braz Acad Sci [2006], 78, 791-807. Asea, PEA; Kucey, RMN, Stewart, JWB. Inorganic phosphate solubilization by two Penicillium species in solution culture and soil. Soil Biol. Biochem [1988], 20, 459-464. Bar-Yosef, B; Rogers, RD; Wolfram, JH; Richman, E. Pseudomonas cepacia mediated rock phosphate solubilization in kaolinite and montmorillonite suspensions, Soil Sci. Soc. Am. J. [1999], 63, 1703-1708. Bishop, ML; Chang, AC; Lee, RWK. Enzymatic mineralization of organic phosphorus in a volcanic soil in Chile. Soil Sci, [1994], 157, 238-243. Biswas, TK; Cundiff, C . Multiple forms of acid phosphatase in germinating seeds of Vigna Sinensis. Phytochemistry [1991]. 30, 77, 2119-2125. Brookes, PC; Powlson, DS; Jenkinson, DS. Phosphorus in the soil microbial biomass. Soil Biol. Biochem. [1984], 16, 169-175. Burgstaller, W; Straser, H; Shiner, F. Solubilization of zinc oxide from filter dust with Penicillium simplicissium: bioreactor, leaching and stoichiometry. Environ. Sci. Technol [1992], 26, 340-346. Chen, YP., Rekha, PD., Arun, AB., Shen, FT., Lai, WA., Young, CC. Phosphate solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities. Appl. Soil Ecol. [2006], 34, 33-41. Chien, SH; Menon, RG. Factors affecting the agronomic effectiveness of phosphate rock for direct application. Fert Res [ 1995], 41, 227-234. Cunningham, JE; Kuiack, C. Production of citric and oxalic acids and solubilization of calcium phosphate by Penicillium bilaji. Appl. Environ. Microbiol, [1992], 58, 14511458. Elizabeth, P; Miguel, S; BALL MM; Andrés, YL. Isolation and characterization of mineral phosphate-solubilizing bacteria naturally colonizing a limonitic crust in the south-eastern Venezuelan region. Soil Biol. Biochem, [2007], 39, 2905-2914. Entz, MH; Gulford, R; Gulden, R. Crop yield and soil nutrient status on 14 organic farms in the eastern portion of the northern Great Plains. Can J. Plant Sci [2001], 81, 351-354. Fenice, M; Selbman, L; Federici, F; Vassilev, N. Application of encapsulated Penicillium variabile P16 in solubilization of rock phosphate. Biores Technol [2000], 73, 157-162. Gerretson, FC. The influence of microorganisms on the phosphorus uptake by plant. Plant Soil [1948], 1, 51-81. Goldstein, AH. Involvement of the quinoprotein glucose dehydrohenase in the solubilization of exogenous phosphates by gran-negative bacteria, pp. 197-203 in A. Torriani-Gorini, E. Yagil, S. Silver [Eds]: Phosphate in Microorganisms: cellular and molecular biology. ASM Press, Washington DC [1994]. Goud, MJP; Goud, JVS; Charya, MAS. Replica plate screening method for detecting phosphatase activity in basidiomycetes using 1-napthyl phosphate as a chromogenic substrate. Sci. World J., [2008], 3, 13-15. Guimarães, LHS; Peixoto-Nogueira, SC; Michelinl, M; Rizzatti, ACS; Sandrim, VC; Zanoelo, F; Aquino, ACMM; Junior, AB; de Lourdes, M; Polizeli, TM. Screening of

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242

Almas Zaidi, Md. Saghir Khan, Mohd. Oves et al.

filamentous fungi for production of enzymes of biotechnological interest. Braz J. Microbiol., [2006], 37, 474-480. Gupta, N., Sabat, J., Parida, R; Kerkatta, D. Solubilization of tricalcium phosphate and rock phosphate by microbes isolated from chromite, iron and manganese mines. Acta Bot Croatica, [2007], 66, 197–204. Gupta, RR; Singal, R; Shanker, A; Kuhad, RC., Saxena, RK. A modified plate assay for secreening phosphate solubilizing microorganisms. Gen Appl. Microbiol, [1994], 40, 255-260 Hameeda, B., Harini, G., Rupela, OP., Wani, SP., Reddy, G. Growth promotion of maize by phosphate-solubilizing bacteria isolated from composts and macrofauna. Microbiol. Res., [2008], 163, 234-242. Henri, F., Laurette, NN., Annette, D., John, Q., Wolfgang, M., François-Xavier, E. Dieudonne N.: Solubilization of inorganic phosphates and plant growth promotion by strains of Pseudomonas fluorescens isolated from acidic soils of Cameroon. Afric J. Microbiol Res, [2008], 2, 171-178. Igual, JM; Valverde, A; Cervantes, E; Velásquez, E. Phosphate solubilizing bacteria as inoculants for agriculture: use of updated molecular techniques in their study. Agronomie, [2001], 21, 561–568. Illmer, P., Schinner, F. Solubilization of inorganic calcium phosphates-solubilization mechanisms soil. Soil Biol. Biochem., [1995], 27, 257-263. Illmer, P., Schinner, F. Solubilization of inorganic phosphates by microorganisms isolated from forest soil. Soil Biol. Biochem., [1992], 24, 389-395. Illmer, PA; Barbato, A; Schinner, F. Solubilization of hardly soluble AlPO4 with Psolubilizing microorganisms. Soil Biol. Biochem., [1995], 27, 260-270. Jackson, ML. [1967]. ‘Soil Chemical Analysis’, Prentice Hall, India. Johri, JK., Surange, S., Nautiyal, CS. Occurrence of salt, pH and temperature tolerant phosphate solubilizing bacteria in alkaline soils. Curr. Microbiol., [1999], 39, 89-93. Khan, MS; Zaidi, A; Wani, PA. Role of phosphate solubilizing microorganisms in sustainable agriculture- A review. Agron Sustain Dev [2007], 27, 29-43. Khan, MS; Zaidi, A; Ahemad, M; Oves, M; Wani, PA. Plant growth promotion by phosphate solubilizing fungi–current perspective. Arch Agron Soil Sci, [2009], DOI: 10.1080/03650340902806469. Kim, YH; Bae, B; Choung, YK. Optimization of biological phosphorus removal from contaminated sediments with phosphate-solubilizing microorganisms. J. Biosci. Bioengg, [2005], 99, 23-29. King, JE. The colorimetric determination of phosphorus. Biochem J, [1932], 26, 292 – 297. Konietzny U, Greiner R [2004] bacterial phytase: potential application, in vivo function and regulation of its synthesis. Braz. J. Microbiol. [2004] 35:11-18. Kpomblekou, AK; Tabatabai, MA. Effect of organic acids on release of phosphorus from phosphate rocks. Soil Sci, [1994], 158, 442-452. Kucey, RMN. Effect of Penicillium bilaji on the solubility and uptake of P and micronutrients from soil by wheat. Can. J. Soil Sci [1988], 68, 261-270. Kumari, A; Kapoor, KK; Kundu, BS; Mehta, RK. Identification of organic acids producedduring rice straw decomposition and their role in rock phosphate solubilisation. Plant Soil Environ., [2008], 54, 72–77.

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Strategies for Development of Microphos…

243

Maliha, R., Samina, K., Najma, A., Sadia, A., Farooq, L. Organic acids production and phosphate solubilization by phosphate solubilizing microorganisms under in vitro conditions. Pak J. Biol. Sci., [2004], 7, 187-196. Mehrvaz, S; Chaichi, MR; Alikhani, HA. Effects of phosphate solubilizing microorganisms and phosphorus chemical fertilizer on yield and yield components of barley [Hordeum vulgare L.]. American-Eurasian J. Agric Environ. Sci. [2008], 3, 822-828. Molla, M; Chaudhury, AA. Microbial mineralization of organic phosphate in soil. Plant Soil [1984], 78, 393-399. Nautiyal, CS. An efficient microbiological growth medium for screening of phosphate solubilizing microorganisms. FEMS Microbiol. Let. [1999], 170, 265-270. Netik, A; Torres, NV; Riol, JM; Kubicek, CP. Uptake and export of citric acid by Aspergillus niger is reciprocally regulated by manganese ions. Biochim. Biophys Acta [1997], 1326, 287-294. Nozawa, M; Hu, HY; Fujie, K; Tanaka, H; Urano, K. Quantitative detection of Enterobacter cloacae strain HO-I in bioreactor for chromate wastewater treatment using polymerase chain reaction [PCR]. Water Res, [1998], 32, 3472 -3476. Oberson, A; Friesen, DK; Rao, IM; Bühler, S; Frossard, E. Phosphorus transformations in an oxisol under contrasting land-use systems: The role of the microbial biomass. Plant Soil, [2001], 237, 197- 210. Oehl, F; Oberson, A; Tagmann, HU; Besson, JM; Dubois, D; Mader, P; Roth, H; Frossard, E. phosphorus budget and phosphorus availability in soils under organic and conventional farming. Nut Cyc Agroecosys, [2002], 62, 25-35. Omar, SA; Abd-Alla, MH. Physiological aspects of fungi isolated from root nodules of faba bean [Vicia faba L.]. Microbiol Res, [2000], 154, 339-347. Omar, SA. The role of rock phosphate solubilizing fungi and vesicular arbuscular mycorrhiza [VAM] in growth of wheat plants fertilized with rock phosphate. World J. Microbiol. Biotechnol, [1998], 14, 211-219. Parks, EJ; Olson, GJ; Brinckman, FE; Baldi, F. Characterization by high performance liquid chromatography [HPLC] of the solubilization of phosphorus in iron ore by a fungus. J. Indust Microbiol. Biotechnol., [1990], 5, 183-189. Patel, DK; Archana, G; Kumar, GN. Variation in the nature of organic acid secretion and mineral phosphate solubilization by Citrobacter sp. DHRSS in the presence of different sugars. Curr. Microbiol., [2008], 56, 168-174. Pikovskaya, RI. Mobilization of phosphorus in soil in connection with vital activity of some microbial species. Microbiol., [1948],.17, 362- 370. Ponmurugan, P., Gopi C. In vitro production of growth regulators and phosphatase activity by phosphate solubilizing bacteria. African J. Biotechnol, [2006], 5, 348-350. Pradhan, N., Sukla, LB. Solubilization of inorganic phosphates by fungi isolated from agriculture soil. African J. Biotechnol., [2005]. 5, 850-854. Ramachandran, K., Srinivasan, V., Hamza, S., Anandaraj, M. Phosphate solubilizing bacteria isolated from the rhizosphere soil and its growth promotion on black pepper [ Piper nigrum L.] cuttings. Dev Plant Soil Sci, [2007], 102, 325-331. Reyes, I; Bernier, L; Simard, RR; Antoun, H. Solubilization of phosphate rocks and minerals by a wild-type strain and two UV-induced mutants of Penicillium rugulosum. Soil Biol. Biochem., [2001], 33, 1741-1747.

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244

Almas Zaidi, Md. Saghir Khan, Mohd. Oves et al.

Reyes, I; Bernier, L; Simard, RR; Antoun, H. Effect of nitrogen source on the solubilization of different inorganic phosphates by an isolate of Penicillium rugulosum and two UV induced mutants. FEMS Micobiol Ecol. [1999a], 28, 281-290. Richardson, AE; Hadobas, PA; Hayes, JE; O’_Hara, CP; Simpson, RJ. Utilization of phosphorus by pasture plants supplied with myo-inositol hexaphosphate is enhanced by the presence of soil micro-organisms. Plant Soil, [2001b], 229, 47–56. Richardson, AE; Hadobas, PA; Hayes, JE. Extracellular secretion of Aspergillus phytase from Arabidopsis roots enables plants to obtain phosphorous from phytate. Plant J., [2001a], 25, 641–649. Rodriguez, H; Fraga, R; Gonzalez, T; Bashan, Y. Genetics of phosphate solubilization and its potential applications for improving plant growth-promoting bacteria. Plant Soil [2006], 287, 15–21. Rodriguez, H., Fraga, R. Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnol Adv, [1999], 17, 319-339. Ruch, DG; Motta, JJ. Ultrastructure and cytochemistry of dormant basidiospores of Psilocybe cubensis. Mycologia [1987], 79, 387-398. Sagoe, CI; Ando, T; Kouno, K; Nagaoka, T. Relative importance of protons and solution calcium concentration in phosphate rock dissolution by organic acids. Soil Sci. Plant Nutr, [1998b], 44, 617-625. Sanchez, PA; Shepherd, KD; Soule, MJ; Place, FM; Buresh, RJ; Izac, AN. Soil fertility replenishment in Africa: an investment in natural resource capital. In: Replenishing soil fertility in Africa, Indianapolice. Proc, SSSA Special publication Number 51. Madison, WI: American Society of Agronomy. 1997. pp. 1-46. Saxena, J; Sharma, V. Phosphate solubilizing activity of microbes and their role as biofertilizer. In; Advances in Microbiology, [Ed.] Trivedi, P.C., Scientific Publ. Jodhpur, India, PP; 59-73. [2003]. Schadeck, RJG; Buchi, DF, Leite, B. Ultrastructural aspects of Colletotrichum graminicola conidium germination, appressorium formation and penetration on cellophane membranes: focus on lipid reserves. J. Submicro Cytol. Pathol. [1998a], 30, 555-561. Schadeck, RJG; Leite, B; Buchi, DF. Lipid mobilization and acid phosphatase activity in lytic compartments during conidium dormancy and appressorium formation of Colletotrichum graminicola. Cell Struc. Func [1998b], 23, 333-340. Sharma, AK. Bio-fertilizers for Sustainable Agriculture. Agrobios Indian Publications. pp.456, [2002]. Shin, W., Ryu, J., Kim, Y., Yang, J., Madhaiyan, M; Sa,, T. Phosphate solubilization and growth promotion of maize [Zea mays L.] by the rhizosphere soil fungus Penicillium oxalicum. 18th World Congress of Soil Science. July 9-15, Philadelphia, Pennsylvania, USA. [2006]. Singal, R., Gupta, R., Saxena, RK. Rock phosphate solubilization under alkaline conditions by Aspergillus japonicus and A. foetidus, Folia Microbiol, [1994], 39, 33-36. Song, OR; Lee, SJ; Lee, YS; Lee, SC; Kim, KK; Choi, YL. Solubilization of insoluble inorganic phosphate by Burkholderia cepacia DA23 isolated from cultivated soil. Braz. J. Microbiol, [2008], 39, 151-156. Souchie, EL, Azcón, R; Barea, JM; Saggin-Júnior, OJ; Silva, EMR. Phosphate solubilization and synergism between P-solubilizing and arbuscular mycorrhizal fungi. Pesquisa Agropecuária Brasileira [2006a], 41, 1405-1411.

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245

Subba Rao, NS. Advances in Agricultural Microbiology. Oxford and IBH Publications Company, India. pp. 229-305, [1982]. To-O, K; Kamasaka, H; Kuriki, T; Okada, S. Substrate selectivity in Aspergillus niger KU-8 acid phosphatase II using phosphoryl oligosaccharides. Biosci. Biotechnol. Biochem. [2000], 64, 1534-1537. To-O, K; Kamasaka, H; Kusaka, K; Kuriki, T; Kometani, K; Okada, S. A novel acid phosphatase from Aspergillus niger KU-8 that specifically hydrolyzes C-6 phosphate groups of phosphoryl oligosaccharides. Biosci. Biotechnol. Biochem. [1997], 61, 15121517. Turan, M. Evaluation of the capacity of phosphate solubilizing bacteria and fungi on different forms of phosphorus in liquid lulture. J. Sustain Agric, [2006], 28, 99-108. Vassilev, N; Fenice M; Federici, F. Rock phosphate solubilization with gluconic acid produced by immobilized Penicillium variabile P16. Biotechnol Tech, [1996a], 20, 585588. Vassilev, N; Vassileva, M; Bravo, V; Fernández-Serrano, M; Nikolaeva, I. Simultaneous phytase production and rock phosphate solubilization by Aspergillus niger grown on dry olive wastes. Indust Crops Prod [2007], 26, 332-336. Vazquez, P; Holguin, G; Puente, ME; Lopez-Cortes, A; Bashan, Y. Phosphate-solubilizing microorganisms associated with the rhizosphere of mangroves in a semiarid coastal lagoon. Biol. Fertil Soils [2000], 30, 460-468. Wani, PA; Khan, MS; Zaidi, A. Synergistic effects of the inoculation with nitrogen fixing and phosphate solubilizing rhizobacteria on the performance of field grown wheat. J Plant Nutr. Soil Sci. [2007a] 170, 283 - 287. Wani, PA; Khan, MS; Zaidi, A. Co-inoculation of nitrogen fixing and phosphate solubilizing bacteria to promote growth, yield and nutrient uptake in chickpea. Acta Agron Hungarica [2007b], 55, 315-323. Welch, SA; Taunton, AE; Banfield, JF. Effect of microorganisms and microbial metabolites on apatite dissolution. Geomicrobiol J. [2002], 19, 343-367. Whitelaw, MA; Harden, TJ; Helyar, KR. Phosphate solubilization in solution culture by the soil fungus penicillium radicum. Soil Biol. Biochem. [1999], 32, 655-665. Whitelaw, MA. Growth promotion of plants inoculated with phosphate solubilizing fungi. Adv Agron [2000], 69, 99-151. Xu, RK; Zhu, YG; Chittleborough, D. Phosphorus release from phosphate rock and an iron phosphate by low molecular weight organic acids. J. Environ Sci [2004], 16, 5-8. Yadav, RS; Tarafdar, JC. Phytase and phosphatase producing fungi in arid and semi-arid soils and their efficiency in hydrolyzing different organic P compounds. Soil Biol Biochem [2003], 35, 745-751. Yi, Y., Huang, W., Ge, Y. Exopolysaccharide: a novel important factor in the microbial dissolution of tricalcium phosphate. World J. Microbiol. Biotechnol., [2008], 24, 10591065. Zaidi, A; Khan, MS. Stimulatory effects of dual inoculation with phosphate solubilizing microorganisms and arbuscular mycorrhizal fungus on chickpea. Aus. J. Exp. Agric [2007], 47, 1016-1022. Zaidi A. Synergistic interactions of nitrogen fixing microorganisms with phosphate mobilizing microorganisms, Ph.D Thesis, [1999] Aligarh Muslim University, Aligarh.

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In: Phosphate Solubilising Microbes for Crop Improvement ISBN 978-1-60876-112-8 Editor: M. S. Khan and A. Zaidi © 2009 Nova Science Publishers, Inc.

Chapter 11

VARIATION IN PLANT GROWTH PROMOTING ACTIVITIES OF PHOSPHATE-SOLUBILIZING MICROBES AND FACTORS AFFECTING THEIR COLONIZATION AND SOLUBILIZING EFFICIENCY IN DIFFERENT AGRO- ECOSYSTEMS Mohammad Oves, Almas Zaidi, Mohd. Saghir Khan∗ and Munees Ahemad

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Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh-202002; U.P., India

ABSTRACT Phosphate-solubilizing microorganisms commonly represents a group of soil microorganisms that forms an important component of phosphorus cycle and release P by solubilizing insoluble phosphorus or by mineralizing organic phosphorus. They predominate in the rhizosphere and non-rhizosphere area of different crops and vary greatly in number, diversity and phosphate-solublizing ability. The solubilization of phosphate by microbes is a multifactor phenomenon and is dependent primarily on the metabolic activities of the organisms. Phosphate-solubilizing microbes including bacteria, fungi and actinomycetes in addition to providing phosphate ions to the plants also synthesize various other biological compounds like, phytohormones, such as auxin and gibberellic acid as well as vitamins and are often used as plant growth promoters. The phosphate-solubilizing organisms further enhance the growth of plants by synthesizing siderophores, antibiotic and cyanide. The implications of such variations in growth promoting activities of different phosphate-solubilizing microorganisms are discussed. The performance of phosphate-solubilizing microorganisms in natural soil environment is however, influenced by several factors. Understanding the factors affecting the survivability and functionality of the phospho-inoculants in soils is likely to lead to ∗

Correspondence to: [email protected]

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Mohammad Oves, Almas Zaidi, Mohd. Saghir Khan et al. predict the behavior and efficacy of the inoculants in different agro-ecological regions. And hence, use of such phosphate- solubilizing microorganisms may be a viable alternative to chemical fertilizers for increasing the productivity of various crops.

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11.1. INTRODUCTION There is more than enough food in the world to feed every person. Yet for more than a billion people whose only food and income comes from the crops they grow, when the crops fail, there is simply no resource to buy food from others. Indeed, in many rural areas, where 70 per cent of the poorest 1.2 billion in the world live and work, agricultural productivity is declining sharply. Much of this is due to destruction of agricultural lands and the deterioration of the environments in which crops are grown. Hence, problems of food scarcity loom in the future, while most countries are trying to attain self sufficiency and food security. While searching solutions to these problems, one could easily identify that the maintenance of soil fertility and quality are two basic concepts that are considered important in optimizing the sustainability of food systems. The conventional agricultural practices like use of chemicals for raising the productivity of crops though are used extensively but they do not offer a clear solution, as the excessive use of agrochemicals make the soil unsuitable for cultivation. Hence, one has to look at alternative source of nutrients which could be supplied to plants in a more sustained manner. In this regard, improvement in agricultural sustainability could be achieved by applying naturally abundant but functionally different groups of microbial communities inhabiting soil ecosystems. Soil micro-organisms play a significant role in providing essential nutrients (e.g., N and P) to plants by decomposing organic substances. To achieve similar objective, microbial inoculants are used as an economic input to increase crop productivity; fertilizer doses can be lowered and more nutrients can be harvested from the soil. Microbial technology thus offers simple, low cost, highly efficient and safe options for improving the productivity of crops. An understanding of microbial variations accompanied by functional diversity is thus essential in order to harness the full potentials of microbes in sustained growth of crops in different agro-ecosystems (Tilak et al. 2005). Of the various major and micro-nutrients required by plants, the deficiency of phosphorus restricts the growth and development of plants severely. However, the content of fixed P in cultivated and fertilized soils increases over time, depending upon the rate of application, the adsorptive capacity of soil matrix surfaces, as well as on microbial activity, particularly that of microorganisms involved in P cycling (Borie and Rubio 2003). Phosphate mobilization/solubilization by microorganisms in cultivated soils and its uptake by plants are hence, required for better management of agro-ecosystems. Such a microbial approach is likely to lead to reduce the application of P fertilizers and, in turn may protect the environmental quality. Thus, exploiting the benefits of microbes especially the P-solubilizing organisms for better plant growth appears to be a more promising approach, assuming that issues of lack of consistency of growth stimulation can be resolved. In this context, microbiological approaches involving phosphate-solubilizing organisms have been reported to enhance the crop productivity in different agro-ecological niches (Linu et al. 2009; Zaidi and Khan 2007; Wani et al. 2007a and 2007b).

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11.2. FUNCTIONAL VARIATIONS AMONG PHOSPHATE-SOL UBILIZING MICROORGANISM

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Functional diversity is an aspect of the overall microbial diversity in soil, and encompasses a range of activities. The relationship between microbial diversity and function in soil is largely unknown but functional variations among microbes have been assumed to influence ecosystem stability, productivity and resilience towards stress and disturbance. The density, composition and activity of microbial community is however, influenced by different soil factors such as, structure, moisture level, nutrient supply, pH, electrical conductivity, redox potential, and stress causing chemicals (Curl and Truelove 1986; Lynch 1990; Nelson and Mele 2007). In addition, environmental factors as temperature, pressure, air composition, surface and spatial relations can change markedly, and therefore so do the microhabitats in the soil (Nanniperi et al. 2003). Knowledge on microbial biodiversity including phosphatesolubilizing microorganisms is therefore, essential to understand their ecological role and application in sustainable agriculture. Phosphate solubilizing microorganisms are though largely known for their ability to provide soluble P to plants in a more sustainable manner, but they also improve the growth of plants by other mechanisms also (Figure 1).

Figure 1. Plant growth promoting substances secreted by phosphate solubilizing bacteria.

For example, Hamdali et al. (2008) reported eight rock phosphate (RP)-solubilizing Actinomycetes isolates inhabiting Moroccan phosphate mines. Actinobacterial isolates were found to colonize plant roots, to excrete substances able to stimulate plant growth (phytohormone, indoleacetic acid, IAA) and to limit the proliferation of common phytopathogenic fungi. In other study, 43 isolates of phosphate-solubilizing rhizobacteria

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(PSRB) were isolated from 37 rhizospheric soil collected from tomato growing regions of Karnataka. Of the 43 isolates, 33 were found to be positive for solubilizing both inorganic and organic forms of P and demonstrated the ability to colonize roots of tomato and to increase the seed quality (Hariprasad and Niranjana, 2008) suggesting that they could be used both as biofertilizers and biocontrol agents (Xiao et al. 2002; Panday et al 2006; Errakhi et al. 2007) for agriculture improvement. Among the other substances produced by P-solubilizing microbes and involved in plant growth is siderophores (Matthijs et al. 2007; Hamdali et al. 2008), Indole acetic acid (IAA) and gibberellic acid (Vikram et al. 2007; Souchie et al. 2007). Siderophores, a low-molecular-weight, iron-chelating ligands help a particular microorganism compete effectively against other organisms for available iron. This enhances the growth of the microorganism while limiting iron availability to the competing microorganisms and consequently restricts their growth (Lemanceau et al. 1985). Cyanide is yet another secondary metabolite synthesized by some Gram-negative phosphate-solubilizing bacteria, such as P. fluorescens, P. aeruginosa, and Chromobacterium violaceum (Siddiqui et al., 2006; Wani et al. 2007c). Though, hydrogen cyanide (HCN) does not directly affect the growth, energy storage, or primary metabolism of plants but is generally considered as a secondary metabolite that has an ecological role and confers a selective advantage to the producer strains (Rudrappa et al. 2008). Moreover, siderophores and cyanide production ability in various phosphate solubilizing microbes are linked to antagonistic and disease suppressing activity against various plant pathogens (Khan et al. 2007). Some of the most commonly identified compounds synthesized by phosphate-solubilizing organisms and affecting plant growth and developments are listed in Table 1.

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Table 1. Phosphate-solubilizing microbes with plant growth promoting activity Phosphate solubilizing bacteria Pseudomonas sp. SRI2, Psychrobacter sp. SRS8 and Bacillus sp. SN9 Streptomyces Pseudomonas sp. Bacillus subtilis Serratia marcescens Pseudomonas fluorescens Acinetobacter sp., Pseudomonas sp. Enterobacter sp. Burkholderia Pseudomonas jessenii Pseudomonas aeruginosa Pseudomonas sp. Azotobacter sp., Mesorhizobium sp., Pseudomonas sp., Bacillus sp. Mesorhizobium loti MP6

Plant growth promoting traits IAA, siderophore, ACC deaminase IAA, siderophores, amylase, catalase, lipase, xylanase ACC deaminase, IAA, siderophore IAA, siderophore, antifungal activity IAA, siderophore, HCN ACC deaminase ACC deaminase, IAA, antifungal activity, N2- fixation ACC deaminase, IAA, siderophore ACC deaminase, IAA, siderophore, heavy metal solubilization ACC deaminase, IAA, siderophore, heavy metal solubilization ACC deaminase, IAA, siderophore ACC deaminase, IAA, siderophore, heavy metal solubilization IAA, siderophore, antifungal activity, ammonia production, HCN

References Ma et al. [2009] [Sousa et al. 2008] Poonguzhali et al. [2008]

Siderophores, IAA, HCN

Chandra et al. [2007]

Singh et al. [2008] Selvakumar et al. [2008] Shahroona et al. [2008] Indiragandhi et al. [2008] Kumar et al. [2008] Jiang et al. [2008] Rajkumar and Freitas [2008] Ganesan [2008] Rajkumar and Freitas [2008] Ahmad et al. [2008]

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11.3. FACTORS AFFECTING P-SOLUBILIZATION AND COLONIZATION

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A change in the environment always lead to a new selection pressure and, with time, a community adapts to the new conditions. In this context, various factors have been suggested that affect composition, structure and functions of microbial communities including PSM in soil environment. The factors affecting the performance of microbial communities in soils include, physico-chemical properties of soils (Bashan et al. 1995), moisture content (Van Elsas et al. 1991), plant age and genotypes, composition of root exudates, presence of environmental pollutants (heavy metals/pesticides) in soils (Khan et al. 2009a ) and the presence of recombinant plasmids (Van Veen et al. 1997). Of these, root exudates consist of a wide array of compounds and simple substrates from low molecular weight compounds such as, sugars, phenolics, amino acids, organic acids, and other secondary metabolites, to higher molecular weight compounds such as, proteins and mucilage. Thus, the understanding of microbial interactions and factors influencing the survivability of the inoculants in soils is very important which could lead to predict the behavior and efficacy of the inoculants in different agro-ecological regions. The solubilization of phosphate by microbes in natural environment is a multifactor phenomenon and is dependent primarily on the metabolic activities of the organisms. Generally, the density of artificially introduced P- solubilizing microbes decline rapidly upon inoculation in soils (Khan et al. 2009b). Therefore, we need a better understanding as to how multiple environmental variables affect the structural and functional variations in soil environment. Furthermore, rate of such responses to environmental changes and the factors impacting the rate of change and how these responses are related to functional properties like, phosphate-solubilizing activity requires further attention.

11.3.1. Temperature and pH Though, microbial solubilization of inorganic phosphates is well recognized (Khan et al. 2007; Khan et al. 2009b); the colonization and establishment and performance of such microbes are affected severely under stress environments, such as, high temperature and desiccation. The effect of temperature on phosphate-solubilizing efficiency of microbes has however, been contradictory. For example, many workers suggest that 20-25 0C is the optimum temperature at which maximum solubiliztion occurs (Sayer and Gadd, 1998) while 28 0C has been found as optimum temperature for P-solubilization by others (Kang, 2002; Varsha, 2002). Moreover, numerous authors have shown that 30 0C is the best temperature for P-solubilization (Fasim et al. 2002; Johri et al. 1999; Rosado et al. 1998; Kim et al. 1997) while solubilization at extreme temperature (at 45 0C in desert soil) is also reported (Nautiyal et al., 2000; Nahas, 1996). In contrast, solubilization at low temperature (10 0C ) has also been reported (Johri et al. 1999). For instance, cold tolerant mutants (grown at 10°C) of Pseudomonas fluorescens (strains GRS1, PRS9 and ATCC13525) have demonstrated Psolubilizing ability and subsequent effect on plant growth promotion under in vitro and in situ conditions. Under in vitro conditions the cold tolerant mutants exhibited increased plant growth indicating their functionality at low temperature. Subsequently, greenhouse trials

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using soil-plant microcosms were conducted which revealed that strain CRPF2 (high Psolubilizer) was a good rhizosphere colonizer showing a significant increase in root (30 and 20%) and shoot length (20 and 24%) of greengram, both in sterilized and unsterilized soil, respectively. On the contrary, CRPF (low P-solubilizer) did not stimulate plant growth. Furthermore, sand experiments indicated that tricalcium phosphate (TCP) served as better P source for CRPF2 treated greengram seeds (Katiyar and Goel 2003). In an experiment, several phosphate-solubilizing microorganisms were tested for their P-efficiency at 35, 40 and 45 °C. Though, a marked variation in their ability to solubilize TCP was observed, yet the effect was more pronounced at 45°C. And hence, two bacterial (Bacillus subtilis and Bacillus circulans ) and one fungal (Aspergillus niger) strain were found to be thermotolerant as they did solubilize a large amount of TCP at the three tested temperatures (Gaind and Gaur 1991). Furthermore, seed inoculation of greengram showed a better establishment of temperature tolerant strains as revealed by the rhizosphere population. In a recent study, thermo-tolerant phosphate-solubilizing microbes including bacteria, actinomycetes, and fungi were isolated from different compost plants and biofertilizers in order to prepare the multi-functional biofertilizer. Of these, except Streptomyces thermophilus J57 (which lacked pectinase), all strains possessed amylase, CMCase, chitinase, pectinase, protease, lipase, and nitrogenase activities and could solubilize calcium phosphate and Israel rock phosphate. Moreover, majority of the isolates solubilized aluminum phosphate, iron phosphate, and hydroxyapatite. During composting, biofertilizers inoculated with the tested microbes had a significantly higher temperature, ash content, pH, total N, soluble P content, and germination rate than non-inoculated biofertilizer; total organic carbon and C:N ratio showed the opposite pattern. Upon inoculation, these microbes shortened the period of maturity, improved the quality, increased the soluble P content, and enhanced the populations of phosphate-solubilizing and proteolytic microbes in biofertilizers. This study suggested that the inoculation of thermotolerant phosphate-solubilizing microbes into agricultural and animal wastes presents a practical strategy for preparing multi-functional biofertilizer (Chang and Yang, 2009). These reports thus clearly suggest that bacteria could adapt to their varying indigenous environment, so their metabolic activities are linked to the temperature of the environment. Moreover, strains of PSM capable of solubilizing insoluble P efficiently at higher temperatures could be used as inoculant for raising the yield and nutrient uptake by plants under extreme of adverse environmental situations. Even though, other mechanisms do operate in P-solubilization (Asea et al. 1988; Darmwal et al. 1989) but acid production by microbes has been suggested to be a major mechanism involved in P-solubilization (Vikram et al. 2007; Chen et al. 2006; Maliha et al. 2004; Fasim et al., 2002) and this results in lowering of pH, a vital factor in solubilization. Generally, the most appropriate pH for P-solubilization by bacteria is 6 while for fungi and actinomycetes it ranges between 5 and 6. However, pH beyond 7 has been found to greatly reduce the P solubilizing activity of fungi (Gaur, 1990). In contrast, Nahas (1996) have reported solubilization even at pH 12 suggesting that insoluble P could be solubilized by mechanism other than acid production and P-solubilization may depend upon a multitude of factors including decrease in pH, microorganisms and the insoluble phosphate used. Furthermore, the buffering capacity of soils could limit solubilization of P by microorganisms as it has been shown that solubilization of Ca-P complexes is mediated mainly by lowering the pH of the medium by secreting organic acids (Kucey et al. 1989; Halder and Chakrabartty 1993). In addition, studies with mineral phosphates have shown that the nature of the organic

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acid was more important than the amount of organic acid released by P-solubilizers [Kpomblekou et al. 1994]. Accordingly, the efficiency of P-solubilization by Acetobacter liquefaciens (PSB12), Acetobacter sp. PSB67) and Pseudomonas gladioli (PSB 73) was decreased in buffered media compared to non buffered media ( Joseph and Jisha 2008). It is also evident that buffering has a more pronounced effect on the growth of bacteria using rock phosphate as a P source [Narsian et al. 1994]. Presumably, a drop in pH in the absence of buffer is rapid and P-solublizing microbes are not severally affected by buffering. Thus, phosphate-solubilizing microorganisms tolerant to high temperature or pH could enhance production of food and forage in semiarid and arid regions of the world.

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11.3.2. Carbon and N Source The role of carbon in phosphate-solubilization is highly important as the production of acid is greatly affected by the source and the nature of carbon which in turn determine the type and quantity of acids (Gaur and Sachar 1980; Dave and Patel 2003). For example, the variations in P-solubilizing activity of the Penicillium were suggested largely due to the differences in types and/or levels of organic acids produced in culture. As an example, P. radicum has been found to produce gluconic acid (Whitelaw et al. 1999), while P. bilaiae produced citric and oxalic acids (Cunningham and Kuiack 1992). The power of organic acids to complex with cations in RPs (thereby liberating P) varies considerably with citric and oxalic acids as the strongest acids forming complex. In contrast, gluconic acid appears to have a very limited ability to release P complexed with Ca2+ (Illmer and Schinner 1992); however, it may have a role in P solubilization where Al3+ is the major cation binding P (Whitelaw et al. 1999). In the presence of different C sources, bacterial strains have shown a drop in pH and could solubilize RP. The released P was maximum with glucose and minimum with cellobiose by S. marcescens (strain EB 67) and Pseudomonas sp. (strain CDB 35), respectively (Hameeda et al. 2006). For both the strains, lactose, arabinose, sorbitol and raffinose were found to be very poor sources of C during solubilization process. Glucose decreased the pH of the medium maximally, followed by other C sources which although supported growth of bacteria but did not affect P-solubilization effectively. This suggests that acidity rather than the growth of bacteria is related to P-solubilization (Halder et al. 1991). In other report, the highest ability to solubilize apatite by microorganisms was associated with glucose in short-term experiments while sucrose was the best in longer experiments (Banik, 1983). Similarly, glucose and sucrose significantly promoted P-solubilization compared to fructose, lactose, galactose, and xylose by Aspergillus tubingensis. In other study, Di Simmine, (1998) reported solubilisation of P only in the presence of glucose and slight solubilization in presence of mannose while no solubilization was detected in presence of gluconate, galactose, glycerol, sorbitol and fructose. However, among different carbon sources tested, fructose, glucose, xylose, sucrose and starch enhanced solubilization more than galactose and maltose (Cerezine et al., 1988). Amount of glucose in the media was shown to be an important factor for solubilization as oxalic acid and other acid production was stimulated by the addition of glucose. Moreover, a gradual increase in sugar concentration can also increase the activity of glycolytic enzymes and pyruate carbooxylase which in turn lead to increase the production of organic acids. Such phenomenon may not occur with all bacteria though oxalic acid production has been found to be promoted under

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carbon limited conditions in Penicilium billai (Cuningham and Kuiak, 1992) and a relatively low sugar concentration was optimal for phosphate-solubilization by Penicillium sp. isolated from forest soil. This was explained by the fact that suboptimal growth conditions are often necessary for the production of secondary metabolites (Ilmer and Schineer, 1992). In other study, Aspergillus aculeatus, a rhizosphere isolate of gram (Cicer ariatenum) invariably solubilized both foreign (China and Senegal) and Indian (Hirapur, Udaipur, Sonrai) rock phosphates (RP). Arabinose proved superior to glucose among 11 carbon compounds tested. All the test carbon sources, except lactose, supported good growth regardless of their phosphate solubilization. However, it is also reported that P solubilization and mineralization could coexist in the same bacterial strain. For example, inorganic P-solubilizing bacteria (IPSB), Bacillus cereus and B. megaterium, and organic P-mineralizing bacterial (OPMB) strains, B. megaterium, Burkholderia caryophylli, Pseudomonas cichorii, and Pseudomonas syringae isolated from soils taken from subtropical flooded and temperate non-flooded soils have shown both P-solubilizing and -mineralizing abilities (Tao et al. 2008). Among the various N sources, potassium nitrate significantly increased P- solubilization compared to other N sources (such as ammonium sulfate, ammonium nitrate, aspargine). The effect of different N sources on P-solubilization revealed maximum solubilization of P in the presence of ammonium sulphate in the wild (39.9%) and mutant strain (67.1%) followed by ammonium nitrate and urea. However, the lowest P- solubilization was recorded with sodium nitrite (NaNO2). Production of inorganic acids by proton exchange mechanism in the presence of ammonium ion has been reported to accelerate P solubilization (Halder et al. 1991; Nautiyal et al. 2000). In another study, ammonium sulphate was the best nitrogen source followed by urea and asparagine. Nitrates, while supported good growth but did not support good phosphate- solubilization (Narsian and Patel, 2000). In a similar study, ammonium salts have been found as the best nitrogen source, followed by asparagine, sodium nitrate, potassium nitrate, urea and calcium nitrate for P-solubilization by a phosphatesolubilizing fungus, Paecilomyces marquandii (Ahuja et al. 2007).

11.3.3. Salinity Salinity is a serious threat to agriculture in arid and semiarid regions (Rao and Sharma 1995). Nearly 40% of the world’s land surface can be categorized as having potential salinity problems (Cordovilla et al 1994); most of these areas are confined to the tropics and Mediterranean regions. Increase in the salinity of soils or water supplies used for irrigation result in decreased productivity of most crop plants and lead to marked changes in the growth pattern of plants (Cordovilla et al 1994) and have a detrimental effect on soil microbial populations as a result of direct toxicity as well as osmotic stress (Tate 1995). In a study, the soil from the saline area of Amravati district, India was investigated for the assessment of phosphate-solubilizing potential of fungi and bacteria. In that study, microbial populations in 30.80% samples showed the ability to solubilize the inorganic insoluble phosphate among a total of 107 samples collected. From the study it was obvious that the fungi (e.g., Aspergillius spp., Penicillium spp. and Fusarium spp.) had more solubilizing ability than bacteria (e.g., B.subtilis, and B.megatherium) [Rajankar et al 2007]. In other study, the fungus Eupenicillium parvum also solubilized North Carolina rock phosphate and Mussoorie rock phosphate, and exhibited high levels of tolerance against desiccation, acidity, salinity,

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aluminum, and iron. Solubilization of inorganic phosphates by the fungus was however poor in the presence of aluminum than iron (Vyas et al. 2007). Johari et al. (1999) reported 18 bacterial isolates out of 57 isolates in presence of 5% sodium chloride while two bacterial isolates lost the ability of phosphate-solubilization in plate assay in absence of sodium chloride. In contrast, Nautiyal (1999) and Rosado et al. (1998) reported solubilization of P in presence of 10% sodium chloride but solubilization activity decreased gradually with the increasing concentration of sodium chloride. The loss in P solubilizing activity of organisms while growing in salt amended medium could possibly be due to restriction in growth and proliferation leading to reduced P-solubilizing efficiency or it might be possible that too much chloride ions may chelates or neutralize proton ions or acid produced in the media.

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11.3.4. Agrochemicals Agrochemicals in general, have played a significant role in the green revolution, but injudicious application of synthetic chemicals has led to a profound decline in physicochemical properties of soils leading to losses in soil fertility, destruction of natural environment, changes in structure and functional properties of microbial communities and consequently the reduction in crop yields in different agro-ecological systems (Wani et al. 2008; Khan et al. 2009a). For example, nickel has shown to severely inhibit aerobic growth of various microorganisms (Babich and Sotzky 1983; Wani et al. 2007d). In a study, an insecticide, phorate, was used to determine its effect on the phosphate- solubilizing and IAA producing potential of the selected phosphate-solubilizing bacteria. Phorate (100 and 500 mg ml-1) in general, stimulated the phosphate-solubilizing activities of the selected bacteria whereas IAA production was significantly reduced (Wani et al 2005). In addition, unfavourable pH and high reactivity of aluminium and iron in soils decrease P availability as well as P-fertilizer efficiency also with high total P contents. The most direct approach to analyze agrochemicals induced community changes is the determination of tolerant microbial communities (Baath et al., 1998; Holtan-Hartvik et al., 2002).

11.3.5. Crop System The distribution pattern and population density of phosphate- solubilizing bacteria was assessed in cultivated soils. The phosphate-solubilizing bacteria were assessed for Psolubilizing capicity, production of growth regulators, phosphatase activity, pH change and titrable acidity. The population levels of PSB were highest in the rhizosphere soil of groundnut (Arachis hypogaea), and lowest in the rhizosphere of ragi (Eleusine Coracana), sorghum (Sorghum bicolour) and maize (Zea mays) (Ponmurgan and Gopi 2006). About half of culturable rhizobacteria associated with perennial ryegrass (Lolium spp.), white clover (Trifolium pretense), oat (Avena spp.) and wheat (Triticum aestivum) were capable of solubilizing P-containing compounds. The rhizosphere of pasture plants (perennial ryegrass and white clover) contained predominantly Na-phytate solubilizers, whereas the Caphosphate solubilizers dominated the oat and wheat rhizosphere (Jorquera et al., 2008). However, an effective interaction between P-solubilizers and plants depends on higher population of P-solubilizers maintained in the rhizosphere over long periods, exudation of

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carboxylates and protons into the rhizosphere by roots and microorganisms, low P uptake by microorganisms and positive synergistic interaction with mycorrhizal fungi or other plant growth promoting rhizobacteria.

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11.3.6. Root Exudates The exudation of large amounts of diverse C compounds by plant roots is of prime ecological importance as it enhances the soil microbial activity. Plant exudes a variety of organic compounds, like, carboxylate anions, phenolics, carbohydrates, amino acids, enzymes, other proteins, etc. and inorganic ions (protons, phosphate and other nutrients, etc.) into the rhizosphere which determine the chemistry and biology of rhizosphere (Crowley and Rengel, 1999). Root exudates are good nutrient source for microorganisms allowing microbial communities to proliferate rapidly in the rhizosphere (Marilley and Aragno, 1999). Furthermore, the amount and composition of root exudates affect microbial community composition which in turn influences nutrient availability. Plants grown in nutrient deficient or sufficient soils often have differential microbial communities in the rhizosphere (Marschner et al., 2005b; 2006; 2007). Nutrient deficiency can influence rhizosphere microorganisms either directly by affecting their nutrition or indirectly by changing root morphology and exudation (Rengel and Marschner, 2005). However, roots can maintain distinct rhizosphere microbial communities even when interwoven with roots of other species (Wang et al. 2007a). Plants and microorganisms increase exudation of P-hydrolysing enzymes under P deficiency. These enzymes break down organic P, thus making P available for uptake. For instance, phytase cleaves phytate, the major form of organic P in soils (Rengel and Marschner 2005). Roots excrete little amount of phytase whereas microorganisms (e.g., Aspergillus niger) exude large amounts (Richardson et al., 2001) and indirectly enable plants to utilise phytate (Osborne and Rengel, 2002). Genetically modifying plants to excrete microbial phytase may allow plants to increase P uptake, but effectiveness of phytase is limited by the low phytate availability in soil and binding of phytase to soil particles (George et al., 2005). Exudation of phosphatases, the other enzyme involved in mineralization of organic P increases when plants are P deficient (Radersma and Grierson, 2004). When grown in an acidic P-deficient soil amended with Fe-P, the P-efficient Triticum aestivum genotype had a greater acid phosphatase activity in the rhizosphere than the inefficient genotype, with phosphatase activity correlating positively with growth and P uptake (Marschner et al., 2005b; 2006).

CONCLUSION In agricultural practices, chemical P fertilizers are generally used in order to achieve higher crop yields. The disproportionate and indiscreet use of phosphatic fertilizers however, destructs the structure, composition and functional ability of microbial communities including phosphate-solubilizing microbes inhabiting soils. And hence, in turn, the altered microbial activities deteriorate the fertility of soils and consequently the yields of various crops. To

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avoid such losses in soil fertility and crop productivity and to reduce dependence on phosphatic fertilizers, the use of such phosphate- solubilizing microbes are being advocated. The protection of soil fertility by the application of phosphate-solubilizing microbes is thus a breakthrough for sustainable crop productivity. However, this technology requires considerable and sustained efforts to identify phosphate-solubilizing microbes possessing multiple growth promoting activities and ways as to how their survival and functional properties could be restored when applied under adverse environmental conditions.

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REFERENCES Ahuja, A., Ghosh, S. B. and D’Souza, S. F. (2007). Isolation of a starch utilizing, phosphate solubilizing fungus on buffered medium and its characterization. BioresourceTechnol., 98, 3408-3411. Asea, P. E. A., Kucey, R. M. N. and Stewart, J. W. B. (1988). Inorganic phosphate solubilization by two Penicillium species in solution culture and soil. Soil Biol. Biochem., 20, 459–464. Baath, E., Frostegard, A., Díaz-Raviña, M. and Tunlid, A. (1998). Microbial communitybased measurements to estimate heavy metal effects in soil: The use of phospholipid fatty acid patterns and bacterial community tolerance. AMBIO, 27, 58-61. Babich, H, Sotzky, G. Toxicity of nickel to microbes: environmental aspects. In: Laskin AI (ed) Advances in applied microbiology, volume 29. New York: Academic Press, pp 1995–1265 (1983). Banik, S. (1983). Variation in potentiality of phosphate-solubilizing soil microorganisms with phosphate and energy source. Zentralbl Mikrobiol., 138, 209–216. Bashan, Y; Puente, ME; Rodriquea, MN; Toledo, G; Holguin, G; Ferrera-Cerrato, R; Pedrin, S. (1995). Survival of Azorhizobium brasilense in the bulk soil and rhizosphere of 23 soil types, Appl. Env. Microbiol, 61, 1938-1945. Borie, F. and Rubio, R. (2003). Total and organic phosphorus in Chilean volcanic soils. Gayana Bot 60, 69–78 Cerezine, P. C., Nahas, E. and Banzatto, D. A. (1988). Soluble phosphate accumulation by Aspergillus niger from fluorapatite, Appl. Microbiol. Technol., 29, 501-505. Chandra, S; Kamlesh, C; Dubey, R.C; Maheshwari, D.K.. (2007). Rhizosphere competent Mesorhizobium loti MP6 induces root hair curling, inhibits Sclerotinia sclerotiorum and enhances growth of Indian mustard (Brassica campestris). Braz J Microbiol., 38:124130. Chang, CH; Yang, SS (2009). Thermo-tolerant phosphate-solubilizing microbes for multifunctional biofertilizer preparation. Biores technol, 100; 1648-1658. Chen, Y. P., Rekha, P. D., Arun, A. B., Shen, F. T., Lai, W. A. and Young, C. C. (2006). Phosphate solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities. Appl. Soil Ecol., 34, 33–41. Cordovilla, M. P., Ligero, F.and Lluch, C. (1994). The effect of salinity on N fixation and assimilation in Vicia faba. J. Exp. Bot., 45, 1483–1488.

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Crowley, D. E. and Rengel, Z. (1999). Biology and chemistry of nutrient availability in the rhizosphere. In Mineral Nutrition of Crops: Fundamental Mechanisms and Implications, Ed Z Rengel. pp. 1-40. Food Products Press, New York. Cuningham, J. E., and Kuiak, C. (1992). Production of citric and oxalic acid and solubilization of calcium phosphate by penicillium billai. Appl. Environ. Microbiol., 58, 1451-1458. Curl, E. A., and Truelove, B. (1986). The Rhizosphere, Springer-Verlag, Berlin, Germany, 288 pp. Darmwal, N. S., Singh, R. B., Rai, R. (1989). Isolation of phosphate solubilizers from different sources. Curr. Sci., 58, 570–571. Dave, A., Patel, H. H. (2003) Impact of different carbon and nitrogen sources on phosphate solubilization by Pseudomonas fluorescens. Indian J. Microbiol., 43, 33–36. Di Simine, C. D., Sayer, J. A. and Gadd, G. M. (1998). solubilization of zinc phosphate by strain of pseudomonas fluorescens isolated from forest soil. Biol. Fertil. Soils, 28, 87-94. Errakhi, R., Bouteau, F., Lebrihi, A .and Barakate, M. (2007). Evidences of biological control capacities of Streptomyces spp. Against Sclerotium rolfsii responsible for damping-off disease in sugar beet (Beta vulgaris L.). World J. Microbiol Biotechnol, 23, 1503– 1509. Fasim, F., Ahmed, N., Parsons, R. and Gadd, G. M. (2002). Solubilization of zinc salts by bacterium isolated by the air environment of tannery. FEMS Micro. Lett., 213, 1-6. Gaind, S., and Gaur, A. C. (1991).Thermotolerant phosphate solubilizing microorganisms and theirinteraction with mung bean. Plant and Soil, 133, 141-149. Ganesan, V. (2008). Rhizoremediation of cadmium soil using a cadmium-resistant plant growth-promoting rhizopseudomonad. Curr. Microbiol., 56, 403-407. Gaur, A. C. Phosphate solubilizing microorganisms as biofertilizers, pp. 176, Omega Scientific Publisher, New Delhi, India. (1990). Gaur, A. C., Sachar, S. (1980). Effect of rock phosphate and glucose concentration on phosphate solubilization by Aspergillus awamori. Curr. Sci., 49, 553–554. George, T. S., Simpson, R. J., Hadobas, P. A. and Richardson A. E. (2005). Expression of a fungal phytase gene in Nicotiana tabacum improves phosphorus nutrition of plants grown in amended soils. Plant Biotechnology Journal, 3, 129-140. Halder, A. K., Mishra, A. K. and Chakrabarty, P. K. (1991). Solubilization of inorganic phosphates by Bradirhizobium. Indian J. Exp. Biol., 29, 28–31. Halder, A. K. and Chakrabarty, P.K. (1993) Solubilization of inorganic phosphates by Rhizobium. Folia Microbiologia, 38, 325- 330. Hamdali, H., Bouizgarne, B., Hafidi, M., Lebrihi, A., Virolle, M. J. and Ouhdouch, Y. (2008). Screening for rock phosphate-solubilizing Actinomycetes from Moroccan phosphate mines. Appl Soil Ecol., 38, 12–19. Hameeda, B; Rupela, O.P; Reddy, G., Satyavani, K. (2006). Application of plant growthpromoting bacteria associated with composts and macrofauna for growth promotion of Pearl millet (Pennisetum glaucum L.). Biol. Fertil Soils, 43; 221-227. Hariprasad, P. and Niranjana,S. R. ( 2008). Isolation and characterization of phosphate solubilizing rhizobacteria to improve plant health of tomato. Plant and Soil , 316, 13-24. Holtan-Hartvik, L., Bechman, H., Hoyas, T. R., Linjordet, R. and Bakken, L. R., (2002). Heavy metals tolerance of soil denitrifying communities: N2O dynamics. Soil Biology and Biochemistry, 34, 1181-1190.

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259

Ilmer, P. and Schineer, F. (1992). Solubilization of insoluble phosphates by microrganisms isolated from forest soils. Soil Biol. Biochem., 24, 389- 395. Indiragandhi, P., Anandham, R., Madhaiyan, M. and Sa, T. M. (2008). Characterization of Plant Growth–Promoting Traits of Bacteria Isolated from Larval Guts of Diamondback Moth Plutella xylostella (Lepidoptera: Plutellidae). Curr. Microbiol., 56, 327-333. Jiang, C., Sheng, X., Qian M. and Wang, Q. (2008). Isolation and characterization of a heavy metal-resistant Burkholderia sp. from heavy metal-contaminated paddy field soil and its potential in promoting plant growth and heavy metal accumulation in metal-polluted soil. Chemosphere, 72, 157-164. Johri, J. K., Surange, S. and Nautiyal, C. S. (1999). Occurrence of salt, pH and temperature tolerant phosphate solubilizing bacteria in alkaline soils, Curr. Microbiol., 39, 89-93. Jorquera, M., Hernández, M., Rengel, Z., Marschner, P. and Mora, M. (2008). Isolation of culturable phosphobacteria with both phytate-mineralization and phosphate-solubilization activity from the rhizosphere of plants grown in a volcanic soil. Biol. Fertil. Soils, 44, 1025–1034. Joseph, S. and Jisha, M. S. (2008). Buffering Reduces Phosphate Solubilizing Ability of Selected Strains of Bacteria. American-Eurasian J. Agric. and Environ. Sci., 4 (1), 110112, 2008. Kang, S. C., Ha, G. C., Lee, T.G. and Maheshwari, D. K. (2002). Solubilization of insoluble inorganic phosphates by a soil inhabiting fungus sp. Ps 102. Curr. Sci., 79(5), 439-442. Katiyar, V. and Goel, R. (2003). Solubilization of inorganic phosphate and plant growth promotion by cold tolerant mutants of Pseudomonas fluorescens. J. Microbiol Res, 158, 163-168. Khan, M. S., Zaidi, A., Wani, P.A. and Oves, M. (2009a). Role of plant growth promoting rhizobacteria in the remediation of metal contaminated soils. Env Chem Lett, 7, 1-19. Khan, M.S; Zaidi, A; Ahemad, M; Oves, M; Wani, P.A. (2009b). Plant growth promotion by phosphate solubilizing fungi–current perspective. Arch Agron Soil Sci., DOI: 10.1080/03650340902806469. Khan, M. S., Zaidi, A. and Wani, P.A. (2007). Role of phosphate solubilizing microorganisms in sustainable agriculture- A review. Agron. Sustain. Dev., 27, 29-43. Kim, K. Y., Jordan, D. and Kirshanan, H. B. (1997). Rahnella aquatilis, a bacterium isolated from soyabean rhizophere, can solubilize hydroxyapatite. FEMS Micobiol. Lett., 153, 273-277. Kpomblekou, A. K. and Tabatabai, M. A. (1994). Effect of organic acids on the release of phosphorus from phosphate rocks. Soil Science, 158, 442-448. Kucey, R. M. N., Janzen, H. H. and Legett, M. E. (1989) Microbially mediated increases in plant-available phosphorus. Advances in Agronomy, 42, 198-228. Kumar, K.V., Singh, N., Behl, H.M., Srivastava, S. (2008). Influence of plant growth promoting bacteria and its mutant on heavy metal toxicity in Brassica juncea grown in fly ash amended soil. Chemosphere, 72, 678-683. Lemanceau, P., Alabouvette, C. and Meyer J. M. Production of fusarinine and iron assimilation by pathogenic and non-pathogenic Fusarium. In: Swinburne TR (ed) Iron, siderophores and plant diseases. Plenum, London, pp 251–259; (1985). Linu, M. S. Stephen, J. and Jisha, M. S. (2009). Phosphate solubilizing Gluconacetobacter sp., burkholderia sp. And their potential interaction with cowpea (Vigna unguiculata (L.) Walp.). Internatl. J. Agric. Res., 4, 79-87.

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260

Mohammad Oves, Almas Zaidi, Mohd. Saghir Khan et al.

Lynch, J. M. (1990). Beneficial interactions between micro-organisms and roots. Biotechnology Advances, 8, 335-346. Ma, Y; Rajkumar, M; Freitas, H. (2009). Isolation and characterization of Ni mobilizing PGPB from serpentine soils and their potential in promoting plant growth and Ni accumulation by Brassica spp. Chemosphere, 75, 719-725. Maliha, R., Samina, K., Najma, A., Sadia, A. and Farooq, L. (2004). Organic acids production and phosphate solubilization by phosphate solubilizing microorganisms under in vitro conditions, Pak. J. Biol. Sci., 7, 187-196. Marilley, L. and Aragno, M. (1999). Phytogenetic diversity of bacterial communities differing in degree of proximity of Lolium perenne and Trifolium repens roots. Appl. Soil Ecol., 13, 127-136. Marschner, P., Solaiman, Z. and Rengel, Z. (2005b). Growth, phosphorus uptake, and rhizosphere microbial-community composition of a phosphorus-efficient wheat cultivar in soils differing in pH. J. Plant Nutrition and Soil Sci., 168, 343-351. Marschner, P., Solaiman, Z. and Rengel, Z. (2006). Rhizosphere properties of Poaceae genotypes under P-limiting conditions. Plant Soil, 283, 11-24. Marschner, P., Solaiman, Z. and Rengel, Z. (2007). Brassica genotypes differ in growth, phosphorus uptake and rhizosphere properties under P-limiting conditions. Soil Biology and Biochemistry, 39, 87-98. Matthijs, S., Tehrani, K. A., Laus, G., Jackson, R. W., Cooper, R.M. and Cornelis, P. (2007). Thioquinolobactin, a Pseudomonas siderophore with antifungal and anti-Pythium activity. Environ Microbiol, 9, 425– 434. Nahas, E. (1996). Factors determining rock phosphate solubilization by microorganisms isolated from soil. World J. Microbiol. Biotechnol., 12, 567-572. Nanniperi, P., Ascher, J., Ceccherini, M.T., Landi, L., Pietramellara, G. and Renella, G. (2003). Microbial diversity and soil functions. European J. Soil Sci., 54, 655-670. Narsian, V., Thakkar, J. and Patel, H. H. (1994). Isolation and screening of phosphate solubilizing fungi. Indian J. Microbiol., 34, 113-118. Narsian, V. and Patel, H. H. (2000). Aspergillus aculeatus as a rock phosphate solubilizer. Soil Biol Biochem, 32, 559-565. Nautiyal, C. S. (1999). An efficient microbiological growth medium for screening phosphate solubilizing microrganisms.FEMS Microbiol. Lett., 170, 265-270. Nautiyal, C. S., Bhadauria, S., Kumar, P., Lal, H., Mondal, R. and Verma, D. (2000). Stress induced phosphate solubilization in bacteria isolated from alkaline soils. FEMS Microbiol Lett., 182,291– 296. Nelson, D. R. and Mele, P.M. (2007). Subtle changes in rhizosphere microbial community structure in response to increased boron and sodium chloride concentrations. Soil Biology and Biochemistry, 39, 340-351. Osborne L D and Rengel Z 2002 Growth and P uptake by wheat genotypes supplied with phytate as the only P source. Aust. J. Agric. Res., 53, 845-850. Pandey, A. Trivedi, P. Kumar, B. and Palni, L. M. S. (2006). Characterization of a Phosphate Solubilizing and Antagonistic Strain of Pseudomonas putida (B0) Isolated from a SubAlpine Location in the Indian Central Himalaya. Curr. Microbiol., 53, 102–107. Ponmurugan, P. and Gopi, C. (2006). Distribution pattern and screening of phosphate solubilizing bacteria isolated from different food and forage crops. J. Agronomy, 5, 600604.

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Variation in Plant Growth Promoting Activities…

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Poonguzhali, S. and Madhaiyan, M. (2008). Isolation and identification of phosphate solubilizing bacteria from chinese cabbage and their effect on growth and phosphorus utilization of plants. J. Microbiol. Biotechnol., 18, 773-777. Radersma, S. and Grierson, P. F. (2004). Phosphorus mobilization in agroforestry: organic anions, phosphatase activity and phosphorus fractions in the rhizosphere. Plant Soil, 259, 209-219. Rajankar, P. N., Tambekar, D. H. and Wate S.R. (2007). Study of Phosphate Solubilization Efficiencies of Fungi and Bacteria Isolated from Saline belt of Purna river basin. Res. J. of Agric. and Biological Sci., 3(6), 701-703. Rajkumar, M. and Freitas, H. (2008). Influence of metal resistant-plant growth-promoting bacteria on the growth of Ricinus communis in soil contaminated with heavy metals. Chemosphere, 71, 834-842. Rao, D. L. N., and P. C. Sharma. 1995. Effectiveness of rhizobial strains for chickpea under salinity stress and recovery of nodulation on desalinization. Indian J. Exp. Biol. 33:500– 504. Rengel, Z. and Marschner, P. (2005). Nutrient availability and management in the rhizosphere: exploiting genotypic differences. New Phytologist, 168, 305-312. Richardson, A. E., Hadobas, P. A. and Hayes. J. E. (2001). Extracellular secretion of Aspergillus phytase from Arabidopsis roots enables plants to obtain phosphorus from phytate. Plant Journal, 25, 641-649. Rosado, A. S., De Azevedo, F.S., da Croz, D.W., Van Elas, J. D. and Seldin, L. (1998). Phenotypic and genetic diversity of Paeni bacillus azatofeixans strains isolated from the rhizophere soil of different grasses. J. Appl. Microbiol. 84, 216-226. Rudrappa, T., Splaine, R.E., Biedrzycki, M.L., Bais, H.P. (2008). Cyanogenic pseudomonads Iinfluence multitrophic interactions in the rhizosphere. Public Library of Science., 3, e2073. Sayer, J.A. and Gadd, G.M. (1998). Solubilization and precipitation of metals by fungi. Minerol. Soc. Bull., 3-5. Selvakumar, G., Mohan, M., Kundu, S., Gupta, A.D., Joshi, P., Nazim, S. and Gupta, H. S. (2008). Cold tolerance and plant growth promotion potential of Serratia marcescens strain SRM (MTCC 8708) isolated from flowers of summer squash (Cucurbita pepo). Let. Appl. Microbiol., 46, 171-175. Shaharoona, B., Naveed, M., Arshad, M. and Zahir, Z. A. (2008). Fertilizer-dependent efficiency of Pseudomonads for improving growth, yield, and nutrient use efficiency of wheat (Triticum aestivum L.). Appl. Microbiol. Biotechnol., 79, 147-155. Siddiqui, I.A., Shaukat, S.S., Sheikh, I.H., Khan, A. (2006). Role of cyanide production by Pseudomonas fluorescens CHA0 in the suppression of root-knot nematode, Meloidogyne javanica in tomato. World J. Microbiol. Biotechnol., 22, 641-650. Singh, N., Pandey, P., Dubey, R. and Maheshwari, D. K. (2008) Biological control of root rot fungus Macrophomina phaseolina and growth enhancement of Pinus roxburghii (Sarg.) by rhizosphere competent Bacillus subtilis BN1. World J. Microbiol. Biotechnol., 24, 1669-1679. Souchie, E. L., Azcon, R., Barea, J. M., Saggin-Júnior, O.J and da Silva, E.M.R. (2007). Indolacetic acid production by P-solubilizing microorganisms and interaction with arbuscular mycorrhizal fungi. Acta Sci. Biol. Sci., 29, 315-320.

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Sousa, C.D.S; Soares, A.C.F; Garrido, M.D.S. (2008). Characterization of streptomycetes with potential to promote plant growth and biocontrol . Sci. agric. (Piracicaba, Braz.) 65, 50-55. Tao, G.C., Tian, S.J., Cai, M.Y., Xie, G.H. (2008). Phosphate-solubilizing and -mineralizing abilities of bacteria isolated from soils. Pedosphere., 18; 515-523. Tate, RL. Soil microbiology (symbiotic nitrogen fixation), p. 307– 333. John Wiley and Sons; Inc., New York, N.Y. (1995). Tilak, K. V. B. R., Ranganayaki, N., Pal, K. K., De, R., Saxena, A. K., Nautiyal, C. S. Mittal, S., Tripathi, C.A. K. and Johri, B. N. (2005). Diversity of plant growth and soil health supporting bacteria. Current Science, 89, 1. Van, Elsas J.D; Van, Overbeek, LS; Fouchier, R. (1991). A specific marker pat for studying the fate of introduced bacteria and their DNA in soil using a combination of detection techniques, Plant Soil,. 138, 49-60. Van Veen, J.A; Leonard, S; Van Overbeek, L.S; Van Ellsas, J.D. (1997). Fate and activity of microorganisms introduced into soil, Microbiol. Mol. Biol. Rev., 61, 121-135. Varsha, N.H.H. (2002). Aspergillus aculeatus as a rock phosphate solubilizer. Soil Biol. Biochem., 32, 559-565. Vikram, A. Hamzehzarghani, H., Alagawadi, A. R., Krishnaraj, P. U. and Chandrashekar, B. S. (2007). Production of plant growth promoting substances by phosphate solubilizing bacteria isolated from Vertisols. J Plant Sci., 2, 326-33. Vyas, P. Rahi, P., Chauhan, A. and Gulati, A. (2007). Phosphate solubilization potential and stress tolerance of Eupenicillium parvum from tea soil. Mycological Research, 111, 931938. Wang, D., Marschner, P., Solaiman, Z. and Rengel, Z. (2007a). Below ground interactions between intercropped wheat and Brassicas in acidic and alkaline soils. Soil Biology and Biochemistry, 39, 961-971. Wani, P. A., Zaidi. A., Khan, A. A. and Khan, M. S. (2005). Effect of Phorate on Phosphate Solubilization and Indole Acetic Acid Releasing Potentials of Rhizospheric Microorganisms. Annals of Plant Protection Sciences, 13, 139–144. Wani, P. A., Khan, M. S. and ZAIDI, A. (2007a). Synergistic effects of the inoculation with nitrogen fixing and phosphate solubilizing rhizobacteria on the performance of field grown chickpea. J. Plant Nutr. Soil Sci., 170, 283-287. Wani, P. A., Khan, M .S. and Zaidi, A. (2007b) Co-inoculation of nitrogen fixing and phosphate solubilizing bacteria to promote growth, yield and nutrient uptake in chickpea. Acta Agron. Hung., 55, 315-323. Wani, P. A., Khan, M .S. and Zaidi, A. (2007c). Chromium reduction, plant growth promoting potentials and metal solubilization by Bacillus sp. isolated from alluvial soil. Curr. Microbiol., 54, 237-243. Wani PA; Khan MS, Zaidi A (2007d) Effect of metal tolerant plant growth promoting Bradyrhizobium sp. (vigna) on growth, symbiosis, seed yield and metal uptake by greengram plants. Chemosphere 70, 36-45. Wani, P. A., Khan, M .S. and Zaidi, A. (2008). Chromium reducing and plant growth promoting Mesorhizobium improves chickpea growth in chromium amended soil, Biotechnol. Lett., 30, 159-163.

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Whitelaw, M. A., Harden, T. J. and Helyar, K. R. (1999). Phosphate solubilization in solution culture by the soil fungus penicillium radicum. Soil Biology and Biochemistry, 32, 655665. Xiao, K., Samac, D.A and Kinkel, L. L. (2002). Biological control of Phytophthora root rots on alfalfa and soybean with Streptomyces. Biol Control 23, 285–295. Zaidi, A.and Khan, M. S. (2007). Stimulatory effects of dual inoculation with phosphate solubilizing microorganisms and arbuscular mycorrhizal fungus on chickpea. Aust. J. Exp. Agric., 47, 1016-1022.

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In: Phosphate Solubilising Microbes for Crop Improvement ISBN 978-1-60876-112-8 Editor: M. S. Khan and A. Zaidi © 2009 Nova Science Publishers, Inc.

Chapter 12

MANAGEMENT OF PLANT DISEASES USING PHOSPHATE-SOLUBILIZING MICROBES Adel Ahmed El-Mehalawy∗ Microbiology Department, Ain Shams University, Faculty of Science, Egypt

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ABSTRACT Phosphorus, which is important for plant growth, occurs in soil in the form of both inorganic and organic compounds. Plants absorb phosphorus only in an available form. Phosphate solubilizing microbes have the ability to solubilize insoluble phosphate, and also release soluble inorganic phosphate into the soil by decomposing phosphate-rich organic compounds. Some genera and species belonging to this group of microorganisms are also characterized by their ability to control plant pathogens causing different diseases by synthesizing antibiosis, mycoparasitism or secretion of cell wall degrading enzymes. This chapter focuses on as to how phosphate solubilizing microbes could be used effectively to control the phytopathogenic microbes and serve as a biocontrol agent.

12.1. INTRODUCTION Phosphorus [P] ranks second after nitrogen with regard to the extent of its requirement by plants and microorganisms and functions most importantly in energy production and transfer. When applied from external sources as phosphatic ferilizer, a greater portion of P becomes insoluble and unavailable to plants due to complex formation with calcium or magnesium salts [in calcareous soils] and iron or aluminum salts [in acid environments]. Of the microbial communities, phosphate solubilizing microorganisms [PSM] including bacteria, fungi and actinomycetes transform insoluble P into soluble forms and make it available to plants resulting in improved growth and yields. Bacteria, like, Pseudomonas striata and Bacillus megaterium are important P-solubilizers [Fallah, 2006] while fungi like, Aspergillus and ∗

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Penicillium, are potential P-solubilizers [Fenice et al., 2000; Narsian and Patel, 2000]. Soil phosphates are rendered available to plants by soil microorganisms through secretion of organic acids. Therefore, P-dissolving soil microorganisms play an important role in correcting P deficiency in soils. They may also release soluble P into soil by decomposing phosphate rich organic compounds. These microbial inoculants can substitute almost 20% to 25% of the phosphorus requirement of plants [Rodriguez and Fraga, 1999 ;Richardson, 2001] Lately, the development and use of bio-control agents for the management of diseases affecting plant production is being advocated. Biological control of plant diseases can be defined as "the involvement of the use of beneficial microorganisms, such as specialized fungi and bacteria, to attack and control plant pathogens and the diseases they cause. These specialized fungi and bacteria normally inhabit most of the soils around the world. In their natural habitat, these organisms compete with other microorganisms for space and food and produce toxic substances that parasitize and/or kill other soil-inhabiting pathogenic organisms such as, Pythium, Phytophthora, Rhizoctonia and others capable of inflicting losses to plants. Numerous studies have shown that these microorganisms play a vital role in the makeup of the soil environment and are part of the normal checks and balances that make up a healthy soil [Jeffries et al., 2003]. Recently, some of the more promising strains of these bio-control agents have been developed and marketed as an alternative to traditional chemical based fungicides for their use against pathogens [e.g., Trichoderma] causing diseases onto ornamental plants [Grinyer et al.,2004b]. Such bio-control products are introduced on regular basis for a wide variety of plant pathogenic organisms, some of which remain active for years while others show its activity for a very short period.

Figure 1. Movement of phosphorus.

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12.2. PHOSPHORUS FORMS IN SOIL Both inorganic and organic P occurs in soil. The inorganic forms are compounds of calcium, iron, and aluminum. Bulk of P in earth exists as apatites [M10 [PO4] 6x,2]. The M refers to calcium and the X is the anion fluorine but it can also be Cl-OH- or CO3 which means that P can exist as flour, chloro, hydroxyl and carbonate apatites. Different substitutions and combinations of M and X may result in about 200 different types of P occurring in nature. Rock phosphates [RP] high in carbonate apatite are commonly mined for fertilizers. The organic P containing compounds derived from plants and microorganisms include nucleic acids, phospholipids and phytin. Organic matter derived from dead and decaying plant debris is rich in organic sources of phosphorus [Figure 1].The deficiency of P may occur in crop plants growing in soils containing inadequate phosphates. This may be partly due to the fact that plants are able to absorb P only in an available form. Soil phosphates are rendered available either by plant roots or by soil microorganisms through secretion of organic acids.

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12.3. PHOSPHATE SOLUBILIZING MICROBES AND P SOLUBILIZATION - AN OVERVIEW Phosphate solubilizing microorganisms as a group form an integral component of rhizosphere, which benefit growth and development of plants by providing it with P. On the other hand, certain microorganisms, through assimilation, may immobilize available phosphates in their cellular material. Such assimilation processes in soil may also contribute to P deficiency of plant crops. Many fungi [Penicillium, Sclerotium, Aspergillus] and bacteria [Achromobacter, Acinetobacter, Aerobacter Bacillus, Enterobacter, Erwinia, Eubacterium, Klebsiella, Mycobacterium, Micrococcus, Mesorhizobium, Pseudomonas, Rhizobium etc.] are potential solubilizers of bound phosphates [Mishra, 1996; Villegas and Fortin, 2001; Bano and Musarrar, 2004; Pandey, 2008]. Although bacteria have been used as commercial inoculants for various crops, fungi seem to be better agents for dissolving insoluble P. Phosphate-dissolving bacteria secretes various organic acids such as, formic, acetic, propionic, lactic, glycolic, fumaric and succinic acids which in turn, affect solubilization of P. Although organic acid production is invariably associated with P solubilization, many workers have not been able to correlate changes in the pH of the medium with the amount of P solubilized [Khan et al. 2007]. Among P solubilizing bacteria, fluorescent pseudomonads has been considered as an important group of bacteria due to their biofertilizing and biocontrol properties [Jha et al. 2009]. In numerous studies, plant growth promoting rhizobacteria have been shown to exhibit the production of antimicrobial metabolites which take part in suppression of diseases caused by soil-borne plant pathogens as well as are involved in the induction of systemic resistance against insects and nematodes [Whipps, 2001]. Growth promotion and availability of P are not the only mechanisms by which these microorganisms exert a positive effect on plants. But microbially mediated solubilization of insoluble P is often combined with production of other metabolites, which take part in the management of soil-borne phytopathogens [Altomare et al. 1999]. In vitro studies show the

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potential of P-solubilizing microorganisms for the simultaneous synthesis and release of pathogen suppressing metabolites, mainly siderophores [a low molecular weight, iron chelating ligands] and lytic enzymes. Siderophore helps microorganisms compete effectively against other organisms by limiting iron availability to the competing one and hence, restrict their growth. Similarly auxins are thought to play a role in host-parasite interaction and are involved in the interaction between a plant pathogen and its host. Various authors have proposed mechanisms of biocontrol action of IAA, which resulted in two main hypotheses: [1] a potential involvement of IAA together with glutathione S-transferases in defense-related plant reactions and [2] an inhibition of spore germination and mycelium growth of different pathogenic fungi. For instance, IAA supply to excised potato [Solanum tuberosum] leaves reduced the severity of the disease provoked by Phytophthora infestans [Vassliev et al. 2006].

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12.4. BIOLOGICAL CONTROL OF PLANT DISEASES The interaction between biocontrol bacteria, actinomycetes, yeast and filamentous fungi and plant pathogens continue to be the focus of large number of researches in order to offset the contamination of the environment caused by the use of agrochemicals. In general, biological control of plant disease is carried out by one of the following modes of action- [a] inhibition of plant pathogens by antimicrobial compounds [antibiosis] [b] competition for iron through the production of siderophores [iii] competition for colonization sites [iv] competition for nutrient supplied by seeds and roots [v] induction of plant resistance mechanism [vi] inactivation of pathogen germination factors present in seed or root exudates [vii] degradation of pathogenicity factors of the pathogen such as toxins and [viii] production of extracellular cell-wall degrading enzymes [e.g., chitinase and β-1,3-glucanase] that can lyze the cell walls of pathogens. Most of these interactive biological control mechanisms occur in the rhizosphere which can be classified as- [1] bacteria-bacteria-pathogen interaction: where biocontrol may operate through antibiosis or compete for space or nutrition in the rhizosphere [2] bacterial-fungal pathogen interaction: where biocontrol operate through antibiosis, compete for iron, exhibit parasitism and production of extracellular enzymes, for instance, actinomycetes parasitize and degrade spores of fungal plant pathogens, induced resistance and [3] fungal-fungal pathogen interaction: where biocontrol may act by synthesizing antibiosis, induced resistance or mycoparasitism [Keel and Dèfago, 1997].

12.5. MANAGEMENT OF PLANT DISEASES BY PHOSPHATE SOLUBILIZING-MICROBES Phosphate-solubilizing microorganisms as a group form an integral component of the soils. In addition to providing P to the plants, PS microorganisms also act as a bio-control agent and promote the growth of plants by suppressing the soil borne phytopathogens. Several in vitro studies show the potential of PS microorganisms for the simultaneous synthesis and release of pathogen-suppressing metabolites, mainly siderophores, and lytic enzymes [Chang and Yang 2009]. Potential application of PS microbes as bio-control agent is reviewed and discussed.

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12.5.1. Disease Suppression by Phosphate-Solubilizing Bacteria Mycoparasites and other antagonists have been extensively explored, but none of them have provided sufficient control of plant diseases. In fact, majority of the researches on the management of pathogens have used either mycoparasites or a limited number of other antagonists but inadequate efforts have been directed towards exploiting the potential of rhizobacteria, especially phosphate-solubilizing microorganisms in disease management. Though, phosphate-solubilizing organisms are well known for their P-solubilizing ability, yet the metabolites released by them possess antimicrobial activity [Leeman et al. 1995]. Bio-control ability of PS bacterium Pseudomonas aeruginosa ID 4365, a bio-control agent of groundnut [Arachis hypogaea] phytopathogens from marine origin, was previously attributed to the production of pyoverdin type of siderophores. However, pyoverdin-rich supernatants of this organism has shown better antifungal activity compared to equivalent amount of purified pyoverdin indicating the presence of undetected metabolite [s] in pyoverdin rich supernatants. In addition to pyoverdin, Pseudomonas aeruginosa ID 4365 strain produced siderophores, [viz. pyochelin and salicylic acid] and two broad spectrum antifungal compounds [viz. pyocyanin and phenazine-1-carboxylic acid] and exhibited antifungal activity [Rane et al. 2008]. A variety of other microbial compounds are involved in the suppression of phytopathogenic growth leading thereby to the reduction in damage to plants. These microbially synthesized compounds include defence enzymes, such as, chitinase, ß-1,3-glucanase, peroxidise, protease and lipase [Karthikeyan et al. 2006]. As an example, phosphate solubilizing and antagonistic bacterial strain designated as BO, showed maximum similarity with Pseudomonas putida, solubilized tricalcium phosphate and demonstrated antifungal activity against phytopathogenic fungi [Alternaria alternate, Fusarium oxysporum and R. solani] in Petri dish assays [El-Mehalawy et al. 2007] by producing chitinase [86.5 n mol min-1 mg-1 of protein], β-1,3- glucanase [98 n mol min-1 mg-1 of protein], salicylic acid [8 μgmL-1], siderophore [13 μ mol benzoic acid ml-1] and hydrogen cyanide [0.06 m OD at 625 nm] [Figure 2] [Pandey et al. 2006]. Though, it caused transparent clearing in case of A. alternata, it suppressed the process of conidia formation in F. oxysporum. Of these, chitinase and ß-1,3-glucanase degrade the fungal cell wall and caused lysis of fungal cell. Furthermore, chitin and glucan oligomers released during degradation of the fungal cell wall by the action of lytic enzymes act as elicitors that induce various defense mechanisms in plants [Karthikeyan et al. 2005]. Such enzymes produced by Pseudomonas stutzeri have shown the lysis of the pathogen Fusarium sp. [Peter et al. 2007]. Furthermore, peroxidase plays a key role in the biosynthesis of lignin which limits the extent of pathogen spread [Bruce and West 1989]. In a study, a rapid increase in peroxidase activity was recorded in coconut [Cocos nucifera L.] treated with a mixture of P. fluorescens, T. viride and chitin which contributed to induced resistance against invasion by Ganoderma lucidum, the causal agent of Ganoderma disease [Karthikeyan et al. 2006]. These findings suggest that phosphate-solubilizing bacteria possessing the ability to synthesize hydrolytic enzymes can also effectively be utilized for managing the plant diseases. The growth promotion and antifungal properties were further demonstrated through a maize-based bioassay under greenhouse conditions. Although the bacterial inoculation resulted in significant increment in plant biomass, it stimulated bacterial and suppressed fungal populations in the rhizosphere.

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Figure 2. Antifungal activity of P. putida against A. alternata [Plate A], F. oxysporum [Plate B] and R. solani in Petri dish bioassay.

In greenhouse experiment, P. putida showed a considerable reduction in disease severity caused by pathogenic fungus Rhizoctonia solani [the causal agent of damping-off of seedling] in cotton [Gossypium barbadense] plants and increased the measured growth parameters [length and biomass of roots and shoots]. When P. putida was grown under optimum cultural condition on broth media and assessed for the production of antifungal compounds, five active components namely, tossendanin, dodecamethyl-hexasiloxane, 1-2, benzenedicarboxylic aci, bis [2-ethylhexyl] ester, 2,6-diterbutyl-4-hydroxy methyl phenol and ethyl-2-hydroxybutyrate were detected using paper chromatography. Another bacterial species, Bacillus subtilis, was shown to have antagonistic activity against Phyophthora capsici, the causal agent of blight in red pepper. Two different in vitro and in vivo assays were employed for screening potential antagonist against the soil plant pathogen. First, the traditional in vitro dual culture assay on PDA was taken as preliminary screening criteria for selecting the antagonist. Secondly, assay was performed on the basis of interaction of pathogen, antagonist and host plant, which resembled the field condition. In dual culture assay, there was no involvement of the host plant, where as in real conditions, the host plant played an important role in suppressing the induced antagonist [Lee et al, 2008]. Phosphate-solubilizing bacteria also help to management the root-knot diseases. for example, treatment of the seeds of mungbean [Vigna radiata [L.] wilczek] with P. flourescens suppressed the disease severity, nematode reproduction and promoted the yield of mungbean by 31%, greater than those obtained by nematicide application. P. flourescens reduced the egg mass by 21%, while B. subtilis reduced the egg mass by 12%. Fecundity of the nematode [number of egg/egg mass] was decreased by 13% due to P. flourescens treatment. Such effects were probably due to the synthesis of toxins such as, phenazin, pyrrolnintrin etc. produced by P. flourescens [ Toohey et al., 1965; Khan et.al. 2003]. In a screening program, P. corrugate, a phosphate-solubilizing bacterium, had antagonistic activity against Botrytis cinerea, the causal agent of grey mildew of tomato. Such inhibitory effects were suggested to be due to synthesis of HCN and cell wall-degrading enzymes, such as, cellulases, proteases and chitinases. In addition, production of IAA and phosphatase by plant beneficial bacteria might have contributed to the enhancement of host plant root system leading to the overall growth of plants [Guo et al. 2007]. Other bacterial communities such as, Pseudomonas aeruginosa, P. mosselii, P. monteilii, P. plecoglossicida, P. putida, P. fulva and P.

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fluorescens have also shown phosphate solubilization, production of plant growth promoting enzymes, hormones and exhibited antagonism against phytopathogenic fungi that attack on various crops [Naik et al. 2008].

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12.5.2. Management of Plant Disease by Phosphate-Solubilizing Actinomycetes The actinomycetes, mainly belonging to the genus Streptomyces form an integral component of soil bacteria. Actinomycetes are Gram positive, aerobic and mycelium forming bacteria. As a group they are known for their activity in nutrient cycling, nitrogen fixation, production of secondary metabolites and plant-growth promotion [Conn et al. 1997; Lazarovitz, and Nowak 1997]. However, they are largely involved in the degradation of complex molecules [e.g., cellulose, lignocellulose, xylan and lignin] and play a significant role in soil organic matter decomposition processes [Petrosyan et al., 2003; Ding et al., 2004; Murashima et al., 2002; Lynd et al., 2002]. In addition, such microbes also acts as a potential biocontrol agent [Hoster et al., 2005; Nassar et al., 2003; Thirup et al., 2001] and hence, facilitate the growth of plants both directly and indirectly. Direct effects include production of [i] plant growth regulating hormones [phytohormones] [ii] phosphate solubilization [iii] nitrogen fixation and [iv] increased nutrient uptake. While indirect effect involves the production of [i] secondary metabolites [ii] competition [iii] parasitism and [iv] induction of resistance [Conn et al. 1997. For instance, genus Streptomyces have shown In vitro cellulolytic, xilanolytic and chitinolytic activity, indolacetic acid production and P solubilization activities and siderophore producing ability [Sousa et al. 2008; Barreto et al. 2008; Hamdali et al. 2008b; Cattelan and Hartel 2000; Cattelan, 1999; Crawford et al., 1993]. Many investigations under in vitro or in vivo environment have showed that various species of Streptomyces and other actinomycete genera inhibited soil-borne pathogens by antibiosis, extracellular enzymes, inhibition of spore germination, oospore parasitism and hyphal interaction [Cook an Baker, 1983; Inbar et al., 2005]. For example, strains of Streptomyces ambofaciens showed antifungal activity against Pythiumn damping-off in tomato plants and Fusarium wilt in cotton plants in an artificially infested soil. Similar root disease suppression was obtained with other streptomycetes when applied in the form of spores, mycelia, or combinations of the two in growth chambers and greenhouse experiments [Bolton, 1980]. Some actinomycetes have also shown to limit the growth of Gram negative bacteria [like, E.coli and P. aureus], Gram positive bacteria [ Micrococcus luteus, Bacillus subtilis and Staphylococcus aureus] and filamentous phytopathogenic fungi [Pythium ultimum and Fusarium oxysporum f. sp. albedinis] [Hamdali et al., 2008a]. In addition, actinomycetes species like Streptomyces aurantiacus, S. microflavus, S. purpurea and S. erumpens demonstrated a remarkable antagonistic activities against Rhizoctonia solani [Figure 3]. When grown under optimum cultural conditions in broth media and observed for the production of antifungal compounds, they produced different active compounds. For example, S. erumpens produced tossendanin, 2-nonadecanone, 2-4-DNPH, 12- trichosanone, octacosane and methyl paraben while S. purpureus produced 2, 3-dihydro-3methoxywisthacnistin acetate, 2-nonadecanone, 2-4-DNPH, 12-trichosanone and isopropyl5[methyl-D3]-1-cyclohexanol-4,4-D2. Other actinomycetes like, S. aurantiacus produced 2,3dihydro-3- methoxywisthacnistin acetate, 1,3-dioxane,4-[hexadecylo]2-pentadecyl, 5-

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hydroxy-2,2-dimethyl-5,6-bis [2-oxo-1-propyl]-1-cyclohexanone and ethyle-2hydroxybutyrate and S. microflavus displayed the synthesis of tossendanin, 2-nonadecanone, 2-4-DNPH, 12- trichosanone and isopropyl-5[methyl-D3]-1-cyclohexanol-4,4-D2.

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Figure 3. Antagonistic activity of S. aurantiacus [A], S. microflavus [B], S. purpuerus [C] and S. erumpens [D] against R. solani.

Figure 4. Antagonistic activity of S. lydicus against a] A. solani, b] C. maydis, c] F. oxysporum and d] P. digitatum.

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Each actinomycete reduced the percentage of diseases plants and led to an increase in the measured growth parameters like, length of root, height of shoot, fresh and dry mass of roots and shoots of cotton [Gossypium barbadense L.] compared to the control [El-Mehalawy et al., 2007]. In other study, four Streptomyces species including S. lydicus, S. ederensis, S. erumpens and S. antimicoticus antagonized the phytopathogens, Alternaria solani, Cephalosporium maydis, Fusarium oxysporum f. sp. lycopersici and Penicillium digitaum [the causal agent of early blight of tomato, late blight of wheat, wilt disease of kidney bean and green mold of orange, respectively] through antibiosis [Abd-Alla et al., 2004]. The antagonistic activity of S. lydicus [Figure 4], S. ederensis [Figure 5], S. erumpns [Figure 6] and S. antimycoticus [Figure 7] against A. solani, C. maydis, F. oxysporum P. digitatum were pronounced.

Figure 5. Antagonistic activity of S. ederensis against a] A. solani, b] C. maydis, c] F. oxysporum and d] P. digitatum.

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Figure 6. Antagonistic activity of S. erumpns against a] A. solani b] C. maydis, c] F. oxysporum and d] P. digitatum.

Figure 7. Antagonistic activity of S. antimycoticus against a] A. solani, b] C. maydis c] F. oxysporum and d] P. digitatum.

In other study, antifungal compounds producing strains of five Streptomyces species caused an inhibition of R. solani and P. megaspermas var. sojae. Of these, S. griseus, S. hygroscpicus var. geldanus and S. nousei produced larger zones of inhibition against R. solani and P. megasperma var. sojae while S. hygroscopicus completely inhibited the growth of R. solani. Several other Streptomyces spp. reduced Phytophthora root rot on soybean when the streptomycetes was added to the soil at the same time as P. megasperma var. sojae or seven days before the pathogen was added [Rothrock and Gottlieb, 1984].

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12.5.3. Management of Plant Disease by Phosphate-Solubilizing Fungi Among phosphate-solubilizing fungi, Trichoderma viride showed antagonistic activity against Alternaria solani, the causal agent of wilt disease of tomato [Figure 8 A and B]. Its antagonistic activity was mediated by antibiosis, or by mycoparasitism involving coiling of Trichoderma hyphae around the mycelia of A. solani and degrading its cell wall leading eventually reduced growth and activity of the plant pathogenic fungus [El-Mehalawy, 1999].

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Figure 8. Antagonistic activity of Trichoderma viride against A. solani representing – [A] antibiosis and [B] mycoparasitism.

Trichoderma viride also showed a remarkable antagonistic activity against R. solani [Figure 9] and produced antifungal compounds under optimum cultural conditions in broth medium. When tested, It reduced the severity of disease produced on tomato plants and subsequently increased the measured growth parameters [length of roots, height of shoots and biomass production in roots and shoots [El-Mehalawy et al., 2007]. Among other fungi, species of Penicillium are known to produce a number of mycotoxins, including agriculturally-important toxins, ochratoxin A, citrinin and proquefortine. For example, a phosphate-solubilizing fungus Penicillium bilaii has been reported to produce gliotoxin, an antibiotic, produced also by Aspergillus fumigatus. For Aspergillus fumigatus, the gliotoxin is considered to be involved in the pathology of aspergillosis [Savard et al., 1994].

Figure 9. Antagonistic activity of T. viride against R. solani.

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Arbuscular mycorrhizal [AM] fungi can improve plant growth by taping relatively immobile nutrients, such as, P and making it available to plants. They are ubiquitous and forms mutualistic relationships with roots of most terrestrial plants [smith and Read 1997]. Their association benefits plant nutrition, growth and survival due to their ability to traverse long distances in soils and the ability to exploit soil nutrients. The ability of AM-fungi to solubilize otherwise insoluble phosphate sources besides their role in protecting plants against environmental and cultural stresses and pathogenic attack is reported [Vassilev et al. 2006]. The efficiency of AM-fungi as biocontrol agent depends largely on the kind of AM-fungi involved, the substrate and the host genotypes. However, protection offered by AM-fungi is not effective against all the plant pathogens and is modulated by soil and other environmental conditions too. Some of the common examples of AM-fungi used to suppress diseases are presented in Table 1.

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Table 1. Effects of AM-fungi on fungal diseases and plant growth AM fungus Glomus intaradices

Pathogenic fungus Fusarium oxysporum f. sp. lycopersici

G. fasciclatum G. etunicatum

Aphanomyces euteiches Pythium ultimum

G. fasciculatum

Verticillium albo atrum

G. fasciculatum

F. oxysporum

G. vesiformae

Verticillium dahliae

G. fasiculatum

F. udum

Glomus sp.

Sclerotium cepivorum

G. mossae G. mossae G. mossae

F. oxysporum Phytophthora nicotiana var. parasitica

G. proliferum G. intraradices G. versiformae G. mossae G. intraradices

Fusarium solani Rhizoctonia solani Cylindraclad-ium spathiphylli. P. parasitica

G. etunicatum G. intraradices G. fasciculatum

R. solani F. oxysporum f.sp. ciceris

Effect AM-fungus significantly reduced Fusarium root rot on tomato Reduced root rot on pea Reduced disease severity on cucumber Reduced incidence of disease in alfalfa Reduced colonization by pathogen and severity of disease on cowpea Reduced disease indices in cotton Reduced wilt indices in pigeon pea Reduced white rot incidence and delayed disease development on onion Significantly reduced Fusarium wilt on tomato and pepper

Reduced root necrosis and necrotic root apices ranged between 63-89%. Reduced disease severity of disease on peanut Increased growth and reduced disease severity in banana Reduced disease symptoms produced by P. parasitica on tomato Reduced disease severity in micropropagated banana Reduced the disease severity in chickpea

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CONCLUSION Phosphate-solubilizing microbes being an important and integral component of soils demonstrate both economic and environmental advantages over environmentally unfriendly chemical fertilizers, if applied in agricultural practices. Moreover, the simultaneous activity of phosphate solubilization and management of pathogenic microbes further adds substantial value to the cropping systems. Therefore, among the functionally diverse groups of microbes inhabiting rhizosphere, phosphate solubilizing microorganisms with multiple innate plant growth promoting activities is likely to attract more attention for their use both as P solubilizer and biocontrol agents in modern day agricultural practices. The information provided in this review is likely to help better understand the role of P solubilizers as biological control agent besides their P solubilizing activity which in turn could lead to reduce the dependence on the use of chemicals to attain similar objectives in crop productivity.

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REFERENCE Abd-Allah, NA; El-Mehalawy, AA; Osman, RM; Abu-Shady, MR. Actinomycetes antagonizing plant and human pathogenic fungi. I: Isolation and identification of actinomycetes and their antagonistic activity. Bull. Fac. Sci. Assiut Univ., [2004], 33, 2140. Akhtar, MS; Siddiqui, ZA. Arbuscular mycorrhizal fungi as potential bioprotectants against plant pathogens. Mycorrhizae: Sustainable Agriculture and Forestry [2008], 3, 61-97. Altmare, C.; Norvell, WA; Björkman, T; Herman, GE. Solubilization of phosphates and micronutrients by the plant-groeth promoting and biocontrol fungus Trichoderma harzianum rifai 1295-22. Appl Environ Microbiol, [1999], 65, 2926-2933. Bano, N; Musarrat, J. Characterization of a novel carbofuran degrading Pseudomonas sp. With collateral biocontrol and plant growth promoting potential. FEMS Microbiol Lett [2004], 231, 13-17. Barreto TR; da Silva ACM; Soares ACF; de Souza JT Population densities and genetic diversity of actinomycetes associated to the rhizosphere of Theobroma cacao. Braz J Microbiol, [2008], 39, 464-470. Bolton, AT. Control of Pythium aphanidermatum in Poinsettia in soilless culture by Trichoderma viride and Streptomyces sp. Can J Plant Pathol [1980], 2, 93-95. Chang CH and Yang SS Thermo-tolerant phosphate-solubilizing microbes for multifunctional biofertilizer preparation. Bioresource Technol, [2009],100, 1648-1658. Cook, RJ; Baker, KF. "The nature and practice of biological control of plant pathogens". The American Phytopathological Society St. Paul, Minnesota, USA, [1983]. Conn, KL; Nowak, J; Lazarovits, GA. Gnotobiotic bioassay for studying interactions between potatoes and plant growth promoting rhizobacteria. Can J. Microbiol, [1997], 43, 801808. El-Mehalawy, AA. Biocontrol of soil-borne plant pathogens by Trichoderma viride. The 9th International Conference: Environmental protection is a must. 4-6 May [1999], Alexandria, ARE.

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Adel Ahmed El-Mehalawy

El-Mehalawy, AA; Hassanin, SM; Hassanin, NM; Zaki, SA. Induction of resistance and biocontrol of Rhizoctonia solani in cotton against damping-off disease by rhizosphere microorganisms. New Egypt J Microbiol, [2007], 17, 148-168. Fenice, M; Seblman, L ; Federici, F; Vassilev, N. Application of encapsulated Penicillum variable P16 in solubilization of rock phosphate. Bioresource Technol, [2000], 73, 157162. Grinyer, J; MckKay, M; Nevalainen, H; Herbert, BR. Fungal proteomics: initial mapping of biological control strain Trichoderma harzianum. Curr. Gent, [2004 b], 45:163-169. Guo, Y.; Zheng, H.; Yang, Y. and Wang, H. Characterization of Pseudomonas corrugate strain P94 isolated from soil in Beijng as a potential biocontrol agent. Curr Microbiol, [2007], 55, 247-253. Hamadali, H. Hafidi, M; Virolle, MJ; Ouhdouch, Y. Rock phosphate solubilizing actinomycetes: Screening for plant growth promoting activities. World J Microbiol Biotechnol, [2008a]. 24, 2565-2575. Hamdali, H; Bouizgarne B; Hafidi, M; Lebrihi A; Virolle, MJ, Ouhdouch, Y. Screening for rock phosphate solubilizing actinomycetes from Moroccan phosphate mines. App. Soil Ecol., [2008b], 38, 12-19. Henis, Y; Inbar, M. Effect of Bacillus subtilis on sclerotium formation by Rhizoctonia solani. Phytopathology, [1968], 58, 933-938. Jeffries, P; Gininazzi, S.; Perotto, S. and Turnau,K. The contribution of arbuscular mycorrhizal fungi in sustainable maintenance of plant health and soil fertility. Biol. Fertil. Soils, [2003], 37, 1-16. Jha, BK; Pragash, MG; Cletus, J; Raman, G; Sakthivel, N. Simultaneous phosphate solubilizing potential and antifungal activity of new fluorescent pseudomonad strains Pseudomonas aeruginosa, P. plecoglossicida and P. mosselii. World J. Microbiol Biotechnol, [2009], 25, 573-581. Khan, MR; Khan, SM; Mohiddin, FA. Effect of certain fungal and biocontrol phosphate solubilizing microorganisms on the fusarial wilt of tomato. First International Meeting on Microbial phosphate solubilizers, [2003], 357-361. Khan, MR; Khan, SM; Mohiddin, FA; Askary, TH. Effect of certain phosphate solubilizing bacteria on root-knot disease of nungbean. First International Meeting on Microbial phosphate solubilization [2003], 341-346. Lee, KJ; Kannan, SK; Sub, HS; Seong, CK; Lee, GW. Biological control of Phytophthora blight in red pepper [Capsicum annuum L.] using Bacillus subtilis. World J Microbiol Biotechnol, [2008], 24, 1139-1145. Leeman, M; VanPelt, JA; Hendrickz, MK; Scheffe, RJ.; Barker, PAHM; Schippers, B. Biocontrol of Fusarium wilt of raddish in commercial green house trials by seed treatment with Pseudomonas fluorescens WCS 374. Phytopathol, [1995], 85, 1301-1305. Lazarovitz, G; Nowak, J. Rhizobacterium for improvement of plant growth aestablishment. Hortscience, [1997], 32, 188-192. Mishra, RR. [1996]. Soil Microbiology. pp 110-120. Naik PR; Raman G; Narayanan KB, Sakthivel, N. Assessment of genetic and functional diversity of phosphate solubilizing fluorescent pseudomonads isolated from rhizospheric soil. BMC Microbiology, [2008], doi:10.1186/1471-2180-8-230. Nair, MG; Burke, BA. A new fatty acid methyl ester and other biologicallty active compounds from Aspergillus niger. Phytopathol, [1988], 27, 3169-3173.

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Narsian, V; Patel, HH. Aspergillus aculeatus as a rock phosphate solubilizers. Soil Biol. Biochem, [2000], 32, 559-565. Nautiyal, CS. An efficient microbiology growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiology Lett, [1999], 17, 265-270. Pandey, A; Das, N; Kumar, B; Rinu, K; Trivedi, P. Phosphate solubilization by Penicillium spp. isolated from soil samples of Indian Himalayan region. World J. Microbiol Biotechnol, [2008], 24, 97-102. Pandey, A; Trivedi, P; Kumar, B; Palni, LMS. Characterization of phosphate solubilizing and antagonistic strain of Pseudomonas putida [BO] isolated from a sub-alpine location in the Indian Central Himalayn. Curr. Microbiol, [2006] 53, 102-107. Richardson, AE. Prospects for using soil microorganisms to improve the acquisation of phosphorus by plants. Aust. J. Plant Physiol. [2001], 28, 879-906. Rodriguuez, H; Fraga, R. Phosphate solubilizating bacteria and their role in plant growth promotion. Biocontrol. Adv, [1999], 17, 319-339. Rothrock, CS; Gottlieb, D. Role of antibiosis in antagonism of Streptomyces hygroscopicus var. geldanus to Rhizoctonia solani in soil. Can. J. Microbiol, [1984], 30, 1440-1447. Savarad, ME; Miller, JD; Blais, LA; Seifert, KA; Samson, RA. Secondary metabolites of Penicillium bilaii strain PB-50. Mycopathologia, [1994], 127, 19-27. Toohy, JI; Neston, CD; Krotkove, G. Isolation and identification of two phenazines from a strain of Pseudomonas aureofaciens . Cand. J.. Bot. [1965], 43, 1055-1062. Vassliev, N; Vasslieva, M; Nikolaeva, I. Simultaneous P-solubilizing and biocontrol of microorganisms: Potential and future trends. Appl. Microbiol., [2006], 61, 137-144. Villegas, J; Fortin, JA. Phosphorus solubilization and pH changes as a result of the interaction between soil bacteria and arbuscular mycorrhizal fungi on a medium containing NO3- as nitrogen source. Can. J. Bot, [2001], 80, 571-576. Whipps, JM Microbial onteractions and biocontrol in the rhizosphere. J. Exp. Bot., [2001], 52, 487-511.

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In: Phosphate Solubilising Microbes for Crop Improvement ISBN 978-1-60876-112-8 Editor: M. S. Khan and A. Zaidi © 2009 Nova Science Publishers, Inc.

Chapter 13

PHOSPHATE SOLUBILIZING MICROBES: POTENTIALS AND SUCCESS IN GREENHOUSE AND FIELD APPLICATIONS Diriba Muleta∗ College of Agriculture and Veterinary Medicine, Jimma University, Ethiopia

ABSTRACT

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Phosphorus is an essential macronutrient for the growth of plants. However, in most soils a large portion of phosphorus becomes insoluble and consequently, unavailable to plants. This problem is worsened in tropical soils where low N content, high P fixation, and low organic matter content seriously limit the plant growth. As a consequence, agrochemicals have been extensively applied to attain optimum yields in order to feed the ever increasing human populations. The current agricultural systems suffer both in terms of cost and environmental integritiy by the use of agrochemicals. Thus, it becomes important to search novel ways to increase food production while maintaining environmental quality. Microbial fertilizers in this context could contribute to reversing the trend of loss of soil fertility and facilitate plants to grow better. The use of phosphatesolubilizing microorganisms [PSMs] among naturally abundant and diverse microbes, as biofertilizers, is an emerging area of interest since it provides an ecologically sound and safe method for restoration of soil fertility. Several studies have been conducted using various PSMs including bacteria, actinomycetes and fungi, as bioinoculants, for a range of agriculturally important crops. The major concern of all these efforts is to evolve efficient and competent strains that could adapt to the local conditions to exploit the full potential of this technology for crop production. This chapter highlights the potentials and success achieved following PSMs inoculation under both greenhouse and field applications along with phytobeneficial traits of such microbial biofertilizers. The results recorded when PSMs interact with themselves or with other beneficial soil microbes in the presence of cheap and locally available materials such as, rock phosphate [RP] is

∗

Correpondence to: E-mail: [email protected].

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13.1. INTRODUCTION Chemical fertilizers have played a significant role in the green revolution because their application is considered the quickest and assured way of increasing crop production. Nevertheless, its cost and environmental hazards discourage farmers from using them in in economically disadvantaged countries [Sagoe et al., 1998]. On the other hand, the progressive revitalization of interest for organically grown produce which is closely coupled to apprehension for environmental health and biodiversity [Wikström, 2003; van der Vossen, 2005; Arcand and Schneider, 2006] emphasize the need for better and environmentally friendly agricultural inputs. The pressing need for economically and ecologically acceptable fertilizer sources has prompted the search for new approach to farming [suitable agriculture]. Sustainable agriculture being a major strategy to counteract the rapid decline in environmental quality requires agricultural practices that are affable to the environment and that maintain the long term ecological balance of the ecosystems [Vessey, 2003; Khan et al., 2006]. In this context, use of microbial inputs [microbial biofertilizers] in agronomic practices epitomize an environmentally-pleasant alternative to reduce the dependence on synthetic fertilizers. Plant growth promoting rhizobacteria [PGPR] can affect plant growth by wide arrays of mechanisms such as, solubilization of inorganic P, synthesis of phytohormones, production of siderophores, lowering of plant ethylene levels, N2 fixation and others [Vessey, 2003]. The use of such beneficial bacteria as biofertilizers has increased interest worldwide, to attain sustainability in agriculture, forestry and horticulture [Lucy et al., 2004]. Phosphorus [P] is second only to nitrogen whose deficiency restricts the growth of plants across the globe particularly in highly weathered tropical soils [Vessey, 2003; Khan et al., 2006]. Most natural ecosystems in tropical and subtropical areas are predominantly acidic and often extremely phosphorus-deficient [Khan et al., 2006]. While most mineral nutrients in soil solution are present in millimolar amounts, phosphorus is only available in micromolar quantities or less [Goldstein, 1994]. The majority of applied phosphorus is rapidly fixed by forming compex with soil constituents and becomes poorly available to plants. Therefore, chemical P fertilizers are frequently used to alleviate P deficiency and consequently to attain higher yields. However, the excessive and repeated application of soluble P fertilizers may pose serious problem to groundwater besides economic constraint. Emphasis has therefore, been directed to the use of unavailable P forms by employing phosphate mobilizing microorganisms [PSMs]. Phosphorus biofertilizers can assist in increasing the availability of accumulated phosphates for plants by solubilization [Khan et al. 2007]. P supply through such biological means is, therefore, a realistic alternative to lower the risk of eutrophication and enhanced productivity of ecosystems. Evidence is increasing that phosphate-solubilizing bacteria, fungi and actinomycetes play a central role in conversion of insoluble P to bioavailable form [Pal, 1998]. The microbial biomass in soil also contains a significant quantity of immobilized P [organic P] that is unavailable to plants [Oberson et al., 2001] unless mineralized by microbes. Phosphate-solubilizing microbes have received substantial diligence as inoculants for crops because microbial availability of plants nutrients is non-

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controversial [Pandey et al., 2006; El-Azouni, 2008; Sarma et al., 2009]. The possible use of PSMs as inoculants to increase P-availability to plants and yield increment for various agricultural crops under both greenhouse and field trials has been studied [Abd-Alla and Omar, 2001; Reyes et al., 2002; Zaidi and Khan 2007; Wani et al 2007]. Therefore, solubilization of fixed or the insoluble P by PSMs assumes greater importance for augmenting crop productivity, especially in developing countries.

13.2. PROBLEMS OF PHOSPHORUS FIXATION

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Phosphorus is important for plant growth because it stimulates growth of young plants, promotes a vigorous start and hastens maturity. Alternatively, plant growth is diminished, maturity is delayed and yield is reduced when P is inadequately present in soil. For instance, in sub-Saharan Africa and in similar tropical soils, P deficiency is considered to be one of the main constraints to food production [Sale and Mokwunye 1993; Chien and Menon 1995]. P has the lowest mobility rate among the essential nutrients due to its adsorption and precipitation ability. Consequently, soil P concentration is generally low despite high total P content in soils [Miyasaka and Habte, 2001]. And hence, a greater part of soil P [approximately 95-99%] is present in the insoluble form and cannot be uptaken by the plants [Vassileva et al., 1998]. Theoretical estimates have suggested that the accumulated P in agricultural soils due to fixation is sufficient to sustain maximum crop yields world-wide for about 100 years [Goldstein et al., 1993]. However, although P is abundant in soils in both inorganic form and organic form, it is still one of the major plant growth-limiting nutrients. On average, most nutrients in the soil solution are present in millimolar amounts, but P is present only in micromolar or lesser quantities. These low levels of P are due to the high reactivity of soluble P with calcium [Ca], iron [Fe] or aluminium [Al], which leads to P precipitation [Figure1].

13.3. MICROORGANISMS RESPONSIBLE FOR PHOSPHATE MOBILIZATION: AN OVERVIEW The effects of microbial activities on the biogeochemical cycling of plant nutrients are essential for sustainable ecosystems [Oberson et al., 1993; Jeffries and Barea. 1994; Toro et al., 1997]. Microorganisms are central to the soil P cycle and play a significant role in mediating the transfer of P between different inorganic and organic soil P fractions, subsequently releasing available P for plant acquisition [McLaughlin, 1988]. Soil microorganisms are involved in [1] the transformation of different forms of P [2] mineralization of organic P and [3] solubilization and immobilization of inorganic P [McLaughlin, 1988]. A wide range of microorganisms from autotrophs to heterotrophs, diazotrophs to phototrophs and mycorrhizas are known to have the ability to solubilize inorganic P from insoluble sources. Phosphate solubilizing microorganisms include largely bacteria and fungi, which can grow in media containing tricalcium, iron and aluminium phosphate, hydroxyapatite, bonemeal, rock phosphate and similar insoluble phosphatic compounds as the sole P source.

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Figure 1. Phosphorus channels in soil. [Source: Bagyaraj et al., 2000; Muleta 2007].

Such microbes not only assimilate P but a large portion of soluble P is released in quantities in excess of their own requirement [Gaur, 1990]. The involvement of microorganisms in solubilization of inorganic phosphates dates back to 1903 [Kucey et al., 1989]. It is estimated that P solubilizing microorganisms may constitute 20 to 40% of the culturable population of soil microorganisms and that a significant proportion of these can be isolated from rhizosphere soil [Kucey, 1983; Chabot et al., 1993]. Most phosphate solubilizing microbes are isolated from the rhizosphere of various plants and are known to be metabolically more active than those isolated from sources other than rhizosphere [Vazquez et al., 2000]. For example, in natural coffee forests, over 72% of the rhizobacteria [both Gram-negative and Gram-positive] associated with wild Arabica coffee rhizospheres were shown to be able to solubilize mineral P [Muleta et al. unpubl. data]. The commonly reported genera include Pseudomonas, Bacillus, Rhizobium, Burkholderia, Achromobacter, Agrobacterium, Microccocus, Aereobacter, Flavobacterium, Erwinia, Vibrio, Xanthobacter, Enterobacter, Kluyvera, and Chryseomonas [Chabot et al., 1996; Vazquez et al. 2000; Alikhani et al., 2006]. Rodriguez and Fraga [1999] compared 13 bacterial strains of different genera for their solubilizing abilities on different insoluble mineral phosphate substrates and

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indicated that Pseudomonas, Bacillus and Rhizobium species were among the most powerful P solubilizers. Studies have confirmed that many actinomycete isolates exhibit a very high inherent potential in solubilizing rock phosphate [Mba, 1994; El-Tarabily et al., 2008; Hamdali et al., 2008]. In addition, cyanobacteria, viz. Anabaena sp., Calothrix brauni, Nostoc sp., Scytonema sp. and Tolypothrix ceylonica can solubilize mineral phosphates [Gupta et al., 1998]. Among the phosphate solubilizing fungi, Aspergillus niger, A. flavus, A. nidulans, A. awamori, A. carbonum, A. fumigatus, A. terreus and A. wentii have been reported from the rhizosphere of maize, soybean, chilli, tista soils, acidic lateritic soils and compost [Prerna et al., 1997; Omar, 1998; Seshadri et al., 2004]. Paeciliomyces fusisporus, Penicillium digitatum, P. simplicissimum, P. aurantiogriseum, Sclerotium rolfisii and species of Cephalosporium, Alternaria, Cylindrocladium, Fusarium and Rhizoctonia are other potential solubilizers of insoluble phosphate. Amongst yeasts, Torula thermophila, Saccharomyces cerevisiae and Rhodotorula minuta can solubilize inorganic phosphate [Varsha-Narsian et al., 1994; Asea et al., 1988]. These PSMs occur in both fertile and P-deficient soils and the fastest initial rates of P incorporation were observed in P-deficient soils [Oehl, 2001]. The majority of the PSMs mobilize Ca-P complexes and only a few can solubilize Fe-P and Al-P complexes [Kucey et al., 1989]. In general, P-solubilizing bacteria commonly outnumber P-solubilizing fungi [Kucey, 1983; Kucey et al., 1989]. Fungi, however, exhibit greater P solubilizing ability than do bacteria in both liquid and solid media [Nahas, 1996]. The P solubilizing ability in bacteria may be lost upon repeated sub-culturing but no such lossess has been observed in the case of P-solubilizing fungi [Kucey, 1983]. In addition, soil fungi can solubilize aluminium phosphate and has shown tolerance to Al+3 toxicity [Souchie et al., 2006].

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13.4. OTHER PHYTOBENEFICIAL TRAITS EXHIBITED BY PSMS Besides the phosphate solubilization, PSMs also exhibit other traits beneficial to plants [Figure 2]. Such traits include production of siderophores, hydrogen cyanide, phytohormones, plant growth promoting enzymes, antifungal metabolites, vitamins and 1-aminocyclopropane-1-carboxylate deaminase [Poonguzhali et al., 2006; Muleta et al. 2007, Muleta et al., 2009] and by increasing the efficiency of biological nitrogen fixation and enhancing the availability of other trace elements [Gyaneshwar et al., 2002]. It is reported that the formation of nodule is limited by the unavailability of P and consequently legumes show a high positive response to P supplementation [Gupta and Namdeo, 1997; Jain et al., 1999]. In addition, many PSMs promotes root colonization in association with mycorrhizal fungi by producing specific metabolites [Barea et al., 1997; Toro et al., 1997] and behave as mycorrhiza helper bacteria [Garbaye, 1994; Frey-Klett et al., 1997]. Indirectly, PSMs promote growth of plants by reducing pathogen infection [Antoun et al., 1998; Rosas et al., 2006]. For instance, inoculation of a phosphate solubilizing and efficient biocontrol strain of Pseudomonas putida [B0] resulted in statistically significant increment in roots and shoot biomass of maize [Pandey et al., 2006]. Furthermore, the inoculation stimulated the rhizosphere bacterial flora but suppressed the pathogenic fungal population in the rhizosphere. These results are in line with the findings reported by others [Pandey et al., 1998; Pandey et al., 1999].

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Figure 2. Mechanisms of plant growth promotion by PSMs. [Source: Khan et al., 2007; Muleta, 2007].

Likewise, inoculations of a local landrace of rice with phosphate-solubilizing bacteria Bacillus megaterium, B. subtilis and Pseudomonas corrugata under field based assays stimulated the rhizosphere associated bacterial and actinomycetal populations, improved mycorrhizal colonization and suppressed the fungal flora [Trivedi et al., 2007]. On the other hand, Wang et al. [2007] have investigated the effect of inoculation with phosphatesolubilizing fungal [PSF] isolates Aspergillus niger strain P39 and Penicillium oxalicum strain P66 on the bacterial communities in the rhizospheres of maize [Zea mays L. ‘Haiyu 6’] and soybean [Glycine max Merr. ‘Heinong 35’] using culture-dependent methods and polymerase chain reaction-denaturing gradient gel electrophoresis [PCR-DGGE]. The number of culturable microbes for soybean was notably greater with P39, whereas for maize, the same was significantly greater with P66 compared with the control. In addition, a greater number of microbes were found in the rhizosphere of maize compared with soybean. The fingerprint of DGGE for 16S rDNA indicated that inoculation with PSF also increased bacterial communities. The authors strongly emphasized that complex interactions between plant species and exotic PSMs affected the structure of the bacterial community in the rhizosphere. In addition, improved plant tolerance to water deficit stress has been reported due to co-

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inoculation of PSMs and AMF [Ehteshami et al., 2007]. Likewise, a novel stress tolerant phosphate solubilizing bacteria have been encountered in different agroecosystems of the world [Pal, 1998; Sharan et al., 2008]. Recently, Chang and Yang [2009] have also isolated thermo-tolerant phosphate-solubilizing microbes for multi-functional biofertilizer preparation that could elaborate a range of biotechnologically important enzymes such as amylase, CMCase, chitinase, pectinase, protease, lipase, and nitrogenase. These microbes after inoculation can shorten the period of maturity, improve the quality, increase the soluble phosphorus content, and enhance the populations of phosphate-solubilizing and proteolytic microbes in biofertilizers. The authors concluded that inoculating thermo-tolerant phosphatesolubilizing microbes into agricultural and animal wastes represents a practical strategy for preparing multi-functional biofertilizer. Vassilev et al [2006] have made a thorough review on simultaneous P-solubilizing and biocontrol activity of microorganisms with regard to their potentials and future trends.

13.5. INOCULATION OF PSMS AND PLANT GROWTH RESPONSES Several studies have reported a substantial increase in plant growth following single, dual or tripartite combinations of rhizospheric microorganisms in soils of low P availability [Toro et al. 1996; Zaidi et al., 2003; Zaidi and Khan 2007]. This effect, however, was not observed in soils with adequate P supply [Domey, 1996]. In the following sections, an attempt is made to address and discuss the role of P solubilizing microbes in the overall performance of plants grown in diverse agro-ecosystems.

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13.5.1. Greenhouse Application of PSMs: Potentials and Success Results from greenhouse trials have indicated improvement in growth responses of various crops to PSMs inoculations. Accordingly, El-Tarabily et al. [2008] studied the effect of an efficient phosphate-solubilizing non-streptomyces actinomycetes strain Micromonospora endolithica on bean plants [Phaseolus vulgaris L] in different phosphate sources under a greenhouse setup. The application of Micromonospora endolithica to soil amended with either single super-phosphate [SSP] or powdered RP significantly promoted the growth of plant organs [roots and shoots] compared with those observed for noninoculated soil amended solely with SSP or powdered RP. The inoculant also significantly increased the concentration of available P in the soil and other elements [e.g., N, P, K, S, Mg, Fe and Zn] in the roots and N, P, K, S, Mg and Fe in the shoots of test crop. Plant growth regulators [IAA, indole-pyruvic acid, gibberellic acid, isopentenyl adenine, isopentenyl adenoside or zeatin] tested did not stimulate the growth because neither Micromonospora endolithica nor M. olivasterospora produced detectable levels of these growth regulating substances under in vitro conditions. These results strongly highlighted the exceptional role played by Micromonospora endolithica in mobilizing insoluble P. In other trial, glasshouse experiments were conducted on maize [Zea mays L.] using two efficient phosphatesolubilizing bacterial strains Serratia marcescens EB 67 and Pseudomonas sp. CDB 35 [Hameeda et al., 2008]. Increase in plant biomass [dry weight] was 99% with EB 67 and 94%

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with CDB 35. Similarly, inoculation of a phosphate-solubilizing Pseudomonas putida [B0] resulted in statistically significant increment in roots and shoot biomass of maize after 42 days of growth [Pandey et al., 2006]. Sarma et al. [2009] also studied the effect of bioinoculation of phosphate-solubilizing fluorescent pseudomonad strains R62 and R81 on growth responses of Vigna mungo under pot conditions. It was found that the combined bioinoculation of these two organisms significantly increased the pod yields by 300% in comparison to the control crop. There was also substantial increment in the other plant growth responses such as dry root weight, dry shoot weight, shoot length and number of branches per plant. Experiments on phosphate-solubilizing fungi also present more encouraging results. For instance, Penicillium regulosum strains utilized RP and increasd dry matter yields of maize plants by 3.6 to 28.6% in a low fertility soil at pH 6.25 [Reyes et al., 2002]. The effect of six phosphate-solubilizing fungi [two strains of Aspergillus. awamori, and four of Penicillium citrinum] on growth and seed production of chickpea [Cicer arietinum L. cv. GPF2] plants in pot experiments was evaluated [Mittal et al., 2008]. Highest stimulatory effect was observed with two strains of A. awamori. This treatment resulted in 7-12% increase in shoot height, nearly three-fold increase in seed number and two-fold increase in seeds weight as compared to the control [un-inoculated] plants. Lesser stimulatory effect was recorded for the four strains of P. citrinum. These strains increased the shoot height by 7%, seed number by twofold and seeds weight by 87% as compared to the control plants. Lack of additive effect, however, was observed when all the six fungal isolates were inoculated. Several reasons have been suggested for such variations [cf. section 5.3]. Growth promotion of plants by phosphate-solubilizing microbes however, may not necessarily be associated with P mobilization suggesting the presence of other phytobeneficial traits that can augment the overall plant growth responses [Chabot et al., 1993; Jeon et al. 2003; see section 4]. Shin et al. [2007] studied the impact of the rhizosphere soil fungus Penicillium oxalicum CBPS-3F-Tsa on growth and N and P accumulation in maize plants in the presence of fused and RP under pot culture conditions. P. oxalicum CBPS-3F-Tsa was inoculated to maize plants alone or along with inorganic P in the form of fused phosphates [FP] and RP. Inoculation of P. oxalicum CBPS-3F-Tsa increased the plant growth, and N and P accumulation in plants compared to control plants and also had positive effects when applied with RP. The increase in dry weight and N and P accumulation in shoots and roots highlights the importance of adding P at seeding time and the influence of fungal isolate in playing a role in the release of P in soil. P. oxalicum CBPS-3F-Tsa actively solubilized inorganic RP and increased the P level in maize plants. But this effect was not observed when applied with FP which could most likely be due to the repression of P solubilization by microbes, when exposed to P rich environments. The results, however, clearly suggest that, under greenhouse conditions, it is possible to benefit plants from the P-solubilizing activity of P. oxalicum when RP is also used. In another study, Rudresh et al. [2005a] examined nine isolates of Trichoderma spp. for their ability to solubilize insoluble P using Pikovskaya broth and compared them with an efficient phosphate-solubilizing reference bacterial strain Bacillus megaterium subsp. phospaticum PB. All the nine Trichoderma strains solubilized insoluble TCP to various degrees. Pot cultures experiments with Trichoderma harzianum [PDBCTH 10], Trichoderma viride [TV 97], and Trichoderma virens [PDBCTVs 12] using chickpea 'Annegeri-1' as the test plant and RP as the P source significantly increased P uptake in plants treated with Trichoderma harzianum [PDBCTH 10] followed by Trichoderma virens [PDBCTVs 12] and

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Trichoderma viride [TV 97]. The fungal inoculation also increased growth and yield parameters compared with the uninoculated controls.

13.5.1.1. Plant Growth Responses to Dual/Multi-Strain Inoculation Growing bodies of evidence indicate that multiple inoculations with rhizospheric microorganisms can promote plant growth and grain yield and increase concentrations and uptake of N and P by crop plants better than single inoculation of PSMs [Wani et al., 2007; El-Azouni, 2008]. As an example, El-Azouni [2008] observed the in situ effect of Psolubilizing fungi [Aspergillus niger and Penicillium italicum] on the growth of soybean plants grown in soil amended with TCP. The pot study revealed that the co-cultures of A. niger and P. italicum significantly increased the height and dry weight of plant up to 81% and 105%, respectively, compared to the non-inoculated TCP soil or plants grown in soil treated only with super-P. A considerable increase in number of pods/plant and weight of seeds was also recorded with the application of single or dual inoculation of the tested strains. The increase in protein [57.5%] and oil [29.5%] content of soybean was mainly attributed to the beneficial effect of fungal inoculation. Moreover, an increase in N and P content of the plant was also recorded. Soil analysis showed that the available P and organic C levels were significantly increased while pH was lowered compared to the initial pH of the soil. The authors conclude that the amendment of soil with TCP along with P-solubilizing fungi could be considered as a sustainable way for increasing biological and chemical properties of plants besides improving the physico-chemical properties of the soil.

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13.5.1.2. Interactions of PSMs with Other Phytobeneficial Microbes 13.5.1.2.1. Interactions of PSMs with N2-Fixing Bacteria Biological nitrogen fixation depends appreciably on the available form of phosphorus. So the combined inoculation of nitrogen fixers and phosphate solubilizing microbes may benefit the plant better than either group of organisms alone. Stimulatory effect of combined inoculation of Rhizobium and phosphate-solubilizing bacteria with simultaneous application of RP has led to a of saving of 10 kg P fertiilizer suggesting that superphosphate could be replaced entirely by RP and PSMs inoculation [Sharma, 2006]. Composite inoculation of Rhizobium and PSMs has also shown a considerable increase in biomass, symbiotic properties, grain yield, and nutrient status of legumes [Gupta and Namdeo, 1997; Jain et al., 1999]. The composite inoculation of A. chroococcum, P. striata and A. awamorii significantly increased yield and nutrient uptake of rice [Oryza sativa], grown under greenhouse conditions [Kundu and Gaur, 1984]. The ability of phosphatesolubilizing bacteria to promote growth in greengram crop was investigated under greenhouse conditions [Vikram and Hamzehzarghani, 2008]. Greengram plants inoculated with Serratia sp. [PSBV-14] displayed the highest nodule number and its dry mass and total dry matter accumulation in plants 45 days after sowing. Similarly, treatment receiving the inoculation of another Serratia sp. [PSBV-13] showed the highest root length, root dry matter, P content and P uptake in root and shoot in greengram plants.

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13.5.1.2.2. Interactions of PSMS with Arbuscular Mycorrhizal Fungi In a study by Kim et al. [1998], a pot trial was set up to investigate the effect of phosphate-solubilizing bacterium and arbuscular mycorrhizal fungi [AMF] on tomato growth and soil microbial activity. A control pot along with Enterobacter agglomerans [EA], Glomus etunicatum [GE] and a combination of EA and GE were included. The composite inoculation of EA and GE showed a significantly greater population of PSB compared to sole application of EA. It further enhanced soil microbial biomass C compared to other treatments. Moreover, significantly increased phosphatase activities were observed in the rhizospheres of inoculated tomato plants compared to control plants that may have been contributed by AMF hyphae [Ezawa and Yoshida, 1994]. The Fresh and dry matter accumulation in plant organs [shoots and roots] were substantially higher in inoculated plants compared to the control. The greatest increase in plant weight occurred in the EA with GE treatment. Total N and P uptake by tomato plants was considerably greater in EA, GE, and when EA was inoculated with GE relative to control. The overall improvement in growth of the bio-primed plants could be attributed to compatibility and the synergistic effect of the microbial co-cultures. However, no significant difference in soluble P concentration in the rhizosphere with respect to times among treatments was observed which could probably be linked to uptake by plants and leaching of P. Singh and Kapoor [1999] tested the effect of phosphate-solubilizing Bacillus circulans and Cladosporium herbarum and the AM-fungus Glomus sp. with or without Mussoorie rock phosphate [MRP] on wheat grown in nutrient-deficient natural sandy soil under greenhouse conditions. Maximum root colonization and increase in grain and straw yields was observed following the inoculation of B. circulans, C. herbarum and Glomus sp. with MRP amendment while the lowest reponse was observed with B. circulans without MRP. The uptake of both P and N improved significantly when soil was amended with MRP. Inoculation of both PSMs and AM-fungus further augmented P and N uptake when soil was treated with MRP as compared to single inoculation of PSMs or AM-fungus. Eventhough the population density of PSMs increased substantially in the presence of Glomus sp., the addition of MRP further stimulated the growth of PSMs in the wheat rhizosphere. Such increment in bacterial populations may have been due to high metabolic activities of PSMs for a longer period in the rhizosphere of these plants due to inoculation with the AM-fungus [Jones and Sreenivasa 1993; Singh and Singh 1993]. In other study, Souchie et al. [2006] investigated the effect of synergism between P-solubilizing fungus and AM-fungi on clover [Trifolium pratense L.] under a controlled environmental chamber in the presence of aluminum phosphate as P source. The results show that shoot dry matter of the experimental plant was significantly higher when clover plants were co-challenged with P-solubilizing fungus [Aspergillus sp., PSF7] and two mixed species of AM-fungi [Glomus clarum and Glomus geosporum]. The synergism also increased shoot N and P quantity by 110 and 225%, respectively. From these findings, it can be concluded that PSMs and AM-fungus together can be developed as a composite inoculant which when applied with MRP is likely not only to improve crop yields in nutrient-deficient soils but may also help to reduce the chemical inputs. Increased uptake of N and P as well as substantial plant growth stimulation due to dual inoculation of AM-fungus and PSMs has been demonstrated [Piccini and Azcon 1987; Toro et al., 1997; Kim et al., 1998]. At present, the use of PSMs is not restricted only to agronomic practices but has expanded further to the forestry that is higly important for alleviating desertification. The effect of PSMs and mycorrhizal fungi was studied under greenhouse environment [Scotti et

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al., 2007]. The results showed that the number of PSMs and mycorrhizal fungi spores was significantly higher in the inoculated Eucalyptus rhizosphere, when compared to the native species and also to the noninoculated Eucalyptus plants. The composite application of PSMs and AMF increased the height and diameter, dry matter production and phosphorus content of plant. The authors suggested that dual inoculation of PSMs and mycorrhizal fungi could be used to facilitate the growth and nutrient status of Eucalyptus plants cultivated in semiarid land.

13.5.1.3. Plant Responses to Combination of PSMs, N2-Fixing Bacteria and AM-Fungi The stimulatory effect of phytobeneficial microbes on performance of chickpea was investigated in a pot experiment using sterilized sandy clay loam soil, deficient in available P [Zaidi and Khan, 2007]. Generally, plant growth was improved following inoculation with Mesorhizobium ciceri and P-solubilizing bacterium [Serratia sp.] or P-solubilizing fungus [Penicillium sp.]. Composite application of M. ciceri with Serratia and AM-fungus [Glomus fasciculatum] had maximum positive effect on biological and chemical properties of chickpea plants. Likewise, the same microbial consortium significantly enhanced the symbiotic properties, density of PSMs, root infection by AM- fungus and spore density of the AMfungus. Such improvement in overall growth of chickpea plants was due to the additive effects of these organisms, which might have supplied a more balanced nutrition [N by Mesorhizobium and P by phosphate- solubilizer] to the plants or improved nutrient absorption. Interestingly, besides, P solubilization, many P solubilizers can upregulate mycorrhizal functioning by synthesizing specific metabolites such as, vitamins, amino acids and hormones [Barea et al., 1997]. Such bacteria genereally referred to as mycorrhizal helper bacteria [Duponnois and Plenchette, 2003], could assist the colonization and establishment of AM-fungus in the plant rhizosphere. Synergistic interactions between P-solubilizing bacteria and AM- fungus and their consequent effect on plant growth have been reported by numerous workers [Duponnois and Plenchette, 2003; Zarei et al., 2006]. The soluble P released from soil constituents by the activity of PSMs is actively taken up by mycorrhizal plants [Kucey et al., 1989] because legumes are generally more mycotrophic than other plants [Plenchette et al., 2005]. Arbuscular mycorrhizal fungi do not increase P uptake by dissolving complex soil phosphates [Hayman and Mosse, 1972]. The enhanced uptake of P in plants with colonized roots, however, could be due to an increase in the number of uptake sites per unit area of roots and a greater ability for these roots to exploit the soil for nutrients [Kim et al., 1998]. This has been mainly ascribed to the fact that the AM- fungal mycelium, which extends out from the roots- [i] absorbs soluble P in large volumes of soil and transfers it to the plant roots [Smith and Read, 1997] and [ii] mobilizes P directly from organic matter through the excretion of organic acids [Landeweert et al., 2001]. Moreover, combined inoculation with AM- fungi and PSMs has been shown to result in better uptake of native soil P as well as P from RP [Kucey, 1987] leading to a substantial improvement in plant nutrients. The symbiosis between AMfungi and PGPR induce not only physiological changes in the inoculated plant, but also modify the architecture of the roots [Atkinson et al., 1994]. However, the total N yield in aerial organ [shoots] of plants could arise from an increase in translocation of soil N to the plants mediated by AM- fungus [Read and Perez-Moreno, 2003]. The responses of lentil [Lens culinaris cv. ‘Ziba’] to co-inoculation with AM-fungi and some indigenous rhizobial strains varying in P-solubilizing ability in a calcareous soil with high pH and low amounts of available P and N were investigated under controlled greenhouse

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conditions [Zarei et al., 2006]. The treatments consisted of [i] three inoculants of Rhizobium leguminosarum bv. viciae strains and a mixed rhizobial inoculant with an effective Psolubilizer strain of M. ciceri, [ii] two AM-fungal species Glomus mosseae and Glomus intraradices and [iii] two P sources, superphosphate and phosphate rock. The effects of AMfungi, rhizobial strains and P fertilizers were highly significant for all the measured parameters. The rhizobial strain with P-solubilizing ability demonstrated a more pronounced effect on growth and nutrient uptake than the strain without this ability, although both strains had similar effectiveness for N2-fixation. From these results the authors concluded that synergistic relationships were observed between AM fungi and some rhizobial strains that related to the compatible pairing of these two microsymbionts. The P-uptake efficiency was also increased when P fertilizers was applied along with AM-fungi and/or P-solubilizer rhizobial strains. It has also been reported that AM-fungi improve the P uptake by the plants, which in turn enhance nodulation and N2 -fixation [Abdel-Fattah, 1997].

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13.5.2. Field Application of PSMs: Potentials and Success Field trials on various types of agriculturally important crops have shown that application of PSMs considerably increased yields [Toro et al., 1997]. Accordingly, field experiments were conducted using two efficient strains Serratia marcescens EB 67 and Pseudomonas sp. CDB 35 [Hameeda et al., 2008]. Increase in plant biomass at 48 and 96 days after sowing was 66% and 50% with EB 67 and 51% and 18% with CDB 35 under field conditions. Seed treatment with EB 67 and CDB 35 increased the grain yield of field-grown maize by 85% and 64% compared to the uninoculated control. Trivedi et al. [2007] have also tested the phosphate solubilizing bacteria Bacillus megaterium, B. subtilis and Pseudomonas corrugata for their ability to increase the growth and P nutrition of a local landrace of rice under field based assays. Out of the three treatments, B. subtilis gave the best performance resulting in 1.55 fold increase in grain yield of rice. Inoculations also stimulated the rhizosphere associated bacterial and actinomycetes populations and suppressed the fungal flora. Colonization of roots by mycorrhizal fungi improved in all the treatments. Bacterial treatments also resulted in higher values for phosphorus in shoots and grains in inoculated rice plants. Correspondingly, Kumar et al. [2007] investigated the effect of bacterial inoculations [Bacillus megaterium, Bacillus subtilis and Pseudomonas corrugata] on growth and yield related parameters of maize in field experiments conducted for three consecutive years under rainfed conditions of Himalayan region. Depending on the first year results, P. corrugata was chosen for inoculation in the subsequent experiments. The bacterial inoculations by B. megaterium, B. subtilis and P. corrugata resulted in an increment in grain yield of maize up to 122.4%, 135.2% and 194.3%, respectively, as compared to respective control. In 2nd and 3rd year experiments, P. corrugata increased the grain yield up to 147.28% and 149.93%, respectively, as compared to control. The best performance and consistent trend of P. corrugata to increase plant yields was credited to its initial isolation from rhizosphere of maize growing under temperate conditions. In general, the beneficial effects of bacterial inoculations on maize were due to [1] the colonization and survival of the introduced bacteria, and [2] stimulation of the indigenous microflora in the rhizosphere. From the overall enhanced measured growth parameters, the authors strongly suggested Pseudomonas

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corrugate as an efficient bacterial inoculant for maize grown under rainfed conditions of Himalayan region. In a field experiment, Wasule et al. [2007] determined the effect of phosphate solubilizing bacteria on role of Rhizobium on nodulation, nodule dry weight, dry matter of plant, 1000 seed weight and yield with eight treatments namely, Rhizobium + PSB, Rhizobium, PSB, full fertilizer dose, half fertilizer dose, full fertilizer dose + Rhizobium + PSB, half fertilizer dose + Rhizobium + PSB and control. The results show that Rhizobium + PSB yielded maximum number of nodules [67.13] and nodule dry weight [107.73 mg]. Rhizobium alone showed maximum production of dry matter [3.63 gm]. Full fertilizer dose + Rhizobium + PSB gave the highest 1000 seed weight [109.92 gm]. Half fertilizer dose + Rhizobium + PSB gave maximum yield [10.67 q/ha] which was equivalent to yield recorded with Full fertilizer dose + Rhizobium + PSB [10.66 q/ha] and Rhizobium + PSB [10.63 q/ha]. From the observed results, it can be suggested that bioagents can preferably replace chemical fertilizers. This work is in agreement with that of Son et al. [2006] who studied the effect of bradyrhizobia [Bradyrhizobium japonicum] and phosphate solubilizing bacteria [Pseudomonas spp.] on soybean in rotational system [soybean-rice-rice]. The treatments composed of different combination level of inorganic nitrogen fertilizer levels [namely 20, 40, 60 kg N/ha] and biofertilizer [Bradyrhizobium japonicum+ Pseudomonas spp.] as compared to conventional farmers’ fertilizer level [80 N – 60 P2O5 – 30 K2O kg/ha]. The results show that application of Bradyrhizobium japonicum and Pseudomonas spp. enhanced the number of nodules, dry weight of nodules, yield components, grain yield, soil nutrient availability and uptake of soybean crop. Moreover, the economic efficiency could be increased in term of reducing the production cost for soybean from 785,000 to 1,000,000 VND/ha. Likewise, Babana and coworker [2006] have also reported promising results on wheat performance in a field trial by co-challenging of AMF and an efficient indigenous P mobilizer and known AMF helper, Pseudomonas species, in the presence of naturally occurring RP as main P source. In 45-day-old wheat plants, the highest root length AM colonization [62%] was observed with Tilemsi phosphate rock fertilized wheat inoculated with Glomus intraradices and Pseudomonas sp. Inoculation of wheat Tetra fertilized with Tilemsi phosphate rock with a combination of a commercial isolate of the arbuscular mycorrhizal [AM] fungus Glomus intraradices, Pseudomonas sp., and Aspergillus awamori produced the best grain yield with the highest P concentration. In another study, the response of neem [Azadirachta indica A. Juss] to indigenous arbuscular mycorrhizal fungi [G. intraradices Schenck and Smith and G. geosporum [Nicol, and Gerd.] Walker], asymbiotic nitrogen-fixing bacteria [Azospirillum brasilense] and PSB individually or in various combinations in unsterile soil under nursery conditions was evaluated [Muthukumar et al., 2001]. Microbial inoculation resulted in increased mycorrhizal colonization, greater plant height, leaf area and number, root collar diameter, biomass, phosphorus, nitrogen and potassium content, and seedling quality. Microbial inoculation effects were greatest when seedlings were inoculated with a combination of microbes rather than individually. This clearly indicates that the tested bioagents act synergistically when inoculated simultaneously, with maximum response being when both AM-fungi were coinoculated with A. brasilense and PSB. The results emphasize the importance of microbial inoculations for the production of robust, rapidly growing seedlings in nurseries and illustrate the advantage of inoculating soils of a low microbial population with indigenous microbes. Very recently, Sarma et al. [2009] studied the effect of bio-inoculation of fluorescent pseudomonad strains R62 and R81

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[a potent plant growth promoting and biocontrol agent] on growth responses of Vigna mungo under field conditions. The findings reveal that the combined bioinoculation of these two organisms significantly increased the pods yield by 300% in comparison to the control crop. There was also considerable increment in the other plant growth responses such as dry root weight, dry shoot weight, shoot length and number of branches per plant [Figure 3]. Pal [1998] tested interactions of an acid tolerant strain of phosphate solubilizing bacteria with a few acid tolerant crops. The highest response was observed with frenchbean [Phaseolus vulgaris] followed by fingermillet [Elosine coracana], buckwheat [Fagopyrium esculentum], amaranth [Amaranthus hypochondriacus] and maize [Zea mays]. Available phosphate was also highest in frenchbean cultivated plot indicating better growth responses by legume plants to inoculated PSMs. Similarily, field experiments were carried out at the Indian Agricultural Research Institute, New Delhi during 1996–97 to 1998–99 to study the effect of PSB and incorporation of wheat and rice residues on the relative efficiency of diammonium phosphate [DAP] and Mussoorie rock phosphate [MRP] in three cycles of rice–wheat cropping system [Sharma and Prasad, 2003]. Application of MRP did not show significant effect on grain and straw yield and P uptake by both crops. When MRP was, however, inoculated with PSB, it increased grain and straw yields as well as P uptake of both rice and wheat. Efficiency of MRP+PSB was further improved when rice and wheat residues were incorporated and this practice made MRP on a same level with DAP. Available P in soil after three cycles of rice– wheat cropping was more after MRP+PSB and incorporation of rice and wheat residues than after DAP. The results of this work have a strong positive agronomic implication of low grade rock phosphate such as MRP in the presence of phosphate solubilizing microbes and cheap organic residues for ecofriendly sustainable agriculture.

Figure 3. The filed experimentation results of Vigna mungo. [Source: Sarma et al., 2009].

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A study was conducted in order to evaluate the effects of two N2-fixing [OSU-140 and OSU-142] and a strain of P-solubilizing bacteria [M-13] in single, dual and three strains combinations on sugar beet and barley yields under field conditions in 2001 and 2002 [Sahin et al., 2004]. All inoculations and fertilizer applications significantly increased leaf, root and sugar yield of sugar beet and grain and biomass yields of barley over the control. Single inoculations with N2-fixing bacteria increased sugar beet root and barley yields by 5.6–11.0% depending on the species while P-solubilizing bacteria alone gave yield increases by 5.5– 7.5% compared to control. Dual inoculation and mixture of three bacteria gave increases by 7.7–12.7% over control as compared with 20.7–25.9% yield increases by NP application. Mixture of all three strains, dual inoculation of N2-fixing OSU-142 and P-solubilizing M-13, and/or dual inoculation of N2-fixing bacteria significantly increased root and sugar yields of sugar beet, compared with single inoculations with OSU-140 or M-13. Dual inoculation of N2-fixing Bacillus OSU-140 and OSU-142, and/or mixed inoculations with three bacteria significantly increased grain yield of barley compared with single inoculations of OSU-142 and M-13. In contrast with other combinations, dual inoculation of N2-fixing OSU-140 and Psolubilizing M-13 did not always significantly increase leaf, root and sugar yield of sugar beet, grain and biomass yield of barley compared to single applications both with N2-fixing bacteria. The beneficial effects of the bacteria on plant growth varied significantly depending on environmental conditions, bacterial strains, and plant and soil conditions. Soybean benefited from the co-inoculation of B. japonicum and the P-solubilizing bacterium Pseudomonas striata; dry weight of nodules, dry matter of plant, and yield were increased significantly compared to an uninoculated control in a neutral pH Indian soil with 22 to 40 kg available P ha-1 [Wasule et al., 2003]. In another field trial, Ehteshami et al. [2007] studied the effect of phosphate solubilizing microorganisms and AMF on growth responses of maize under water deficit stress. Co-inoculation of Pseudomonas fluorescens and Glomus intraradices significantly increased grain yield, yield components, harvest index, grain N and P, soil available P and root colonization percentage under water deficit stress. On the other hand, the effect of a combined inoculation of Rhizobium, Bacillus megaterium sub sp. phosphaticum strain-PB and a biocontrol fungus Trichoderma spp. on growth, nutrient uptake and yield of chickpea were studied under field conditions. Combined inoculation of these three organisms showed increased germination, nutrient uptake, plant height, number of branches, nodulation, pea yield, and total biomass of chickpea compared to either individual inoculations or an uninoculated control [Rudresh et. al, 2005b]. Similarily, the synergistic effects of multi-strain inoculants [i.e., N2-fixing and phosphate solubilizing rhizobacteria] on chickpea plant [Cicer arietinum L.] growth, yield, grain protein, and nutrient uptake were assessed in a sandy clay-loam soil. The inoculants included Mesorhizobium ciceri RC4, A. chroococuum A10, Pseudomonas PSB 5 and Bacillus PSB9. Legume grain yield and concentration and uptake of nitrogen [N] and phosphorus [P] were significantly increased as a result of co-inoculation with Mesorhizobium and P-solubilizing Pseudomonas and Bacillus spp. The inoculation with Mesorhizobium ciceri RC4 + Azotobacter chroococuum A10 + Bacillus PSB9 tripled the seed yield and resulted in highest grain protein [295 mg g–1] at 145 d after sowing [DAS]. An 8% increase in P concentration above the uninoculated control was observed in case of a single inoculation with Pseudomonas PSB 5, while the P uptake was highest [2.14-fold above the uninoculated control] with a combined inoculation with [M. ciceri RC4 + A. chroococcum A10 + Bacillus PSB 9] at 145 DAS. The highest N concentration and N uptake at 145 DAS [81% and 16% above the uninoculated control,

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respectively] were observed with the triple inoculation of [M. ciceri RC4 + A. chroococcum A10 + Pseudomonas PSB 5]. The authors strongly remarked the rewarding advantages associated with multiple inoculations with rhizospheric microorganisms in the augmentation of overall plant growth promotion [Wani et al., 2007].

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13.5.3. Reasons for Lack of Synergistic Effect/Failure of PSMs Sometimes it is not uncommon to observe lack of additive effect among consortia of inoculated phosphate solubilizing microorganisms [Kohler et al., 2007; Mittal et al., 2008]. For instance, Souchie et al. [2006] observed a negative effect on some of the measured growth parameters when A. awamori was either used alone or added to the consortium. Similarly, the application of Mesorhizobium and AM fungus together with Penicillium showed a comparatively poor response on overall growth of chickpea compared to M. ciceri with Serratia and G. fasciculatum or dual inoculation treatments of M. ciceri and Serratia or M. ciceri and G. fasciculatum [Zaidi and Khan, 2007]. The reduction in legume growth was possibly due to the decline in rhizobial populations. The decrease in rhizobial population could be due to the release of higher amounts of organic acids [Venkateswarlu et al., 1984] since most rhizobia are sensitive to pH change. In turn, this may have altered the microenvironment, affecting the survivability of rhizobia and the legume–Rhizobium symbiosis [Downey and Kessel 1990]. Consequently, the availability of N to the plants might have been reduced so the growth and yields of the plants were decreased. Similar evidence of negative interaction between Aspergillus awamori and G. fasciculatum [Sattar 1982] and the effect of negatively interacting microorganisms on chickpea [Zaidi et al. 2003], greengram [Zaidi et al. 2004] and Lactuca sativa [Kohler et al., 2007] was previously reported. Other suggested reasons for the lack of additive or synergistic effects include [i] presence of competition for root exudates among rhizotrophic organisms [Kohler et al., 2007] [ii] inadequate solubilization of P together with poor availability of P to the growing plants or [iii] a low content of organic substrates in soil for the production of organic acids by the phosphate mobilizing microbes [Dwivedi et al., 2004]. Despite the beneficial influences by the PSMs, some cases of inconsistent results have been reported. For example, Bacillus megaterium var. phosphaticum performed inconsistently in soils as inoculant in India, former Soviet Union and the United States [Rodriguez and Fraga 1999] while P. bilaiae, active agent in JumpStart ® have also shown variable field results from positive to no response or even a negative response [Jakobsen et al., 2005]. This problem is further worsened especially when the grower has little time or knowledge to deal with bacterial inoculation [Okon and Labandera-Gonzalez 1994]. This is discouraging for both the growers and the commercial industry. Several explanations have been suggested for inconsistent results associated with phosphate solubilizing microorganisms. Firstly, the variability in field performances by the PSMs is due to the laboratory screening process in which the parameters employed for the isolates do not reflect soil conditions [Nautiyal et al., 1999; Leggett et al., 2001]. Laboratory screening for PSMs with selected media is conducted under controlled environmental conditions whereas conditions in soil are uncertain. The performance of microbes is strongly influenced by soil conditions such as pH [buffering] and temperature. Secondly, coating of seeds/direct application in soils with sufficiently lower rates of inoculants; the effectiveness of a microorganism depends on the initial inoculum

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density [Jjemba and Alexander, 1999]. In general, the population size of the introduced microorganism declines rapidly upon inoculation in soils [Ho and Ko, 1984] and thirdly, instability of P solubilizing character even under the laboratory conditions and the problem is more serious in bacteria than fungi [Kucey, 1983].

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13.5.4. Agronomic Effectiveness of Rock Phosphate in Presence of PSMs Natural phosphate rocks have been recognized as a valuable alternative source for P fertilizer, especially for acid soils. Rock phosphate [RP] is widely distributed throughout the world, both geographically and geologically [Zapata and Roy, 2004]. Compared with chemical treatment, microbial solubilization of RP is an alternative environmentally mild approach. The use of RP as a P fertilizer and its solubilization by microbes [Kang et al., 2002], through the production of organic acids [Maliha et al., 2004], have become a valid alternative to chemical fertilizers. In conjugation with P- solubilizing microorganisms, RP provides a cheap source of P fertilizer for crop production. For instance, Penicillium bilaii, and Aspergillus niger a significant citric acid producer have been shown to be effective RP solubilizers [Cunningham and Kuiack 1992, Kucey, 1988; Abd-Alla and Omar 2001; Omar 1998]. Numerous studies have indicated the vanity of finely ground RP use due to the low solubility of its P contents necessitating for appropriate dissolving agents with pretreatments of organic wastes [Vassilev et al. 1996; Goenadi, et al., 2000]. Consequently, a number of studies have recommended utilization of high carbon crop wastes for use as fermentation substrates in the microbial solubilization of RP before application to the field [Vassilev et al. 1995, Vassilev et al. 1996]. This method has the advantage of creating more optimal conditions for microbial organic acid production compared with conditions present in soil [Arcand and Schneider, 2006]. For instance, the pretreatment of Moroccan RP with the A. niger inoculated sugar beet [SB] [Beta vulgaris] waste significantly improved clover [Trifolium repens] dry weight and shoot P content compared to plants supplied with the same amount of untreated RP and SB waste [Vassilev et al. ,1996]. Vassileva et al. [1998] investigated various combinations of olive cake and RP, previously treated or untreated by the fungus, and introduced them into a calcareous, P-deficient soil to improve the growth of T. repens in a greenhouse experiment. It was observed that synergistic action of both the A. niger and G. deserticola caused considerable improvement in growth and plant P uptake. Greater growth and P uptake of mycorrhizal and non-mycorrhizal plants were achieved when microbe-treated olive cake and RP were applied to soil compared with all other treatments. Similarily, in a field experiment, Sharma and Prasad [2003] have demonstrated the agronomic effectiveness of low grade RP such as Mussoorie rock when applied with PSB inoculation and incorporation of rice and wheat residues. An application of efficient RP dissolving actinomycete in poultry manure-treated field plots resulted in yield increases of 43% and 17% with soybeans and 19% and 33% with egusi, respectively [Mba, 1994]. The soil available P increased at the five-leaf stage with overall improvement in other soil properties. The effect of P-solubilizing microorganisms like, A. niger, P. vermiculosum, Bacillus sp. and Pseudomonas stutzeri on yield and nutrient uptake of soybean were studied under a glass house conditions in the presence of different P sources [Sandeep et al.,2008]. The maximum straw yield [39.51 g], seed yield [18.18 g/pot] and pods yield [32.29 g/pot] were obtained with microbial inoculation of P.vermiculosum along with RP treatments. Inoculations along

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with RP application further increased the yield and were comparable with the treatments receiving super phosphate [SP] alone. The highest number and dry weight of nodules were obtained in the treatments of P. vermiculosum with RP. The authors strongly claimed the possibility of replacing the super phosphate fertilizer with eco-friendly P source (RP). Babana and coworker [2006] have also reported the agronomic suitability of naturally occurring RP on wheat crop plants in the presence of AM-fungi and an efficient P-solubiling bacterium Pseudomonas species. The RP did not reduce the level of root infection with AM-fungi compared to the addition of soluble chemical fertilizers. Similarly, a significant increase in the dry matter yield of wheat plants with co-inoculation of RP solubilizing fungi A. niger and P. citrinum, and G.constrictum has been demonstrated [Omar, 1998] suggesting that PSMs especially in combination with AM-fungi could be very effective in increasing the agronomic efficacy of RP [Barea et al., 2002].

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13.5.5. Rhizosphere Competence of Phosphate Solubilizing Microbes In a pot experiment, Sandeep et al. [2008] evaluated available P content and count of Psolubilizing microbes. The highest accumulation of available P content in the rhizospheres of inoculated plant indicated that introduced microorganisms got themselves well established and proliferated. The maximum count [47.33x104 cells/g] was observed for the combination of Bacillus sp. and RP. Furthermore, colonization and survival of the inoculated bacteria (B. megaterium, B. subtilis and Pseudomonas corrugata) in rhizosphere of maize grown under rainfed conditions of Himalayan region showed good competence giving high inoculum numbers [Kumar et al., 2007]. In a study by El-Tarabily et al. [2008], an efficient Psolubilizing bacterium Micromonospora endolithica showed an exceptional rhizosphere competence and a strong ability to colonize bean roots up to a depth of 14 cm by causing a significant growth promotion in the inoculated plants. In comparison, non-phosphatesolubilizing, non-rhizosphere-competent, non-streptomycete actinomycetes isolate of M. olivasterospora failed to increase available soil P, nutrient levels in roots and shoots or to promote plant growth emphasizing the remarkable rhizosphere competence of P-solubilizing microorganisms. A high proportion of P-solubilizing microbes is concentrated in the rhizosphere and such bacteria are more metabolically active than those isolated from other sources [Vazquez et al., 2000], which is of great relevance to plants, particularly in Pdeficient soils. Singh and Kapoor [1999] have demonstrated that a larger population of PSMs was recorded in the presence of AM-fungi, and addition of MRP further stimulated the growth of the population of PSMs in the wheat rhizosphere. This may have been due to high metabolic activities of PSMs for a longer period in the rhizosphere of these plants due to inoculation with the AM-fungus [Singh and Singh 1993; Jones and Sreenivasa 1993]. Khan et al. [2007] investigated the effect of certain phosphate-solubilizing bacteria [Bacillus subtilis, B. polymyxa, Pseudomonas fluorescens and P. stutzeri] on root-knot nematode disease of mungbean and evaluated the rhizosphere populations of the inoculated microbes in course of time. It was found that rhizosphere population of all tested PSMs was significantly increased over time irrespective of nematode presence or absence. Evidently, a P-solubilizer and a streptomycin-resistant marker (MP6strep+) of Mesorhizobium loti has been verified to efficiently colonize roots of mustard seedlings [Chandra et al., 2007]. Several research results strongly recommend the suitability of indigenous phytobeneficial microbial strains when

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producing and using bio-fertilizers due in part to their better competence and persistence in the rhizosphere than introduced ones [Requena et al., 1997; Kızılkaya, 2008]. Phytobeneficial microbes show considerable viariations to fungicides application on seeds. Accordingly, survival of M. ciceri [SP4] and A. chroococcum [CBD-15 and M4] was tested on chickpea seeds treated with fungicides bavistin [methyl N-[1H-benzimidazol-2yl] carbamate] and thiram [tetramethyl-thiuram disulfide], whereas survival of PSB, Pseudomonas striata [27] and B. polymyxa [H5] was examined on two cultivars [Arkel and BV] of pea [Pisum sativum] seeds treated with thiram [Gaind et al., 2007]. Viability of A. chroococcum [W5] was also examined on wheat seeds treated with bavistin, captan [cis-Ntrichloromethyl thio-4 cyclohexane-1, 2-dicarboximide] and thiram under laboratory conditions using standard dilution and the plate count technique. The viable population of all the tested strains of diazotrophs and PSB declined on prolonged contact with fungicides. However, the viability of PSB varied even with the cultivars. Cultivar of pea (BV) showed better recovery of P. striata in the presence of thiram, whereas the Arkel cultivar of pea resulted in better recovery of viable B. polymyxa. Azotobacter chroococcum [W5] showed better survival in the presence of bavistin, compared to thiram and captan. Higher viable population of M. ciceri [SP4] and A. chroococcum [M4] was recovered from chickpea seeds treated with bavistin compared to thiram. However, thiram-treated seeds resulted in a greater number of A. chroococcum [CBD-15]. Under field conditions, adverse effect of thiram was reflected on the performance of M. ciceri [SP4] and A. chroococcum [M4] strains, resulting in reduced root and shoot biomass and grain yield, compared to bavistin treated and culture inoculated treatment. CBD-15 showed better performance in the presence of thiram compared to bavistin.

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CONCLUSION Phosphorus is one of the important macronutrients and plays an imperative role in metabolism of crop plants. Most soils around the world are P deficient and pose sever problems in crop productivity. To alleviate such a problem, agrochemicals are highly helpful in maximizing productivity and economic returns. The cost and environmental hazards associated with the use of agrochemicals, however, are the major drawbacks accompanied with this kind of production system. Thus, there is a strong need to find alternative methods that may increase productivity of crops under the principle of low cost sustainable agriculture. The unconventional crop management systems that will reduce off-farm inputs are being developed to offset rising production costs, decrease environmental and health hazards associated with the use of such agricultural chemicals and maintain soil productivity levels. One of the most important means to achieve the goals of sustainable agriculture is to use phosphate-solubilizing microbes because microbial availability of plant nutrients is noncontroversial. The use of efficient PSMs may be agronomically important since they could lessen the problem of P deficiency in many soils with deposits of insoluble P sources. Application of phosphate-solubilizing microbes as inoculants simultaneously increases P uptake by the plant and crop yield. These microbial biofertilizers are gaining increased attention mainly due to [1] the environment friendly nature of bioinoculants [2] it can prevent the long term hazards associated with the continued use of chemical fertilizers and [3] its

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increased acceptability as natural “organic” products at global level. There is accumulated evidence that multi-strain inoculants are in a better position to compete with indigenous microorganisms and enhance plant growth. Accordingly, various trials corroborated the positive interactions between PSMs, N2-fixing bacteria and AM-fungi and establish their role in enriching soil P pool. Thus, it has been suggested that combination of these phytobeneficial microbes with simultaneous application of rock phosphate may broaden the spectrum of phosphate solubilizers with a possibility of saving substantial amounts of N and replacing entire superphosphate in organic agriculture. Insoluble phosphate solubilization per se does not account for all of the plant growth-promoting effect but PSMs besides P solubilization carry other multiples of phytobeneficial traits. Consequently, PSMs exhibit functional and genetic diversity due to their innate potential of producing an array of plant growth promoting traits and are considered to play a vital role in plant growth promotion, disease suppression and subsequent enhancement of yield. Further research should also focus on the selection of effective and competitive multifunctional biofertilizers for a variety of crops, to develop quality control system for the production of inoculants and their delivery in the field, to ensure and explore the benefits of plant-microorganism symbiosis, and how the persistence of microbial biofertilizers could be enhanced under both conventional and stressed soil environment. The success of inoculants application will depend on how we address and solve these problems. In this context, the advent of molecular tools has however, provided some insight for enhancing our ability to understand and manage the rhizosphere which is likely to lead to develop new products with improved efficiency. Furthermore, transfer of technology from lab to land in a big way is urgently required to boost the crop productivity via P-solubilizing microbes. Studies conducted to provide a deeper insight into this biosystem will be of great practical interest to multidisciplinary sectors, agronomists, microbiologists, industries, and the farmers and will be crucial in obtaining rhizosphere competent microbial biofertilizers, which can be used to solve various environmental problems in different agro-ecological niches.

ACKNOWLEDGMENTS The financial support by research and publication office [RPO] of Jimma University is highly acknowledged. The author would like to thank Prof. M. Leggett and Dr. Ararso Etana for their unfailing material support. The author would also like to thank Prof. Md. Saghir Khan for his prompt and kind initiation to write this chapter and for his unreserved material support.

REFERENCES Abd-Alla, MH; Omar, SA. [2001]. Survival of rhizobia/bradyrhizobia and a rock-phosphatesolubilizing fungus Aspergillus niger on various carriers from some agro-industrial wastes and their effects on nodulation and growth of faba bean and soybean. J. Plant Nutr., 24, 261– 272.

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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Phosphate Solubilizing Microbes

301

Abdel-Fattah, GM. [1997]. Functional activity of VA-mycorrhiza [Glomus mosseae] in the growth and productivity of soybean plants grown in sterilized soil. Folia Microbiologica, 42, 495–502. Alikhani, HA., Saleh-Rastin, N; Antoun, H.[2006]. Phosphate solubilization activity of rhizobia native to Iranian soils. Plant Soil, 287,35–41. Antoun, H., Beauchamp, CL; Goussard, N., Chabot, R; Roger, L. [1998]. Potential of Rhizobium and Bradyrhizobium speciess as plant growth promoting rhizobacteria on nonlegumes: Effect on radishes [Raphanus sativua L.]. Plant Soil, 204, 57-67. Arcand, MM; Schneider, KD. [2006]. Plant- and microbial-based mechanisms to improve the agronomic effectiveness of phosphate rock: a review. An Acad Bras. Cienc, 78 , 791-807. Asea, PEA., Kucey, RMN; Stewart JWB. [1988]. Inorganic phosphate solubilization by two Penicillium species in solution culture and soil. Soil Biol. Biochem., 20, 459– 464. Atkinson, D., Berta, G; Hooker, JE. [1994]. Impact of Mycorrhizal Colonization on Root Architecture, Root Longevity, and the Formation of Growth Regulators. In: Gininazzi, S. and Scheupp, H. [eds.]. Impact of Arbuscular Mycorrhizas on Sustainable Agriculture and Natural Ecosystems. Basel: Birkhauser Verlag, pp.89-99. Babana, AH; Antoun, H. [2006]. Biological system for improving the availability of Tilemsi phosphate rock for wheat [Triticum aestivum L.] cultivated in Mali. Nutr. Cycl. Agroecosys., 76, 285–295. Bagyaraj, DJ., Krishnaraj, PU; Khanuja, SPS. [2000]. Mineral phosphate solubilisation: Agronomic implications, mechanism and molecular genetics. Proceedings of the Indian National Science Academy (PINSA) B66, 69-82. Barea, JM., Azcón-Aguilar, C; Azcón, R. [1007]. Interactions between mycorrhizal fungi and rhizosphere microorganisms within the context of sustainable soil-plant systems. In: Gange, A.C.and Brown, V.K. [eds.]. Multitrophic interactions in terrestrial systems. Cambridge: Blackwell Science, pp.65-77. Barea, JM., Azcón, R., Azcón -Aguilar, C. [2002]. Mycorrhizosphere interactions to improve plant fitness and soil quality. Antonie van Leeuwenhoek 81, 343-351. Chabot, R., Antoun, H; Cescas, MP. [1993]. Stimulation of the growth of corn and lettuce by microorganisms which solubilize inorganic phosphorus. Can. J. Microbiol., 39, 941–947. Chabot, R., Antoun, H., Cesas, MP. [1996]. Growth promotion of maize and lettuce by phosphate solubilizing Rhizobium leguminosarum biovar phaseoli. Plant Soil, 184, 311321. Chandra, S., Choure, K., Dubey, RC; Maheshwari, DK. [2007].Rhizosphere competent Mesorhizobium loti mp6 induces root hair curling, inhibits Sclerotinia sclerotiorum and enhances growth of indian mustard [Brassica campestris]. Braz. J. Microbiol., 38, 124130. Chang, CH; Yang, SS. [2009]. Thermo-tolerant phosphate-solubilizing microbes for multifunctional biofertilizer preparation. . Bioresource Technol., 100, 1648-58. Chien, SH; Menon, RG. [1995]. Factors affecting the agronomic effectiveness of phosphate rock for direct application. Fert Res, 41, 227– 234. Cunningham, JE; Kuiack, C. [1992]. Production of citric and oxalic acids and solubilization of calcium phosphate by Penicillium bilaii. Appl. Environ. Microbiol., 58, 1451– 1458. Domey, S. [1996]. Occurrence, activity and root-colonizing capacity of phosphate-mobilizing bacteria in relation to P content of soil. In: Dogra, R.C., Behl, R.K. and Khurana, A.L.

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

302

Diriba Muleta

[eds.]. Resource management in agriculture. New Delhi: CCS Haryana Agricultural University, Hisar and MMB, pp 66–74. Downey, J; Kessel, CV. [1990]. Dual inoculation of Pisum sativum with Rhizobium leguminosarum and Penicillium bilaji. Biol. Fertil. Soi,l 10, 194–196. Duponnois, R; Plenchette, C. [2003]. A mycorrhiza helper bacterium enhances extomycorrhizal and endomycorrhizal symbiosis of Australian Acacia species. Mycorrhiza, 13, 85-91. Dwivedi, BS., Singh, VK; Dwivedi, V. [2004]. Application of phosphate rock with or without Aspergillus awamori inoculation to meet phosphorus demands of rice-wheat systems in the Indo-Gangetic plains of India. Aust. J. Exp. Agr., 44, 1041– 1050. Ehteshami, SMR., Aghaalikhani, M., Khavazi, K., Chaichi, MR. [2007]. Effect of phosphate solubilizing microorganisms on quantitative and qualitative characteristics of maize [Zea mays L.] under water deficit stress.Pakistan J. Biol. Sci., 10, 3585-3591. El-Azouni, IM. [2008]. Effect of phosphate solubilizing fungi on growth and nutrient uptake of soybean [Glycine max L.] plants. J. Appl. Sci. Res., 4, 592-598. El-Tarabily, KA., Nassar, AH; Sivasithamparam, K. [2008]. Promotion of growth of bean [Phaseolus vulgaris L.] in a calcareous soil by a phosphate-solubilizing, rhizospherecompetent isolate of Micromonospora endolithica. Appl. Soil Ecol. 39, 161-171. Ezawa, T; Yoshida, T. [1994] Characterization of phosphatase in marigold roots infected with vesicular-arbuscular mycorrhizal fungi. Soil. Sci. Plant Nutr., 40, 255–264. Frey-Klett, P., Pierrat, JC; Garbaye, J. [1997]. Location and survival of mycorrhiza helper Pseudomonas fluorescens during establishment of ectomycorrhizal symbiosis between Laccaria biocolor and Douglas fir. Appl. Environ. Microbiol., 63, 139–144. Gaind, S., Rathi, MS., Kaushik, BD., Nain, L; Verma, OP. [2007]. Survival of bio-inoculants on fungicides-treated seeds of wheat, pea and chickpea and subsequent effect on chickpea yield. J. Environ. Sci. Health B, 42, 663 – 668. Garbaye, J. [1994]. Helper bacteria: a new dimension to the mycorrhizal symbiosis. New Phytol., 128, 197-210. Gaur, A. C. [1990]. Phosphate Solubilizing Microorganisms as Biofertilizers. New Delhi, Omega Scientific Publishers. Goenadi, DH., Siswanto; Sugiarto, Y. [2000]. Bioactivation of poorly soluble phosphate rocks with a phosphorus-solubilizing fungus. Soil Sci. Soc. Am. J., 64, 927–932. Goldstein, AH. [1994]. Involvement of the quinoprotein glucose dehydrogenase in the solubilization of exogenous phosphates by Gram-negative bacteria. In: Torriani-Gorini, A, Yagil, E, Silver, S [eds.]. Phosphate in Microorganisms: Cellular and Molecular Biology. Washington, D.C: ASM Press, pp 197-203. Goldstein, AH; Rogers, RD; Mead, G. [1993]. Mining by microbe. Bio/Technol. 11, 12501254. Gupta, RP., Vyas, MK; Pandher, MS. [1998]. Role of phosphorus solubilizing microorganisms in P-economy and crop yield. In: Kaushik, B. D[ed.]. Soil–Plant– Microbe Interaction in Relation to Nutrient Management. New Delhi: Venus Printers and Publishers, pp. 95–101. Gupta, SC; Namdeo, SL.[1997]. Effect of Rhizobium, phosphate solubilizing bacteria and FYM on nodulation, grain yield and quality of chickpea. Indian J. Pulses. Res., 10, 171– 174.

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Phosphate Solubilizing Microbes

303

Gyaneshwar, P., Kumar, GN., Parekh, LJ; Poole, PS. [2002]. Role of soil microorganisms in improving P nutrition of plants. Plant Soil, 245, 83-93. Hamdali, H., Bouizgarne, B., Hafidi, M., Lebrihi, A., Virolle, MJ; Ouhdouch, Y. [2008a]. Screening for rock phosphate solubilizing actinomycetes from Moroccan phosphate mines. Appl. Soil Ecol. 38, 12-19. Hameeda, B., Harini, G., Rupela, OP., Wani, SP; Reddy, G. [2008]. Growth promotion of maize by phosphate-solubilizing bacteria isolated from composts and macrofauna. Microbiol. Res., 163, 234-242. Hayman, DS; Mosse, B. [1972]. The role of vesicular-arbuscular mycorrhiza in the removal of the phosphorus from soil by plant roots. Rev. Ecol. Biol. Sol., 9,463–470. Ho, WC; Ko, WH. [1984]. Soil microbiostasis: Effects of environmental and edaphic factors. Soil Biol. Biochem., 17, 167-170. Jain, PC., Kushawaha, PS., Dhakal, US., Khan, H; Trivedi, SM. [1999]. Response of chickpea [cicer arietinum L.] to phosphorus and biofertilzier. Legume Res., 22, 241–244. Jakobsen, I., Leggett, M., Richardson, AE. [2005]. Rhizosphere microorganisms and plant phosphorus uptake. In: Sims, J.T. and Sharpley, A.N. [eds.]. Phosphorus Agriculture and the Environment. Wisconsin: Agronomy Monograph No 48. ASAC, Crop Science Society of Agronomy Inc: and Soil Science Society of Agronomy Inc. Madison, pp. 437496. Jeffries, P; Barea, JM. [1994]. Biogeochemical cycling and arbuscular mycorrhizas in the sustainability of plant-soil systems. In: Gianinazzi, S.and Schu¨epp H. [ed.]. Impact of arbuscular mycorrhizas on sustainable agriculture and natural ecosystems. Basel: Birkha¨user Verlag, pp. 101–115. Jeon, JS., Lee, SS, Kim, HY., Ahn, TS; Song, HG. [2003]. Plant growth promotion in soil by some inoculated microorganisms. J. Microbiol., 41, 217–276. Jjemba, PK; Alexander, M. [1999]. Possible determinants of rhizosphere competence of bacteria. Soil Biol. Biochem. 31, 623-632. Jones, NP; Sreenivasa, MN. [1993]. Effect of inoculation of VA mycorrhiza and/or phosphate solubilizing bacteria on rhizosphere microflora of sunflower. II. Azotobacter and phosphate solubilizing bacteria. J. Ecotoxicol. Environ. Monit., 3, 55–58. Kang, SC; Ha, CG; Lee, TG; Maheshwari, DK. [2002]. Solubilization of insoluble inorganic phosphates by a soil fungus Fomitopsis sp. PS 102. Curr. Sci., 82, 439-442. Khan, MR., Khan, SM., Mohiddin, FA; Askary, TH. [2007]. Effect of certain phosphatesolubilizing bacteria on root-knot nematode disease of mungbean.In: Vela´zquez, E and Rodrı´guez-Barrueco, C. [eds.]. First International Meeting on Microbial Phosphate Solubilization. The Netherlands: Springer, pp. 341–346. Khan, MS., Zaidi, A; Wani, PA. [2006]. Role of phosphate-solubilizing microorganisms in sustainable agriculture - A review. Agron Sustain Dev 26, 1-15. Kim, KY., Jordan, D; McDonald, GA. [1998]. Effect of phosphate-solubilizing bacteria and vesicular-arbuscular mycorrhizae on tomato growth and soil microbial activity. Biol. Fertil. Soils., 26, 79–87. Kızılkaya, R. [2008]. Yield response and nitrogen concentrations of spring wheat [Triticum aestivum] inoculated with Azotobacter chroococcum strains. Ecol. Eng., 33,150-156. Kohler, J., Caravaca F., Carrasco, L; Rolda, A. [2007]. Interactions between a plant growthpromoting rhizobacterium, an AM fungus and a phosphate-solubilizing fungus in the rhizosphere of Lactuca sativa. Appl. Soil Ecol., 35, 480–487.

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

304

Diriba Muleta

Kucey, RMN. [1983]. Phosphate-solubilizing bacteria and fungi in various cultivated and virgin Alberta soils. Can. J. Soil Sci., 63, 671–678. Kucey, RMN. [1987]. Increased phosphorus uptake by wheat and field beans inoculated with a phosphorus solubilizing Penicillium bilaji strain and vesicular-arbuscular mycorrhizal fungi. Appl. Environ. Microbiol., 53, 2699–2703. Kucey, RMN., Janzen, HH; Leggett, ME. [1989] Microbially mediated increases in plant available phosphorus. Adv Agron 42:199–221. Kucey, RMN. [1988]. Effect of Penicillium bilaii on the solubility and uptake of micronutrients from soil by wheat. Can. J. Soil Sci., 68, 261– 270. Kumar, B., Trivedi, P; Pandey, A. [2007]. Pseudomonas corrugata: A suitable bacterial inoculant for maize grown under rainfed conditions of Himalayan region. Soil Biol. Biochem., 39, 3093-3100. Kundu, B. S; Gaur, AC. [1984]. Rice response to inoculation with N2-fixing and Psolubilizing microorganisms. Plant Soil, 79, 227-234. Landeweert, R., Hoffland, E., Finlay, R., Kuyper, TW; van Breemen, N. [2001]. Linking plants to rocks: ectomycorrhizal fungi mobilize nutrients from minerals. Trends Ecol. Evol., 16, 248–254. Leggett, ME., Gleddie, SC; Holloway, G. [2001]. Phosphate-solubilizing microorganisms and their use. In: Ae, N., Arihara, J., Okada, K. and Srinivasan, A. [eds.]. Plant Nutrient Acquisition: New perspectives. Tokyo: Spring-Verlag, pp.299-318. Lucy, M., Reed, E; Glick, BR. [2004]. Applications of free living plant growth-promoting rhizobacteria. Antonie van Leeuwenhoek , 86, 1-25. Maliha, R., Samina, K., Najma, A., Sadia, A; Farooq, L. [2004]. Organic acids production and phosphate solubilization by phosphate solubilizing microorganisms under in vitro conditions. Pakistan J. Biol. Sci., 7, 187-196. Mba, CC. [1994]. Field studies on two rock phosphate solubilizing actinomycete isolates as biofertilizer sources. Environ. Manage, 18, 263-269. McLaughlin, MJ; Alston, AM; Martin, JK. [1988]. Phosphorus cycling in wheat-pasture rotations. The role of microbial biomass in phosphorus cycling. Aust. J. Soil Res., 26, 333-342. Mittal, V., Singh, O., Nayyar, H., Kaur, J; Tewari, R. [2008]. Stimulatory effect of phosphatesolubilizing fungal strains [Aspergillus awamori and Penicillium citrinum] on the yield of chickpea [Cicer arietinum L. cv. GPF2]. Soil Biol. Biochem., 40, 718-727. Miyasaka, SC; Habte, M. [2001]. Plant mechanisms and mycorrhizal symbioses to increase phosphorus uptake efficiency. Commun. Soil Sci. Plant Anal., 32, 1101-1147. Muleta, D. [2007]. Microbial inputs in coffee production systems, Southwestern Ethiopia: Implication for biofertilizers and biocontrol agents (Doctoral Thesis), Department of Microbiology, SLU, Sweden. Muleta, D., Assefa, F; Granhall, U. [2007]. In vitro antagonism of rhizobacteria isolated from Coffea arabica L. against emerging fungal coffee pathogens. Eng. Life Sci., 7, 577–586. Muleta, D., Assefa, F., Hjort, K., Roos, S; Granhall, U .[2009]. Characterization of rhizobacteria isolated from wild Coffea arabica L. [In press]. Eng. Life Sci. Muthukumar, T., Udaiyan, K; Rajeshkannan, V. [2001]. Response of neem [Azadirachta indica A. Juss] to indigenous arbuscular mycorrhizal fungi, phosphate-solubilizing and asymbiotic nitrogen-fixing bacteria under tropical nursery conditions. Biol.Fertil. Soils, 34, 417-426.

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Phosphate Solubilizing Microbes

305

Nahas, E. [1996]. Factors determining rock phosphate solubilization by microorganisms. World J. Microbiol. Biotechnol., 12, 567-572. Nautiyal, CS. [1999]. An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol. Letters, 170, 265-270. Oberson, A., Friesen, DK., Rao, IM., Buhler, S; Frossard, E. [2001]. Phosphorus transformations in an oxisol under contrasting land-use system: The role of the microbial biomass. Plant Soil, 237,197-210. Oberson, A., Fardeau, JC., Besson, JM; Sticher, H. [1993]. Soil phosphorus dynamics in cropping systems managed according to conventional and biological agricultural methods. Biol. Fert. Soils, 16,111–189. Oehl, F., Oberson, M., Probst, A., Fliessbach, H., Roth, R; Frossard, E. [2001]. Kinetics of microbial phosphorus uptake in cultivated soils. Biol. Fertil. Soils., 34, 31-41. Okon, Y; Labandera-Gonzalez, CA. [1994]. Agronomic applications of Azospirillum: an evaluation of 20 years worldwide field inoculation. Soil Biol. Biochem., 26, 1591-1601. Omar, SA. [1998]. The role of rock-phosphatesolubilizing fungi and vesicular-arbuscular mycorrhiza [VAM] in growth of wheat plants fertilized with rock phosphate. World J. Microbiol. Biotechnol., 14, 211-218. Pal, SS. [1998]. Interactions of an acid tolerant strain of phosphate solubilizing bacteria with a few acid tolerant crops. Plant Soil, 198, 167–77. Pandey, A., Trivedi, P., Kumar, B; Palni, LMS. [2006]. Characterization of a Phosphate Solubilizing and Antagonistic Strain of Pseudomonas putida [B0] isolated from a SubAlpine location in the Indian Central Himalaya.Curr. Microbiol., 53,102–107. Pandey, A., Durgapal, A., Joshi, M; Palni, LMS. [1999]. Influence of Pseudomonas corrugata inoculation on root colonization and growth promotion of two important hill crops. Microbiol. Res., 154, 259–266. Pandey, A., Palni, LMS. [1998]. Isolation of Pseudomonas corrugate from Sikkim Himalaya. World J. Microbiol. Biotechnol., 14,411–413 17. Piccini, D; Azcon, R. [1987]. Effect of phosphate solubilizing bacteria and vesiculararbuscular mycorrhizal fungi on the utilization of Bayovar rockphosphate by alfalfa plants using a sand vermiculite medium. Plant Soil, 50,45–50. Plenchette, C., Clermont-Dauphin, C., Meynard, JM; and Fortin, JA. [2005]. Managing arbuscular mycorrhizal fungi in cropping systems. Can. J. Plant Sci., 85, 31-40. Poonguzhali, S., Madhaiyan, M; Sa, T. [2006]. Cultivation-dependent characterization of rhizobacterial communities from field grown Chinese cabbage Brassica campestris ssp pekinensis and screening of traits for potential plant growth promotion. Plant Soil, 286, 167–180. Prerna, A., Kapoor, KK; Akhaury, P. [1997]. Solubilization of insoluble phosphate by fungi isolated from compost and soil. Environ. Ecol., 15, 524–527. Read, DJ; Perez-Moreno, J. [2003]. Mycorrhizas and nutrient cycling in ecosysytem a journey towards relevance. New Phytol., 157, 475–492. Requena, N., Jimenez, I., Toto, M; Barea, JM. [1997]. Interactions between plant growthpromoting rhizobacteria [PGPR], arbuscular mycorrhizal fungi and Rhizobium spp. in the rhizosphere of Anthyllis cytisoides, a model legume for revegetation in Mediterranean semi-arid ecosystem. New Phytol., 136, 667-677.

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

306

Diriba Muleta

Reyes, I., Bernier, L; Antoun, H. [2002]. Rock phosphate solubilization and colonization of maize rhizosphere by wild and genetically modified strains of Penicillium rugulosum. Microbial Ecol., 44, 39-48. Rodriguez, H.and Fraga, R. [1999]. Phosphate solubilizing bacteria and their role in plant growth promotion. Biotech. Adv., 17, 319–339. Rodriguez, R, Vassilev, N; Azcon, R. [1999]. Increases in growth and nutrient uptake of alfalfa grown in soil amended with microbially-treated sugar beet waste. Appl. Soil Ecol., 11, 9– 15. Rosas, SB., Andres, JA., Rovera, M; Correa, N. [2006]. Phosphate-solubilizing Pseudomonas putida can influence the rhizobia-legume symbiosis. Soil Biol. Biochem., 38, 3502-3505. Rudresh, DL., Shivaprakash, MK; Prasad, RD. [2005a]. Tricalcium phosphate solubilizing abilities of Trichoderma spp. in relation to P uptake and growth and yield parameters of chickpea [Cicer arietinum L.]. Can. J. Microbiol., 51, 217–222. Rudresh, DL., Shivaprakash, MK; Prasad, RD. [2005b].Effect of combined application of Rhizobium, phosphate solubilizing bacterium and Trichoderma spp. on growth, nutrient uptake and yield of chickpea [Cicer aritenium L.]. Appl. Soil Ecol., 28, 139-146. Sagoe, CI., Ando, T., Kouno, K; Nagaoka, T. [1998]. Residual effects of organic acid-treated phosphate rocks on some soil properties and phosphate availability. Soil Sci. Plant Nutr., 44, 627– 634. Sahin, F., Çakmakçi, R; Kantar, F. [2004].Sugar beet and barley yields in relation to inoculation with N2-fixing and phosphate solubilizing bacteria. Plant Soil, 265, 123–129. Sale, PWG; Mokwunye, AU. [1993]. Use of phosphate rocks in the tropics. Fertil. Res. 35, 33– 45. Sandeep, AR., Joseph, S; Jisha, MS. [2008].Yield and nutrient uptake of soybean [Glycine max [L.] Merr] as influenced by phosphate solubilizing microorganisms. World J. Agr. Sci., 4, 835-838. Sarma, MVRK; Saharan, K., Prakash, A., Bisaria, VS; Sahai, V. [2009]. Application of fluorescent pseudomonads inoculant formulations on Vigna mungo through field trial. Intl J. Biol. Life Sci., 1,1 2009. Sattar, MA. [1982]. Phosphate solubilization and production of growth promoting substances by phosphomicroorganisms. Ph.D. thesis, Indian Agricultural Research Institute, New Delhi, India. Scotti, MR., Sa´, N., Marriel, I., Carvalhais, LC., Matias, SR., Corrêa, EJ., Freitas, N., Sugai, MA; Pagano, MC. [2007]. Effect of plant species and mycorrhizal inoculation on soil phosphate-solubilizing microorganisms in semi-arid Brazil: Growth promotion effect of rhizospheric phosphate-solubilizing microorganisms on Eucalyptus camaldulensis. In: Vela´zquez, E and Rodrı´guez-Barrueco, C. [eds.]. First International Meeting on Microbial Phosphate Solubilization. The Netherlands: Springer, pp. 167–172. Seshadri, S., Ignacimuthu, S; Lakshminarasimhan, C. [2004]. Effect of nitrogen and carbon sources on the inorganic phosphate solubilization by different Aspergillus niger strains. Chem. Eng. Commun., 191, 1043-1052. Sharan, A., Shikha, Darmwal, S; Gaur, R. [2008]. Xanthomonas campestris, a novel stress tolerant, phosphate-solubilizing bacterial strain from saline–alkali soils. World J. Microbiol. Biotechnol., 24, 753-759. Sharma, AK. [2006]. Biofertilizers for Sustainable Agriculture. Agrobios [India].

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Phosphate Solubilizing Microbes

307

Sharma, SN; Prasad, R. [2003]. Yield and P uptake by rice and wheat grown in a sequence as influenced by phosphate fertilization with diammonium phosphate and Mussoorie rock phosphate with or without crop residues and phosphate solubilizing bacteria. The Journal of Agricultural Science, 141:359-369. Shin, W., Ryu, J., Kim, Y., Yang, J., Madhaiyan, M; Sa, T. [2006]. Phosphate Solubilization and Growth Promotion of Maize [Zea mays L.] by the Rhizosphere Soil Fungus Penicillium oxalicum. 18th World Congress of Soil Science, July 9-15, 2006 Philadelphia, Pennsylvania, USA. Singh, HP; Singh, TA. [1993]. The interaction of rockphosphate, Bradyrhizobium, vesiculararbuscular mycorrhizae and phosphate-solubilizing microbe on soybean grown in a sub Himalayan mollisol. Mycorrhiza, 4, 37–43. Singh, S; Kapoor, KK. [1999]. Inoculation with phosphate-solubilizing microorganisms and a vesicular-arbuscular mycorrhizal fungus improves dry matter yield and nutrient uptake by wheat grown in a sandy soil. Biol. Fertil. Soils, 28, 139–144. Smith, SA; Read, D. [1997]. Mycorrhizal symbiosis [2nd ed.]. London: Academic Press. Son, TTN., Diep, CN; Giang,. TTM. [2006]. Effect of bradyrhizobia and phosphate solubilizing bacteria application on soybean in rotational system in the mekong delta. Omonrice, 14, 48-57. Souchie, EL., Azcón, R., Barea, JM., Saggin-Júnior, OJ; da Silva EMR. [2006]. Phosphate solubilization and synergism between P-solubilizing and arbuscular mycorrhizal fungi. Pesq. agropec. bras., 41, 1405-1411. Toro M., Azcon, R., Herrera, R. [1996]. Effects on yield and nutrition of mycorrhizal and nodulated Pueraria phaseoloides exerted by P-solubilizing rhizobacteria. Biol. Fertil. Soils, 21, 23-29. Toro, M., Azco´n, R; Barea, J. [1997]. Improvement of arbuscular mycorrhiza development by inoculation of soil with phosphate-solubilizing rhizobacteria to improve rock phosphate bioavailability [32P] and nutrient cycling. Appl. Environ. Microbiol., 63, 4408– 4412. Trivedi, P., Kumar, B. Pandey, A; Palni, LM.S. [2007]. Growth promotion of rice by phosphate solubilizing bioinoculants in a Himalayan location. In: Vela´zquez, E and Rodrı´guez-Barrueco, C. [eds.]. First International Meeting on Microbial Phosphate Solubilization. The Netherlands: Springer, pp. 291-299. van der Vossen, HAM. [2005]. A critical analysis of the agronomic and economic sustainability of organic coffee production. Exp. Agr., 41, 449-473. Varsha-Narsian, J., Thakkar; Patel, HH. [1994]. Inorganic phosphate solubilization by some yeast. Indian J. Microbiol., 35, 113–118. Vassilev, N, Baca, MT., Vassileva, M., Franco, I; Azcon, R. [1995]. Rock phosphate solubilization by Aspergillus niger grown on sugar-beet waste medium. Appl. Microbiol. Biotechnol. 44, 546– 549. Vassilev, N., Franco, I., Vassileva, M; Azcón, R. [1996]. Improved plant growth with rock phosphate solubilized by Aspergillus niger grown on sugar-beet waste. Bioresource Technol., 55, 237– 241. Vassilev, N., Vassileva, M; Nikolaeva, I. [2006].Simultaneous P-solubilizing and biocontrol activity of microorganisms: potentials and future trends. Appl. Microbiol. Biotechnol., 71, 137–144.

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308

Diriba Muleta

Vassileva, M., Vassilev, N; Azcón, R. [1998]. Rock phosphate solubilization by Aspergillus niger on olive cake-based medium and its further application in a soil–plant system. World J. Microbiol. Biotechnol., 14, 281-284. Vazquez, P., Holguin, G., Puente, M., Elopez-Cortes, A; Bashan, Y. [2000]. Phosphate solubilizing microorganisms associated with the rhizosphere of mangroves in a semi arid coastal lagoon. Bio. Fertil. Soils, 30, 460-468. Venkateswarlu, B., Rao. AV; Raina, P. [1984]. Evaluation of phosphorus solubilization by microorganisms isolated from arid soil. J. Indian Soc. Soil Sci., 32, 273–277. Vessey, JK. [2003]. Plant growth promoting rhizobacteria as biofertilizers. Plant Soil, 255, 571-586. Wang, GH., Jin, J., Xu, MN, Pan, XW; Tang, C. [2007]. Inoculation with phosphatesolubilizing fungi diversifies the bacterial community in rhizospheres of maize and soybean. Pedosphere, 17, 191-199. Wani, PA., Khan, MS; Zaidi, A. [2007]. Synergistic effects of the inoculation with nitrogenfixing and phosphate-solubilizing rhizobacteria on the performance of field-grown chickpea. J. Plant Nutr. Soil Sci., 170, 1–5. Wikström, D. [2003]. Willingness to pay for sustainable coffee: a choice experiment approach. M. Sc. thesis, Luleål University of technology, Sweden. Wasule, DL; Wadyalkar, SR; Buldeo, AN. [2007]. Effect of phosphate solubilizing bacteria on role of Rhizobium on nodulation by soybean. In: Velázquez, E. and RodríguezBarrueco, C.[eds.]. First International Meeting on Microbial Phosphate Solubilization. The Netherlands: Springer, pp.139-142. Wasule, DL., Wadyalkar, SR; Buldeo, AN. [2003]. Effect of phosphate solubilizing bacterial on role of Rhizobium on nodulation by soybean. In: Velázquez, E. [ed.]. First International Meeting on Microbial Phosphate Solubilization. Salamanca, Spain: Springer, pp.139-142. Vikram, A; Hamzehzarghani, H. [2008]. Effect of phosphate solubilizing bacteria on nodulation and growth parameters of green gram [Vigna ratiata L. Wilczek]. Res. J. Microbiol., 3, 62-72. Zaidi, A; Khan, MS. [2007]. Stimulatory effects of dual inoculation with phosphate solubilizing microorganisms and arbuscular mycorrhizal fungus on chickpea. Aust. J. Exp. Agr., 47, 1016–1022. Zaidi, A; Khan, MS. [2006].Co-inoculation effects of phosphate solubilizing microorganisms and Glomus fasciculatumon green gram- Bradyrhizobium symbiosis. Turk. J. Agr. For., 30, 223-230. Zaidi, A., Khan, MS; Aamil, M. [2004]. Bioassociative effect of rhizospheric microorganisms on growth, yield and nutrient uptake of greengram. J. Plant Nutr., 27, 599-610. Zaidi, A., Khan, MS; Amil, M. [2003]. Interactive effect of rhizotrophic microorganisms on yield and nutrient uptake of chickpea [ Cicer arietinum L.]. Eur. J. Agron., 19, 15-21. Zapata, F; Roy, RN. [2004]. Use of phosphate rocks for sustainable agriculture. In: Fertilizer and Nutrition Bulletin No. 13. A joint production of the FAO land and water development division agency. Food and Agriculture Organization of the United Nations,Rome. Zarei, M., Saleh-Rastin, N., Alikhani, HA; Aliasgharzadeh, N. [2006]. Responses of lentil to co-inoculation with phosphate-solubilizing rhizobial strains and arbuscular mycorrhizal fungi. J. Plant Nutr., 29, 1509–1522.

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Chapter 14

GENETIC AND PHENOTYPIC CHARACTERIZATION OF PHOSPHATE-SOLUBILIZING BACTERIA AND THEIR EFFECTS ON GROWTH AND SYMBIOTIC PROPERTIES OF ALFALFA PLANTS Lorena B. Guiñazú1, Javier A. Andrés∗1,2, Nicolás A. Pastor1, Marisa Rovera1 and Susana B. Rosas1

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1

Laboratorio de Interacción Microorganismo - Planta, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto Campus Universitario. X5804BYA, Río Cuarto, Córdoba, Argentina 2 Microbiología Agrícola, Facultad de Agronomía y Veterinaria, Universidad Nacional de Río Cuarto. Campus Universitario. X5804BYA, Río Cuarto, Córdoba, Argentina.

ABSTRACT Alfalfa is the most important forage legume in the semiarid Argentinean Pampas because of the quality nutrients that it provides. Furthermore, the effect that this plant has on soil fertility and improvement and conservation of the soil structure is very important. The alfalfa productivity could be increased if it is inoculated by to high efficiency rhizobial strains as well as other plant growth-promoting rhizobacteria. The use of phosphate-solubilizing bacteria as inoculants increases phosphorous uptake and yield of crops. The objective of this work is to phenotypically and genetically characterize alfalfa rhizosphere bacteria and to evaluate the effect of single or mixed inoculation upon nodulation and biological nitrogen fixation [BNF] efficiency. More than forty isolated rhizobacteria showed tricalcium phosphate solubilization in vitro assays and four of them caused greater solubilization than the control strain Pseudomonas putida SP22. The comparison of the 16S rDNA sequences indicated that the strains were phylogenetically related to Bacillus spp. and Pseudomonas spp. A beneficial effect of these isolates on the alfalfa growth was observed in the coinoculation assays. The inoculation of ∗

Correspondence to: e-mail: [email protected]; [email protected]

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Lorena B. Guiñazú, Javier A. Andrés, Nicolás A. Pastor et al. Sinorhizobium meliloti B399 and Pseudomonas sp. F10 caused a significant increase in fresh and dry biomass of plant organs [roots and shoots], length and surface area of roots and symbiotic properties of alfalfa plants. The plants co-inoculated with S. meliloti B399 and Bacillus sp. M7 showed a significant increase in the measured parameters. The coinoculation of Pseudomonas sp. U14 and Bacillus sp. B11 with S. meliloti B399 also showed increases in some of the physiological parameters studied. A single or mixed inoculant containing these strains could be developed in order to be used in agricultural practices.

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14. 1. INTRODUCTION After nitrogen [N], phosphorus [P] is the major plant growth-limiting nutrient despite being abundant in soils in both organic and inorganic forms. Most agricultural soils contain large reserves of total Pn ranging from 200 to 5000 mg P kg-1 with an average of 600 mg P kg-1. A part of P accumulation depends on regular application of chemical fertilizers or sludge from wastewater treatment [De-Bashan and Bashan, 2004]. However, many soils throughout the world are P-deficient because the free phosphorus concentration [the form available to plants] even in fertile soils is generally not higher than 10 µM even at pH 6.5 where it is most soluble. These low levels of P are due to high reactivity of soluble P with calcium [Ca], iron [Fe] or aluminum [Al] that lead to P precipitation. Inorganica P in acidic soils is associated with Al and Fe compounds, whereas calcium phosphates are the predominant form in calcareous soils [Gyaneshwar et al., 2002]. The most abundant phosphates in the Latinoamerican Pampas are bound to calcium where the continuous agriculture practiced for more than 25 years caused considerable decreases in the soluble P and to a smaller extent it also decreased the total P [Urioste et al., 1996]. Phosphorus is one of the most affected minerals because of the soil degradative processes. Moreover, it is added to soil as soluble inorganic phosphates that after forming complex with soil constituents may become insoluble and unavailable to plants [Singh and Kapoor, 1994]. A great number of bacterial species, associated to plant rhizosphere, can cause beneficial effects upon plants [Glick, 1995]. Such group of bacteria is referred to as plant growth-promoting rhizobacteria [PGPR] [Kloepper and Schroth, 1978] which also include phosphate-solubilizing bacteria [PSB]. Interest has been generated recently on the inoculation of phosphate-solubilizing microorganisms into the soil so as to increase the availability of native fixed P and to reduce the use of chemical phosphatic fertilizers [Subba Rao, 1993; Rodríguez and Fraga, 1999]. Secretion of organic acids, inorganic acids and phosphatase enzymes are common mechanisms that facilitate the conversion of insoluble forms of phosphorus to plant available forms [Richardson, 2001]. Solubilization of insoluble phosphorus to accessible forms like orthophosphate is one of the important traits of PGPR. The main strains able to perform this conversion belongs to the genera Pseudomonas, Mycobacterium, Micrococcus, Bacillus, Achromobacter, Erwinia, Agrobacterium, Burkholderia, Flavobacterium, Rhizobium and Sinorhizobium [Rodríguez and Fraga, 1999; Fernández et al., 2007]. Co-inoculation of leguminous seeds with nitrogen fixing symbiotic bacteria and plant growth promoting genera has acquired an increasing interest. Nitrogen fixation is a Prequiring process and it is known that every aspect of the process of formation of the N2

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fixing nodule is limited by the availability of P [Schulze et al., 2006; Kouas et al., 2005]. Legumes as alfalfa [Medicago sativa L.], clover [Trifolium pratense], common bean [Phaseolus vulgaris], cowpea [Vigna unguiculata and pigeon pea [Cajanus cajan] show a high positive response to P supplementation [Gyaneshwar et al., 2002; Khan et al., 2007]. Nodules of P-sufficient plants have a higher P concentration than shoots and roots, and may contain up to 1.5% of the total plant P [Schulze et al., 2006]. P-deficient plants exhibit reduced carbohydrate supply to nodules and in turn restricts nodule initiation, development and growth and decrease nitrogenase activity [Remans et al., 2007]. Moreover, the nodule bacteroids may be P limited even though the plant is well supplied whit P [Al-Niemi et al., 1997]. Numerous reports suggest a positive effect of combined inoculation of rhizobia with free bacteria of the genera Pseudomonas [Knigth and Langson-Unkefer, 1988; Rosas et al., 2006] and Bacillus [Sindhu et al., 2002] on legume growth including early nodulation, an increase in the number of nodules, higher nitrogenase activity and root respiration, a superior uptake of water and nutrients by the roots and an overall increase in plant health. Coinoculation with Azospirillum and phosphate-solubilizing bacteria increased growth, yield and uptake of nitrogen, phosphorus and other minerals of sorghum [Sorghum bicolor] and barley [Hordeum vulgare] plants [Bashan and Holguín, 1997]. Alfalfa is the most important forage legume in the Latinoamerican Pampas due to its quality of nutrients it provides in the dietary system. In Argentina, alfalfa has traditionally been the basis of livestock production and together with wheat [Triticum aestivum], one of the founding crops of Argentinean agriculture. Presently, it is one of the main forage resources because of its vast adaptability to different soils and climates. Moreover, this species plays a fundamental part in sustaining the structure and nitrogen fertility of the soils when it is correctly inoculated by specific rhizobial strains [Muslera Pardo et al., 1984]. Therefore, the development of biotechnological products involving different rhizosphere microorganisms [multiple inoculants] is of great practical interest in order to achieve together with an optimal biological nitrogen fixation, an increased capacity of incorporation of water and nutrients. The focus of this chapter is to characterize alfalfa rhizosphere bacteria phenotypically and genetically and to evaluate the effect of single or mixed inoculation of phosphate-solubilizing bacteria and nitrogen fixing organisms on growth, nodulation and biological nitrogen fixation efficiency of alfalfa plants.

14.2. MATERIALS AND METHODS 14.2.1. Origin of the Bacterial Isolates and Colony Count The isolates were collected from soil samples of the experimental fields of the Instituto Nacional de Tecnología Agropecuaria [INTA], located in Manfredi [Córdoba province, 63° 44’ O 31° 50’ S] and Balcarce [Buenos Aires province, 58° 15’ O 37° 50’ S] Argentina, Faja Maisán, Chile [72° 55’ O 39° 05’ S] and Punta Espinillo, Uruguay [56° 25’ O 34° 49’ S]. Neither alfalfa nor other Sinorhizobium meliloti host was cultivated in the last five years in these areas. Each soil sample was homogenized and serially diluted using normal saline. A 0.1 ml aliquot was plated in 25% Tryptic Soy Agar [TSA; Britania®] and plates were incubated at 28 °C for 24-48 h. Each individual experiment was repeated three times and

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resulting colonies were expressed as colony forming units per gram of soil [CFU/g soil]. Isolated colonies were maintained in 25% TSA medium until use.

14.2.2. Phenotypical Characterization of Bacterial Strains Bacterial strains were characterized for color, shape, elevation, margins, diameter, surface, opacity and texture [Zinniel et al., 2002]. Each isolate was Gram stained and was subjected to 3% KOH test [Suslow et al., 1982].

14.2.3. In Vitro Assay of Inorganic Phosphorus Solubilization The ability of the bacterial isolates to solubilize tricalcium phosphate was tested using the YGP medium [pH 7] containing yeast extract [2 g/l], glucose [20 g/l], tricalcium phosphate [2 g/l], actidione [60 mg/l] and agar [15 g /l] [Rosas et al., 2006]. This medium was inoculated with the relevant strains and incubated at 28 °C for 5 days. Experiments were performed in triplicate. Bacterial colonies forming clear zones were considered as phosphate solubilizers. Pseudomonas putida SP22 [Rosas et al., 2006] served as a positive control.

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14.2.4. Genotypic Characterization and Identification Genotypic identification was performed by amplification and partial nucleotide sequencing of the 16S ribosomal DNA [16S rDNA] of the isolates that showed an equal or higher phosphate-solubilization capacity than the control. The partial nucleotide sequences of the 16S rRNA gene [rDNA] were determined by direct sequencing of appropriate PCR products. A DNA region corresponding to nucleotides 20 to 338 of Escherichia coli 16S rDNA was amplified from each strain with the universal primers Y1 [59-TGG CTC AGA ACG AAC GCT GGC GGC-39] and Y2 [59-CCC ACT GCT GCC TCC CGT AGG AGT39] as previously described for proteobacteria [Young et al., 1991]. A 25 µl PCR mixtures contained: 0.5 µM of each primer, 200 µM dNTPs, 3 mM MgCl2, PCR reaction buffer [50 mM KCl, 20 mM Tris HCl, pH 8.0], 1 U Taq DNA polymerase [Promega Corp.] and 2 µl of template DNA, previously obtained by heating freshly isolated bacterial colonies in 50 µl of distilled water to 100°C for 15 min. The amplifications were carried out in Thermo Cycler [I Cycler-BioRad]. The cycling conditions were: 94°C for 2 min, followed by 35 cycles at 94°C for 20 sec, at 52°C for 20 sec, at 72ºC 45 sec, and at 72°C for 2 min. After the reaction, 10 µl of the PCR reaction were analyzed in 1.5%-agarose gels containing 1 µg/ml of ethidium bromide and photographed with a Kodak DC290 Digital camera. The nucleotide sequence of the PCR products was determined for both strands with an Automatic Laser Fluorescent DNA Sequencer. The partial sequences of the 16S rDNA genes have been deposited in the GenBanK, and compared against the complete database using the BLAST algorithm.

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14.2.5. Single and Mixed Inoculation Assay In order to evaluate coexistence in 25% TSA and yeast extract mannitol agar [YEMA] [Vincent, 1970], the characterized isolates were confronted in vitro with S. meliloti B399 [strain recommended by INTA for alfalfa inoculation in Argentina]. Each strain was streaked on one side of a Petri plate and incubated at 28 °C for 48-72 h. Rhizobacteria that showed normal growth in the presence of the rhizobial strain in both culture media were selected for further assays of single and mixed inoculation studies.

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14.2.5.1. Bacterial Inoculum Preparation The selected bacterial strains including phosphate-solubilizing strains of Bacillus [M7 and B11] and Pseudomonas [F10 and U14] and symbiotic nitrogen fixer [S. meliloti B399] were used either alone or in combination to evaluate their effect on growth and symbiotic properties of alfalfa plants. The phosphate-solubilizing strain and S. meliloti strains were used to prepare a small amount of inoculant using steam-sterilized Tierra del Fuego peat [pH 6.5]. This material was conditioned according to the method described by Roughley [1970] and saturated with each of the strains of interest grown in 25% Tryptic Soy Broth [TSB]. Growth curves were determined in order to standardize the appropriate bacterial inoculum for each microorganism according to Koch [1981]. The inoculant formulation was carried out with an approximate concentration of 108-109 viable cells/ml. The inoculants were allowed to mature in test tubes [CIAT Manual, 1988] at room temperature for 7 days and kept at 4 °C. Colony counts were counted during formulation and seed inoculation. 14.2.5.2. Treatment of Alfalfa Seeds The seeds of alfalfa cv. Bárbara SP [INTA Manfredi, Argentina] were disinfected with 70% ethanol for 3 min. and washed with several changes of distilled water [SDW] [Andrés et al., 1998]. Then, 2 g of seeds were bacterised with 1 g of inoculant [approximately 103-104 cells/g seeds]. Seeds were coated with a 1:1 mix of inoculants [phosphate-solubilizing isolate and Rhizobium] for co-inoculation assays. The assay included the following treatments: uninoculated control [C], uninoculated control + N [CN], single inoculation with S. meliloti B399, single inoculation with the phosphate-solubilizing isolate, and mixed inoculation: strain S. meliloti B399 + phosphate-solubilizing isolate. Eight seeds were sown in polyethylene pots [6.5 cm diameter, 15 cm height] containing a soil:sand:perlite [2:1:1] sterile mix. Growth parameters were measured 45 days after sowing [DAS]. Pots were kept in a growth chamber at 28 ± 2 ºC with light-dark cycles [16 h light:8 h dark] under a light intensity of 220 μE/m2/s and plants were alternatively watered with SDW and a modified N free Jensen solution [Vincent, 1970], in which the P source was replaced by tricalcium phosphate. The treatments inoculated only with a phosphate-solubilizing isolate and the CN treatment was watered in the same manner, but whit the addition of KNO3 [0.05 g/l] to the nutrient solution. The length and fresh and dry biomass of shoots and roots, root surface area [Carley and Watson, 1966] and number and dry mass of nodules [and their position in the roots] were determined for each treatment.

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14.2.5.3. Determination of Nitrogenase Activity The acetylene reduction assay [Hardy, 1968] was used to assess the nitrogenase activity of nodules. The nodules from each plant were placed in 10 ml vials and 10% of their atmosphere was substituted with acetylene. After 30 min. incubation, 100 μl of the sample was injected in a Hewlett Packard 5890 Series II Plus Gas Chromatograph at constant pressure. The column used was Parapak N [polyvinylpyrolidone], 1/8, 2mm DI which allows the analysis of gases of up to 6 carbons. Acetylene is reduced to ethylene and the evolution of both gases is quantified if nitrogenase activity occurs. 14.2.5.4. Determination of Total N Content Nitrogen content in shoots was determined using a modified Kjeldahl method [Baker and Thompson, 1992]. For this assay, 1.25 g of catalytic mix [potassium sulfate:mercuric oxide, in a 24:1 relation] and 2.5 ml of concentrated sulfuric acid were added to a 100 mg sample. The mixture was digested for 40 min. The ammonia liberated as a product of ammonium sulfate decomposition by a strong alkali [NaOH] was separated by means of distillation and was collected [as ammonium borate] in a 4 % boric acid. Ammonium was determined by titrating against 0.02 N HCl. An automatic analyzer of nitrogen, Kjeltec Auto 1030 [Tecator, Sweden], was used. 14.2.5.5. Statistical Analyses Results were analyzed statically by analysis of variance [ANOVA] using the InfoStat computer software [Universidad Nacional de Córdoba, Argentina, 2000]. When analysis of variance showed significant treatment effects, the Di Rienzo-Guzmán-Casanoves [DGC] test was applied to make comparisons among the means at the P ≤ 0.05.

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14.3. RESULTS 14.3.1. In Vitro Assays The number of bacterial isolates was in the order of 106 CFU/g soil for the different soils. In this study, more than 40 phosphate-solubilizing rhizobacteria were isolated. Of these, bacterial isolates including two strains each of Pseudomonas [F10 and U14] and Bacillus [M7 and B11] were selected because of their greater phosphate-solubilizing activity [Table 1]. The zone of solubilization [halo] among selected bacterial strains ranged between 20.2±1.8 [Pseudomonas F10] to 25.1±2.6 [Bacillus M7]. Interestingly, Bacillus M7 showed an increase of 19% in halo size compared to the reference strain [P. putida sp. 22]. Moreover, all of the strains showed coexistence ability when grown with S. meliloti B399 in YEMA and 25% TSA media. While comparing the genetic properties of the selected bacterial strains with those of the A 16S rDNA sequences available in the GenBank database, the bacterial strains were found phylogenetically related to Bacillus sp. [B11 and M7] and Pseudomonas sp. [F10 and U14].

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Table 1. Phosphate-solubilizing activity of bacterial strains on YGP medium Bacterial strain Pseudomonas putida SP22 Sinorhizobium meliloti B399 Pseudomonas sp. F10 Pseudomonas sp. U14 Bacillus sp. M7 Bacillus sp. B11

Phosphate solubilization [halo size in mm] 21.1 ± 3.2 0 20.2 ± 1.8 22.3 ± 0.9 25.1 ± 2.6 20.5 ± 1.3

Source of bacteria Rosas et al. 2006 INTA, Argentina Faja Maisán, Chile Punta Espinillo , Uruguay Manfredi, Argentina Balcarce, Argentina

Values are mean of three independent replicate for each strain; ± indicates standard deviation.

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14.3.2. Single and Mixed Inoculation Assay The coinoculation treatments of S. meliloti B399 with Pseudomonas F10 demonstrated a maximum increase in the measured parameters compared to all single inoculation or the uninoculated treatments [Table 2]. Among single inoculation, Bacillus B11 showed highest increase in dry matter accumulation in roots of alfalfa plants compared to uninoculated control [59% increase] or uninoculated but N amended treatment [89% increase]. Moreover, while comparing the effects of single inoculations, Bacillus B11 displayed a substantial increase of 40% over other best performing bacterial strain [U14] of Pseudomonas. However, when S. meliloti B399 was used with Bacillus M7 or Pseudomonas F10, increased the dry matter accumulation in roots of alfalfa plants even further by 98% and 132%, respectively over single inoculation of S. meliloti B399. Among the dual inoculation treatments, the effect of S. meliloti B399 with Bacillus B11 and S. meliloti B399 with Pseudomonas U14 was though greater than those observed for single inoculation of S. meliloti B399 but was considerably inferior to those recorded for single inoculation of Bacillus B11 [2.86 mg/plant]. The dual inoculation treatments showed a greater foliar development, reflected mainly in the fresh weight values, since they were significantly superior to those of the single inoculation treatments and the uninoculated controls. The co-inoculation of S. meliloti B399 with Pseudomonas F10 showed highest increase in shoot fresh weight of alfalfa plants compared to uninoculated control [88%] or uninoculated control but N amended treatment [51%]. When analyzing shoot dry weight, it was observed that the coinoculation treatments showed statistically significant differences with the uninoculated controls and the treatments of single inoculation with a phosphate-solubilizing strain, but there were no significant differences with the plants inoculated with S. meliloti B399. Root length was one of the parameters most favored by co-inoculation with phosphate-solubilizing bacteria and S. meliloti B399, since coinoculated plants showed a significant increase with regard to the rest of the treatments, except with the single inoculation of S. meliloti B399. In addition, the dual inoculation treatment of S. meliloti with Pseudomonas F10 showed the highest root surface area [400% increase respect to uninoculated control and 45% respect to S. meliloti B399 and Bacillus M7 treatment]. Although shoot length increased significantly following single inoculation of phosphate solubilizing bacteria and uninoculated controls [data not shown], there were no significant differences between the co-inoculated plants and those treated with S. meliloti B399.

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Table 2. Effect of single and mixed inoculation of phosphate-solubilizing bacteria and nitrogen fixing Sinorhizobium meliloti B399 on biological and chemical properties of alfalfa plants Treatment

Uninoculated control Uninoculated control + N Bacillus spp. M7 Pseudomonas spp. F10 Bacillus spp. B11 Pseudomonas spp. U14 S. meliloti B399 S. meliloti B399 + Bacillus spp. M7 S. meliloti B399 + Pseudomonas spp. F10 S. meliloti B399 + Bacillus spp. B11 S. meliloti B399 + Pseudomonas spp. U14

Root fresh weigth [mg/plant] 9.70a 14.85b 12.80b 12.65b 19.65 b 16.05b 13.70b 23.05c 28.74d 14.50b 22.55c

Root dry weight [mg/plant] 1.80ª 1.51ª 2.18ª 1.64ª 2.86b 2.04a 1.59a 3.15b 3.69b 2.04ª 2.47ª

Shoot fresh weight [mg/plant] 29.45a 36.70a 25.70a 29.15a 45.30 b 39.70b 37.35a 51.10b 55.48c 44.15b 50.00b

Shoot dry weight [mg/plant] 2.03a 3.01a 2.53a 2.24a 3.14 a 2.94a 3.94b 4.18b 4.58b 3.70b 3.67b

Root length [cm] 3.61a 3.38a 3.49a 3.53a 3.67a 3.56a 3.56a 4.56b 4.83b 4.46b 4.65b

Root-surface area [cm3]

Shoot N [mg/g]

0.16ª 0.18ª 0.28b 0.25b 0.38 b 0.32b 0.32b 0.44c 0.64d 0.30b 0.36b

41.9ab 46.2b 41.5ª 41.9ab 43.9c 37.9ª 45.6c 49.6d 42.7b 49.8d 39.3ª

Values are mean of 25 plants for each treatment. Different letters within the same column indicate significant differences [P ≤ 0.05] according to Di RienzoGuzmán-Casanoves [DGC] test.

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When analyzing the N content, it was observed that the alfalfa plants coinoculated with S. meliloti B399 and M7 and S. meliloti B399 co-inoculated with Bacillus B11 presented significantly greater mean values than those of the rest of the inoculation treatments, whereas plants inoculated with S. meliloti B399 and Pseudomonas spp. showed a lower N content than those of the uninoculated control + N [7.5% increase] and the plants inoculated with strain S. meliloti B399 [9% increase]. A significant increase in number and dry mass of nodules was observed with S. meliloti and Pseudomonas F10 with regard to other treatments [Table 3]. The affectivity percentage of nodules was of about 85%, and the majority of nodules were clustered on the secondary roots; in S. meliloti B399 treated plants, nodules were located only on the primary root [data not shown]. Though, nodules collected from each treatment showed nitrogenase activity but no statistically significant [P ≤ 0.05] differences were detected between single and mixed inoculation treatments [Table 3]. Table 3. Effect of single and mixed inoculation of phosphate-solubilizing bacteria and nitrogen fixing Sinorhizobium meliloti B399 on symbiotic properties of alfalfa plants Treatment

S. meliloti B399 S. meliloti B399 + Pseudomonas spp. F10 S. meliloti B399 + Bacillus spp. M7 S. meliloti B399 + Bacillus spp. B11 S. meliloti B399 + Pseudomonas spp. U14

Nodulation No./plant Dry weight [mg/plant] 2.2a 0.17ª 4.6c 0.41b 2.8b 0.18a 3.1b 0.18a 3.2b 0.15a

Nitrogenase activity [μmoles ethylene g nodule fresh-1 h-1] 1.17ª 1.22ª 1.19a 0.856a 1.08a

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Values are mean of 25 plants for each treatment. Different letters within the same column indicate significant differences [P ≤ 0.05] according to Di Rienzo-Guzmán-Casanoves [DGC] test.

14.4. DISCUSSION Bacterial communities form an important and integral component of soil ecosystems and affect the fertility of soils and enhance plant growth by different mechanisms. One of these is the dissolution of insoluble P by the organic acids released by phosphate-solubilizing microbes [Khan et al. 2007]. In this study, four of the bacterial strains showing a greater Psolubilizing activity in vitro assays were identified as Bacillus spp. [M7 and B11] and Pseudomonas spp. [F10 and U14] following amplification and partial nucleotide sequencing of the ribosomal 16S DNA. Numerous reports indicate that the species of the genera Pseudomonas and Bacillus possess the most powerful ability of solubilizing insoluble phosphate [Illmer and Schinner, 1992; Vazquez, 1996; Chen et al., 2006; Wani et al. 2007]. Furthermore, the ability of these strains to solubilize insoluble P prompted us to determine their single or coinoculation growth promoting effects with S. meliloti B399 [this strain does not solubilize phosphates] on alfalfa plants. In general, the combined treatments caused a significant increase in the biomass of roots and shoots, length and surface area of roots and number and dry mass of nodules. The positive effect on plant growth could probably be related, among other factors, to the increase in soluble P availability in soil, as a result of the

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action of Pseudomonas F10. A number of other reports have also shown similar positive effects on nodulation, plant growth and seeds yield while co-inoculating with Pseudomonas spp. [Bolton et al., 1990; Dashti et al. 1998; Sindhu et al., 1999]. The dual inoculation of S. meliloti B399 with Bacillus M7 showed significant increases in root fresh and dry weight, shoot fresh biomass, root length, root surface area and shoot tissue N content, with regard to the plants of single inoculation treatments and uninoculated control. The study of phosphate-solubilizing bacilli as inoculants for several crop plants has generated great interest in the last few years. B. circulans and B. megaterium var. phosphaticum inoculants increased plant weight and P-uptake of millet and pea, respectively, in growth chamber studies [Saber et al., 1977; Raj et al., 1981]. Similarly, Gaind and Gaur [1991] reported that a B. subtilis inoculant increased biomass, grain yield, and P and N-uptake of mung bean grown in a P-deficient field soil amended with rock phosphate. The coinoculation treatments of S. meliloti B399 with Pseudomonas U14 and S. meliloti B399 with Bacillus B11 also showed increases in some of the physiological parameters assessed. Although nitrogenase activity was not significantly increased in this study compared to S. meliloti B399, it was not inhibited by coinoculation with phosphate-solubilizing strains and hence, promoted biological N2 fixation process by alfalfa plants. Several authors concluded that mixed inoculants provide a better nutritional balance for plants and that the improved N and P-uptake are the main mechanisms involved. Among them, Zaidi and Khan [2006] shown that the triple inoculation of the AM fungus Glomus fasciculatum, Bradyrhizobium sp. [vigna] and Bacillus subtilis significantly increased dry matter yield, chlorophyll content and N and P uptake of greengram plants. Legume grain yield and concentration and uptake of N and P in chickpea increased as a result of coinoculation with Mesorhizobium ciceri, Azotobacter chroococcum and P-solubilizing Pseudomonas and Bacillus spp. [Wani et al., 2007]. In our previous report, differences were not observed with respect to inoculation with Sinorhizobium meliloti 3Doh13 [a good solubilizer of phosphorus] alone when alfalfa was coinoculated with S. meliloti 3Doh13 and the phosphorus-solubilizing strain P. putida sp. 22 [Rosas et al., 2006]. In this case, the phosphate solubilization by S. meliloti 3Doh13 was sufficient for plant growth. The isolates selected in this work evidenced a beneficial effect on alfalfa growth, especially in shoot biomass, which is used for forage and shepherding. The nutrient solubilization capacities evidenced in the in vitro assays were presumably the characteristics that enabled such promoting effect; several other potential activities, such as production of phytohormones and antibiotics cannot be ruled out.

CONCLUSION The co-inoculation of alfalfa with the nitrogen-fixing bacteria S. meliloti B399 and phosphate-solubilizing strains of Pseudomonas or Bacillus caused an increase in the biometric parameters analyzed with regard to the plants inoculated with S. meliloti B399 alone or the uninoculated controls suggesting that these strains of Pseudomonas and Bacillus could be promising for the formulation of new inoculants and commercial exploitation.

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ACKNOWLEDGMENTS This research was supported by FONTAGRO [ONU], Secretaría de Ciencia y Técnica de la Universidad Nacional de Río Cuarto, PICTO-Agencia Nacional de Promoción Científica y Tecnológica and Consejo Nacional de Investigaciones Científicas y Técnicas [CONICET, Argentina].

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REFERENCES Al-Niemi, T.S.; Kahn, M.L.; Mc Dermott, T.R. [1997]. P metabolism in the bean-Rhizobium tropici simbiosis. Plant Physiol, 113, 1233-1242. Andrés, J.A.; Correa, N.S.; Rosas, S.B. [1998]. Alfalfa and soybean seed and root exudates treated with thiram inhibit the expression of rhizobia nodulation genes. Phyton – Int. J. Exp. Bot, 62, 47-53. Baker, W.H. and Thompson, T.L. [1992]. Determination of total nitrogen in plant samples by Kjeldahl. In: Plank, C.O. editor. Plant analysis reference procedures for the Southern Region of the United States. The University of Georgia, Series Bulletin 368, pp 13-16. Bashan, Y. and Holguin, G. [1997]. Azospirillum-plant relationships: environmental and physiological advances [1990-1996]. Can J Microbiol. 43, 103-121. Bolton, H.; Elliot, L.F.; Turco, R.F.; Kennedy, A.C. [1990]. Rhizoplane colonization of pea seedlings by Rhizobium leguminosarum and a deleterious root colonizing Pseudomonas spp. and effects on plant growth. Plant Soi,l 123, 121-124. Carley, H.E. and Watson, R.D. [1966]. A new gravimetric method for estimating root-surface areas. Soil Science, 102, 289-291. Chen, Y.P.; Rekha, P.D.; Arun, A.B.; Shen, F.T.; Lai, W.A.; Young, C.C. [2006]. Phosphate solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities. Appl Soil Ecol, 34: 33–41. CIAT Manual [1988] Simbiosis Leguminosa-Rizobio, Manual de Métodos de Evaluación, Selección y Manejo. Programa especial CIAT [Centro Internacional de Agricultura Tropical], Cali, Colombia. Dashti, N.; Zhang, F.; Hynes, R.; Smith, D.L. [1998]. Plant growth promoting rhizobacteria accelerate nodulation and increase nitrogen fixation activity by field grown soybean [Glycine max [L.] Merr.] under short season conditions. Plant Soil 200, 205–213. De-Bashan, L.E. and Bashan, Y. [2004]. Recent advances in removing phosphorus from wastewater and its future use as fertilizar [1997-2003]. Water Res, 38, 4222-4246. Fernández., L.A.; Zalba, P.; Gómez, M.A.; Sagardoy, M.A. [2007]. Phosphate-solubilization activity of bacterial strains in soil and their effect on soybean growth under greenhouse conditions. Biol. Fertil. Soils, 43, 805-809. Gaind, S. and Gaur, A.C. [1991]. Thermotolerant phosphate solubilizing microorganisms and their interaction with mung bean. Plant Soil 133, 141-149. Glick, B.R. [1995]. The enhancement of plant growth by free-living bacteria. Can J Microbiol, 41, 109–117. Gyaneshwar, P.; Naresh Kumar, G.; Parekh, J.L.; Poole, P.S. [2002]. Role of soils microorganisms in improving P nutrition in plants. Plant Soil, 245:83-93.

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320

Lorena B. Guiñazú, Javier A. Andrés, Nicolás A. Pastor et al.

Hardy, R.; Holsten, R.; Jackson, E.; Burns, R. [1968]. The Acetylene-Ethylene Assay for N2 Fixation.Laboratory and field evaluation. Plant Physiol, 43, 1183. Illmer, P. and Schinner, F. [1992]. Solubilization of inorganic phosphates by microorganisms isolated from forest soil. Soil Biol. Biochem, 24, 389–95. InfoStat [2000], Statistical Software. Universidad Nacional de Córdoba [UNC], Argentina. Khan, M.S.; Zaidi, A.; Wani, P.A. [2007]. Role of phosphate-solubilizing microorganisms in a sustainable agriculture – A review. Agron Sustain Dev, 27, 29-43. Kloepper, J.W. and Schroth, M.N. [1978]. Plant growth-promoting rhizobacteria on radishes. In: Proceedings of the IVth International Conference on Plant Pathogenic Bacteria, Vol. 2, Station de Phatologie Végétale et Phytobactériologie, INRA, Angers, France, pp. 879– 882. Knight, T.J. and Langston-Unkefer, P.J. [1988]. Enhancement of symbiotic dinitrogen fixation by a toxinreleasing plan pathogen. Science, 241, 951-954. Kouas, S.; Labidi, N.; Debez, A.; Abdelly, C. [2005]. Effect of P on nodule formation and N fixation in bean. Agron Sustain Dev, 25,389-393. Koch, A.L. [1981]. Growth measurement. In: Gerhardt, P., Murray, R.G.E., Costilow, R.N., Nester, E.W., Wood, W.A., Krieg, N.R., Phillips, G.B. editors. Manual of Methods for General Bacteriology. American Society for Microbiology, Washington, pp. 179–207. Muslera Pardo, E. and Ratera García, C. [1984]. La alfalfa. Ed. Mundi-Prensa, Madrid, España. En: Praderas y forrajes pp 625-694. Raj, J.; Bagyaraj, D.J.; Manjunath, A. [1981]. Influence of soil inoculation with vesiculararbuscular mycorrhiza and a phosphate dissolving bacterium on plant growth and 32P uptake. Soil Biol. Biochem, 13, 105–108. Remans, R.; Croonenborghs, A.; Torrez Gutiérrez R.; Michiels, J. ; Vanderleyden, J. [2007]. Effects of plant growth-promoting rhizobacteria on nodulation of Phaseolus vulgaris L. are dependent on plant P nutrition. Eur. J. Plant Pathol. 119, 341-351. Richardson, A.E. [2001]. Prospects for using soil microorganisms to improve the acquisition of phosphorus by plants. Aust J Plant Physiol, 28, 8797-8906. Rodríguez, H. and Fraga, R. [1999]. Phosphate-solubilizing bacteria and their role in plant growth promotion. Biotech. Adv, 17, 319–339. Rosas, S.B.; Andrés, J.A.; Rovera, M.; Correa, N.S. [2006]. Phosphate-solubilizing Pseudomonas putida can influence the rhizobia–legume symbiosis. Soil Biol. Biochem., 38, 3502-3505. Roughley, R.J. [1970]. The preparation and use of legume seed inoculants. Plant Soil 32, 675-701. Saber, M.S.M.; Yousry, M.; Kabesh, M.O. [1977]. Effect of manganese application on the activity of phosphate-dissolving bacteria in calcareous soil cultived with pea plants. Plant Soi,l 47: 335-339. Schulze, J.; Temple, G.; Temple, S.J.; Beschow, H.; Vance, C.P. [2006]. Nitrogen fixation by white lupin under phosphorus deficiency. Ann. Bot, 98, 731-740. Sindhu, S.S.; Gupta, S.K.; Dadarwal, K.R. [1999]. Antagonistic effect of Pseudomonas spp. on pathogenic fungi and enhancement of growth of green gram [Vigna radiata]. Biol Fertil Soils 29, 62-68. Sindhu, S.S.; Gupta, S.K.; Suneja, S.; Dadarwal, K.R. [2002]. Enhancement of greengram nodulation and growth by Bacillus species. Biol. Plantarum, 45, 117-120.

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321

Singh, S. and Kapoor, K.K. [1994]. Solubilization of insoluble phosphates by bacteria isolated from different sources. Environ Ecol, 12, 51–55. Subba Rao, N.S. [1993]. Biofertilizers in Agriculture and Forestry, Oxford and IBH Publishing Co. Pvt. Ltd. New Delhi. Suslow, T.V.; Schroth, M.N.; Isaka, M. [1982]. Application of a rapid method for Gram differentiation of plant pathogenic and saprophytic bacteria without staining. Phytopathol, 72, 917–918. Urioste, A.M.; Bono, A.A.; Buschiazzo, D.E.; Hevia, G.G.; Hepper, E.N. [1996]. Fracciones de fósforo en suelos agrícolas y pastoriles de la región semiárida pampeana central [Argentina]. Ciencia del Suelo 14: 92-95. Vázquez, P. [1996]. Bacterias solubilizadoras de fosfatos inorgánicos asociadas a la rhizosfera de los mangles: Avicennia germinans [L.] L y Laguncularia racemosa [L.] Gerth. Tesis para el título de Biólogo Marino. Univ. Autónoma de Baja California Sur. La Paz, B.C.S. Vincent, J.M. [1970]. A manual for the practical study of root nodule bacteria. International Biology Programme Handbook Nº 15. Blackwell Scientific Publications, Oxford, U.K. Wani, P.A.; Khan, M.S.; Zaidi, A. [2007]. Synergistic effects of the inoculation with nitrogen-fixing and phosphate -solubilizing rhizobacteria on the performance of fieldgrown chickpea. J. Plant Nutr Soil Sci, 170, 283-287. Young, J.P.W.; Downer, H.L.; Eardly, B.D. [1991]. Phylogeny of the phototrophic Rhizobium strain BTAi1 by polymerase chain reaction-based sequencing of a 16S rRNA gene segment. J. Microbiol. 173, 2271–2277. Zaidi, A. and Khan, M.S. [2006]. Co-inoculation effects of phosphate solubilizing microorganisms and Glomus fasciculatum on greengram-Bradyrhizobium symbiosis. Turk. J. Agric. For, 30, 223-230. Zinniel, D.K.; Lambrecht, P.; Harris, N.B.; Feng, Z.; Kuczmarski, D.; Higley, P.; Ishimaru, C.A.; Arunakumari, A.; Barletta, R.; Vidaver, A. [2002]. Isolation and Characterization of Endophytic Colonizing Bacteria from Agronomic Crops and Prairie Plants. Appl. Environ. Microbiol., 68, 2198–2208.

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In: Phosphate Solubilising Microbes for Crop Improvement ISBN 978-1-60876-112-8 Editor: M. S. Khan and A. Zaidi © 2009 Nova Science Publishers, Inc.

Chapter 15

MICROBIAL FACILITATION OF PHOSPHORUS NUTRITION IN SUGARCANE B. Sundara∗ Agronomy Section, Division of Crop Production, Sugarcane Breeding Institute, Coimbatore-641007, Tamil Nadu, India.

ABSTRACT

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Sugarcane is the chief source of sugar in the world. Phosphorus [P] is the second most important nutrient required for sugarcane growth and yield of cane and sugar. Availability of P, applied as well as native, to the crop is restricted due to fixation and in most soils; it is not more than 15% of the applied dosage. Besides, phosphatic fertilizers are costlier thereby limits the application rates which in turn adversely affect cane production. Hence, there is a strong need to improve P availability and to economize the P application rate without affecting cane yield or juice quality. Research effort to utilize microbes to improve P availability to sugarcane has been limited though a good number of successful applications for other crops are reported. Therefore, a series of field investigations were undertaken at Sugarcane Breeding Institute, Coimbatore, India to use microbes to facilitate P nutrition to sugarcane. Field application of phosphorussolubilizing bacteria [PSB], Bacillus megaterium var. phosphaticum, improved soil available P, plant P level and increased cane and sugar yield by 12.5 and 15%, respectively. A few strains of the N2-fixing bacterium, Glucoacetobacter diazotrophicus also showed P-solubilzing effect, besides fixing N. Inoculation of arbuscular mycorrhiza also showed great promise to improve P nutrition in sugarcane. Combined application of PSB and AM-fungi had greater beneficial effects. With PSB, rock phosphate application was found feasible for sugarcane. The microbes, particularly, PSB and AM-fungi, were more effective at lower fertilizer P application rates and a reduction in the range of 25 to 50 % in the P application rate was found possible. The PSB and AM-fungi applications are gaining momentum in sugarcane agriculture in India as a tool to facilitate and economize P nutrition.

∗

Correspondence to: [email protected]

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15.1. INTRODUCTION Sugarcane [Saccharum officinarum L] is the most important sugar crop of the world contributing around 70 % to the world sugar output, remaining coming from sugar beet. Presently, it is recognized as a multi-utility agro-industrial crop used for the production of sugar, fuel, fiber, power, feed and a number of high-value by-products and many downstream chemicals [Singh and Solomon, 1995]. As a result, ‘sugar complexes’ or ‘sugarcane processing complexes’ are being established in place of stand-alone sugar manufacturing plants. It is a major commercial crop of the world grown in about 105 countries spread over tropical and subtropical regions over an area of 21.98 million hectares with a production of 1557.66 million tonnes of cane at an average productivity of 70.87 tonnes per hectare [FAOSTAT, 2009]. Brazil, India, China, Pakistan, Thailand, Mexico, Columbia, South Africa Australia, the Philippines, Indonesia, Cuba, United States of America, Mauritius are some of the important cane growing countries. Economy of many countries like, Cuba, Mauritius, Fiji, depends largely upon sugarcane agriculture and sugarcane dependent industries. With a C4 photosynthetic pathway, it is the most efficient fixer of solar radiation [Alexander, 1973]. Besides being an important source of calories to human beings, it is also considered as an important renewable source of energy. The Brazilian sugarcane juice derived ethanol programme is well known through out the world. Many more countries are using sugarcane juice to produce ethanol directly. Traditionally, molasses, a by-product of sugar industry, has been an important raw material for ethanol [Singh and Solomon, 1995]. Sugarcane biomass is also considered as a feedstock for ethanol. Sugarcane bagasse as well as trash is extensively used as fuel for cogeneration of power. Press mud [filter cake] is used as manure and also for preparing high value compost. The filter cake and distillery effluents together are used to produce the nutrient rich bio-earth with the help of microbial cultures. Sugar is an important commodity of world trade and commerce. Brazil, Cuba, Mauritius, Thailand, Australia and South Africa are some of the chief exporters of cane sugar. Sugarcane agriculture is a source of livelihood to millions of small and marginal farmers across the continents of Asia, Africa and Latin America. Sugar industries around the world are mostly located in the rural settings and have contributed immensely to the rural prosperity. With such diverse use and economic importance, demand for sugarcane will keep increasing and costeffective productivity improvement will have to be given priority. Sugarcane field duration ranges from 10 to 24 months depending upon the cultivars and growing regions. Sugarcane produces huge amount of dry matter and hence removes large quantities of nutrients. Various estimates of uptake of nutrients are available. On an average, a 100 tonne crop per hectare removes around 200, 75 and 450 kg N, P2O5 and K2O respectively [Clements, 1978; Blackburn, 1984; Husz, 1979]. Among the major nutrients, after nitrogen, phosphorus is the second most important nutrient required for sugarcane production. But its availability to the crop from the native as well as applied sources is extremely limited. In most soils, appropriate management practices are therefore required to augment P supply. Microbial facilitation is an important option to help improve P availability and uptake.

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15.2. ROLE OF PHOSPHORUS IN SUGARCANE

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Phosphorus is a key nutrient required for higher and sustained productivity of sugar from sugarcane. Normal cane development depends greatly on the presence of phosphates in soluble, plant absorbable form in the soil. Phosphorus concentrates in plant parts where physiological activity is greatest. Phosphorus is necessary for the formation of proteins, cell division, metabolism and photosynthesis and thus indispensable for crop growth and yield. It is essential for root growth, tillering and, stalk formation and stalk growth [Humbert, 1968; Clements1980]. Thus, the important yield components, the stalk number and stalk weight, are influenced by phosphorus availability at appropriate stage and in required quantity. Phosphorus interacts with nitrogen and thus influences cane ripening. Deficiency of phosphorus affects root formation and root growth, reduces tillering and delays canopy closure, which facilitates greater weed infestation. Stalk elongation is affected; the stalks become slender and stunted, tapering rapidly towards the top. Due to reduced tillering, there are less of secondary and tertiary stalks. Leaves grow close and leaf color appears greenviolet. Adequate presence of phosphorus in cane juice, about 300-400 ppm, is necessary for proper clarification while processing [Humbert, 1968; Clements, 1978; Blackburn, 1984] Phosphatic fertilizers are expensive. In many countries they are either imported or manufactured using imported raw materials. Due to spiralling cost, there has been a tendency towards discontinuation of P fertilizer application by agrarian communities or inadequate dose of P is applied, particularly in India, one of the largest producers of sugarcane [Sundara and Natarajan, 1997]. This may lead to a decrease in sugarcane productivity as had happened in the past in some sugar factories of tropical India where phosphorus application had been withdrawn for some years [Sundara, 1985].

15.3. PHOSPHORUS AVAILABILITY Most soils contain substantial reserves of total P; most of which remains relatively inert, and only less than 10% of soil P enters the plant-animal cycle [Kucey et.al.1989] Consequently, P deficiency is widespread and P fertilizers are almost universally required to maintain crop production. Although the P present in P fertilizers is initially plant available, it rapidly reacts with the soil constituents, forms a complex [fixation] and becomes progressively less available for plant uptake. The reaction products vary depending upon the soil type, soil environment and the kind of fertilizer applied. In acid soils, the reaction products are aluminum and iron phosphates while in the calcareous soils, the reaction products are calcium phosphates. The products obtained with calcium and magnesium phosphates are dicalcium and dimagnesium phosphates and members of variscite [AlPO4.2H2O], barrandite [AlFe] [PO4.2H2O] and strengite [FePO4.2H2O] isomorphous series. Das and Datta [1967as quoted by Raychaudhury, 1976] reported the formation of breeshite [CaHPO4.2H2O] and moletite [CaHPO4] in black and brown soils when monoammonium phosphate and monocalcium phosphate were added to the soil. In acid soils, is formed with these fertilizers. NH4-taranakite variscite [AlPO4.2H2O] [H6NH4AL5PO4]8.18H2O is formed when soil is treated with monoammonium phosphate. Other reaction products identified are octocalcium phosphate, calcium aluminium phosphate,

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and colloidal ferric aluminium phosphate [Fe, Al, X]PO4.NH2O with monocalcium phosphate. As a result, most of the applied P [often as much as 90%] is rendered unavailable for crop uptake but is retained in insoluble form. In sugarcane, recovery of the applied P is around 10-15% [Humbert, 1968]. Continuous application of phosphate fertilizers for a long period of time renders accumulation of P in insoluble forms [Sharpley et. al. 2004]. Much of the P recovered through the crop is not returned to the soil as most of it goes in the filter mud while processing. Therefore, annual applications are often necessary to maintain adequate labile P. Large reserves of fixed P present in the soil that could support long-term crop requirements, and, the applied P through P fertilizers, will have to be mobilized through appropriate soil management practices involving organic matter additions and/or use of microbes which can facilitate P nutrition either by solubilization of fixed P or aiding P uptake. Phosphorus availability is also affected by immobilization caused by microbes that take up the nutrients to perform their vital functions [Kungk et. al. 1993]. Another important constraint is the extremely limited mobility of the nutrient, applied as well as native, mainly because of fixation. Because of the low mobility of phosphorus in soils, plants only have access to phosphorus a few millimeters around their roots.

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15.4. IMPROVING P AVAILABILITY THROUGH MICROBES Improvement in P availability to crops through microbial means was first demonstrated in Russia. Several groups of microorganisms [fungi, bacteria, actinomycetes] are known to help dissolve fixed P [Subba Rao, 1982; Kucy et.al.1989; Ratti et al, 2001; Gyaneshwar et al, 2002; Deubel and Merbach, 2005]. During the 1950s, farmers in the erstwhile USSR and several east European countries inoculated a large proportion of their agricultural soils with a fertilizer consisting of kaolin impregnated with spores of the bacterium Megatherium viphosphateum. This bacterium was later named Bacillus megaterium var. Phosphaticum and the fertilizer was called phosphobacterin [Cooper, 1959; Menkina, 1963]. Crop yield increases resulting from the addition of B. megaterium to Soviet soils were reported to range from 0-70%. However, experiments in the United States did not show similar increases [Smith et al., 1961]. Experiments in India indicated positive responses to phosphobacterin application [Gaur, 1990; Marwaha, 1995]. Phosphobacterins in general have been found effective in solubilizing inorganic P in the soils. The solubilization effect is generally due to the production of organic acids such as citric, glutamic, succinic, lactic, oxalic, glyoxalic, maleic, fumeric, tartaric and ∝- ketobutyric acids as has been observed in the liquid medium [Banik and Dey, 1981; Geelhoed et. al.1999; Deubel et al, 2000]. The action of organic acids has been attributed to their chelation property. Whitelaw [2000] indicated the production of oxalate, lactate, glycollate, citrate, succinate and tartrate by different P-mobilizing fungi.

15.4.1. Microbes to Improve Phosphorus Nutrition in Sugarcane Reports regarding the use of microbes to improve phosphorus availability to sugarcane are limited. This author at Sugarcane Breeding Institute, Coimbatore, India, has pioneered detailed field studies on microbial means to improve phosphorus availability to sugarcane and

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this chapter largely presents important results obtained in these studies. The improvement of phosphorus nutrition to sugarcane was examined with phosphorus solubilizing bacteria [PSB], the nitrogen fixing bacteria having P solubilization effect besides N fixation, and employing arbuscular mycorrhizal [AM] fungi.

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15.4.2. Phosphorus Solubilizing Bacteria Yadav and Singh [1990] observed increase in germination per cent, tiller number, cane yield and P uptake by inoculating P solubilizer [Bacillus megaterium] with different doses of phosphatic fertilizers in Bihar, India on an alluvial soil. At Lucknow, subtropical India, Parihar et al. [2004] screened 12 endophytic bacteria isolated from sugarcane for phosphate solubilizing activity [PSA]. Nine of them showed PSA, which were further evaluated for their effect on sugarcane plant. Most of them significantly improved plant germination, physiological parameters and total phosphorus content of the plant. In a similar study, Sharma et al. [2003] investigated the influence of PSB, Pseudomonas striata, with press mud in calcareous soils. Basal application of 6 kg PSB with 4 tonnes of pressmud cake per hectare increased shoot population, stalk number and cane yield by 17.2, 5.9, and 12% respectively. Juice quality was not influenced either by sole application of different doses of PSB or combination of PSB and press mud cake [PMC]. Soil available P and total P in soil found to increase in 6 kg PSB applied with 4t PMC by 14.42 and 5.2%, respectively over control. In calcareous soils, 20 kg saving of P was reported. Kumaraswamy et al [1992] recorded an increase in cane yield by the application of PSB along with farmyard manure [FYM] in Tamil Nadu, India. Recently, increase in biological property of sugarcane Venezuela 51–71 variety, on the grower’s oasis substrate following combination of P solubilizing bacteria, Xanthomonas maltophilia and Enterobacter cloacae has been reported [Martinez and Martinez, 2007]. At Coimbatore, India, experiments were performed to assess the impact of phosphate solubilizing bacteria [Bacillus megaterium var. Phosphaticum] applied with various combinations of P fertilizer [Sundara et al., 2002] in order to [i] improve phosphorus availability to the crop [ii] study the bacterial dynamics in the soil [iii] evaluate the effects on sugarcane growth, juice quality and cane and sugar yields and [iv] examine the possibility of using rock phosphate [RP] as source of P to achieve P economy.

15.4.2.1. PSB and P Fertilizer Effects on PSB Population and Available P Status In the experiments with PSB application of 10 kg ha-1 of lignite based PSB biofertilizer [bacterial load of 108cfug-1] without any P fertilizer increased PSB population and soil available P status at different stages of the crop [Table 1]. However, when PSB was used in conjunction with P fertilizer, a much greater effect was observed. All the treatments involving P fertilizer + PSB improved the PSB population and the available P levels in the soil significantly over the sole application of P-fertilizer. The PSB populations in treatments involving PSB inoculations were maintained up to grand growth phase and then declined towards ripening phase. The PSB populations were not significantly influenced by the changes in the P fertilizer rates whether applied with PSB or otherwise. However, the available P levels declined significantly when P rates were reduced to 75 and 50% without PSB addition. But when PSB was supplied with P fertilizers there were no significant changes in the available P levels owing to reduction in the P rate to 75%; but further reduction to 50%

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caused significant decline even with PSB. Sheath P content at different growth stages increased by the applications of PSB, P fertilizers and P fertilizers along with PSB. Table 1. PSB population and available P status as influenced by P-Fertilizer and PSB treatments on sugarcane P-Fertilizer/ PSB treatment

PSB population [x 104g-1]

Soil available P [mg-kg]

Control

TP 20.41

GGP 22.32

RP 20.61

TP 4.4

GGP 4.8

RP 4.0

PSB

26.23

26.36

21.41

5.7

5.6

4.8

100%P

22.31

23.32

20.46

6.8

7.1

5.3

75%P

19.21

20.46

18.36

5.9

5.7

4.9

50%P

19.01

20.21

17.90

5.1

4.9

4.1

100%P+PSB

28.10

29.36

24.62

7.5

7.7

6.2

75%+PSB

30.46

29.02

24.51

7.2

7.0

5.9

50%+PSB

27.60

28.03

23.10

6.5

6.8

4.6

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TP, GGP and RP indicates tillering, grand growth and ripening phase, respectively.

15.4.2.2. PSB and P Fertilizer Application Effects on Cane and Sugar Yields Applications of PSB improved stalk number; stalk weight and cane yield [Table 2]. The recommended P [applied as SSP] rates increased the stalk population, stalk weight and cane yield over control and when PSB was used alone. Addition of PSB to the recommended P rate however, did not further influence the yield and its components. When the P rate was reduced to 75 or 50% without the addition of PSB, significant reductions in the stalk populations, stalk weight and cane yield occurred. But when the P rate was reduced to 75% and applied with PSB, no such reductions were noticed. However, further reduction in the rate to a level of 50% with the addition of PSB reduced the yield. The application of PSB improved the juice brix, sucrose content and purity coefficient as evidenced by the higher commercial cane sugar percent [CCS %]. The CCS [%] was better in treatments where P was applied entirely through SSP or P fertilizers + PSB at 100 % or 75% of the dosage [Table 2]. The mean cane and sugar yields [over six experimental crops, 3 plant + 3 ratoons] of the treatments involving PSB was 121 and 15.58 t ha-1 respectively, in contrast to 107.5 and 13.53 t ha-1 observed for treatments without PSB. Thus the mean increases in cane and sugar yield by PSB application were 12.56 and 15.15%, respectively. This was possibly due to increased PSB activity in the rhizosphere following PSB inoculation and consequent enhanced P solubilization as evidenced by the higher levels of plant available P in the soil at appropriate crop growth stages. Furthermore, the crop utilized more soil P which was evident by the increased levels of tissue [sheath] P observed at various stages of crop growth. P uptake is highly correlated with cane yield [Sundara, 1994]. In a similar study, Kucey et al [1989] have reported increased levels of available P in the soil following inoculation with PS organisms and increase in plant available P and its enhanced uptake by the crop following PSB additions have shown to improve tillering, stalk number and stalk growth leading eventually to higher cane yields.

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Table 2. Effect of PSB and P fertilizer treatments on stalk number, cane yield and commercial cane sugar per cent P-Fertilizer/PSB treatment Control PSB 100%P 75% P 50%P 100%P+PSB 75%P+PSB 50%+PSB

Stalk number 000’/ha 102.3 106.6 113.3 112.7 104.1 120.4 118.7 110.3

Cane yield [t/ha] 101.4 112.1 125.5 113.3 103.6 126.2 126.3 114.6

CCS [%] 12.46 12.63 13.02 12.54 12.51 12.76 12.87 12.56

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Improvement in juice sucrose and purity were significant by PSB additions thus leading to higher CCS [%] and sugar yields. Increased availability of phosphorus facilitates better nitrogen utilization [Humbert, 1968] and thus better crop growth, besides improving juice purity [Clements, 1980]. PSB application enabled the rate of P fertilizer to be reduced by 25 %. This is apparent from the similar cane and sugar yields in those treatments where the recommended rate of P was applied and in treatments where 25% of the rate was reduced but PSB was supplied.

15.4.2.3. Use of Rock Phosphate with PSB Rock phosphate or Phosphate rock is the main raw material used in the manufacture of phosphate fertilizers. However, direct use of rock phosphate for various crops has been practiced for over hundred years [Khasawneh and Doll, 1978]. Finely powdered rock phosphate has been found to be more effective for direct use as a phosphate fertilizer. In general, it is used in acid soils, as in southern China where sugarcane-growing soils are acidic and rock phosphate is extensively applied to supply P. However, being cheap source of P, its use in sugarcane grown in normal soils is possible either with organic manures and/or with Psolubilizing microbes. The long growth duration of sugarcane further facilitates its use. In field studies, rock phosphate [7.86% P] was applied in conjunction with PSB to substitute 50 % of the costly super phosphate. Rock phosphate application with PSB enhanced PSB population and available P levels in the soil. The P uptake by the crop was improved as indicated by increased sheath P level. Substitution of SSP with RP by 50% at different P rates caused significant reduction in cane yield as compared to the corresponding P rates supplied entirely through SSP. But no such reduction was noticed when RP was used along with PSB [Table 3]. The application of RP with PSB in general demonstrated higher CCS [%] and consequently improved sugar yield. The rock phosphate application had residual effect and showed improvement in juice quality.

15.4.3. Phosphate Solubilizing Potential of Nitrogen Fixing Bacterium For sugarcane, Azospirillum spp. is commonly applied as N2 fixing bio-fertilizers and Bacillus spp. as P-solubilizing biofertilizers. It would be more useful if bio-agents with multiple functions like, N2 fixation, P solubilization, and production of phytohormones and

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bio-control properties are identified and used in agricultural practices. This would help to develop cost effective and easy to use bioinoculants. Nitrogen fixing bacterial species such as, Enterobacter cloacae, Bacillus polymyxa, Klebsiella pneumoniae, Azotobacter vinelandii and Azospirillum spp. are commonly isolated from different internal and external parts of sugarcane plants. Recently, association of new bacterium, Gluconacetobacter diazotrophicus [], formerly known as Acetobacter diazotrophicus, with sugarcane is reported [Cavalcante and Dobereiner, 1988] and has been found to fix considerable amount of N2 [Boddey et al., 1991; Urquiaga et al., 1989]. This bacterium has a number of very interesting characteristics such as [i] N2 fixation [ii] tolerance to high acidic pH [iii] ability to grow in high sugar concentration and [iv] production of growth promoting substances. During its growth, it produces high amounts of organic acids which in turn can bring about solubilization of insoluble and fixed phosphates. As an example, Hari [2003] isolated G. diazotrophicus from roots and aerial parts of sugarcane and evaluated their potential for N2 fixation and P solubilization in liquid medium containing tricalcium phosphate [TCP] and Mussorie rock phosphate [MRP]. The P solubilization of G. diazotrophicus was compared with the phosphobacteria, Bacillus megaterium and standard G diazotrophicus. The results indicated that all the G. diazotrophicus strains solubilized the TCP and MRP more effectively than the B. megaterium. All the G. diazotrophicus strains produced organic acids, which reduced the pH of the medium from 6.5 to 3 in seven days while B. megaterium reduced the pH only to a lesser extent during the same period. The results thus indicated that G. diazotrophicus apart from many desirable characteristics could also perform solubilization of insoluble P.

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Table 3. Rock phosphate and PSB effects on sugarcane yield and commercial cane sugar per cent Treatment

Cane yield [t/ha]

CCS [%]

Control [100% P as SSP]

125.5

13.02

100% P[ 50% SSP+50% RP]

106.4

12.56

75% P[50% SSP+50% RP]

101.2

12.47

100% P[50%SSP+50% RP]+PSB

127.1

13.20

75% P [50%SSP+50% RP]+PSB

125.3

13.30

SSP and RP represent single super phosphate and rock phosphate respectively.

15.4.4. Dual Inoculation Efforts have been made to get the benefits of dual inoculation of N-fixing and Psolubilizing bacteria so that N and P fertilizer economy could be realized. Studies in Maharashtra, India, indicated improvements in germination per cent, cane girth, cane height, millable cane number, greater juice purity and higher cane yield with Azotobacter + PSB + 100% NP treatment over individual inoculations [Shinde and Bangar, 2003]. At Coimbatore, combined application of Azospirillum and PSB with 50 or 75% NP and, Gluconacetobacter and PSB with 50 or 75% NP yielded cane and sugar yields at par with the yields obtained at

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100% NP application [Figure.1]. These combinations indicated the possibility of saving N and P fertilizers in the rage of 25 to 50 % [Sundara et al, 2004].

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Figure 1.

15.4.4.1. Arbuscular Mycorrhiza Arbuscular mycorrhizal fungi form mutualistic symbioses with a vast majority of land plants [Smith and Read, 1997; Brundrett, 2002; Allen et al., 2003]. Mycorrhizal fungi are directly involved in the supply of P to plants. Mycorrhizal fungi transport soluble P to the roots but remove plant carbohydrates. It is well known that most plants depend on mycorrhizal activity for their nutrition and are dependent on them for growth. The interaction among mycorrhizal fungi and P-solubilizing microorganisms is direct, as these fungi need soluble P to absorb and transport to the root. In soils with low P content, any decrease in the P solubilizing activity will diminish the P available for mycorrhizal activity. Phosphate acquisition via mycorrhizal pathway begins with the uptake of free phosphates from soil by fungal extra-radical hyphae [Bucher, 2007]. These fungal hyphae extend beyond the host root system, allowing a greater soil volume to be exploited for phosphate uptake. Fungal highaffinity phosphate transporter mediate the uptake at the soil hypha interface [Harrison and Buuren, 1995]. Following the fungal uptake, phosphate is transferred to the fungal vacuole where it is polymerized to form polyphosphate chains and translocated through the vacuolar compartment to the intraradical hyphae. The polyphosphate is then hydrolyzed and phosphate released to the interfacial apoplast. From the interfacial apoplast, plant mycorrhizal transporters guide the phosphate across the periarbuscular membrane. Once in the plant cytosol, phosphate is translocated into the vasculature for delivery to all parts of the plant. In sugarcane, AM fungi are linked with both negative and positive growth responses depending on P supply. For example, a glass house study in Australia with Glomus clarum resulted in 53% increase in yield by the addition of four AM-fungal spores g-1 at 8 mg P kg-1 application

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over those without AM-fungi [Kelly et al 2005]. Root colonization was reduced at nil or 74 mg P kg-1. The AM colonization improved P content in the top visible dewlap [TVD]. Sugarcane is able to achieve maximum yield with lower levels of P in the soil when AM were present suggesting AM does indeed aid P uptake in low P soils. High soil P levels appeared to decrease AM colonization of sugarcane roots [Magarey et al, 2005]. In a pot culture study, Reddy et al [2004] compared four species of mycorrhizal fungi namely, Glomus fasciculatum, G. mosseae, Gigaspora margarita and Acaulospora laevis. Of these, G. margarita and A. laevis were found more efficient in improving plant biomass. Radhika et al. [2005] compared the performance of in vitro and in situ systems for AM- fungi colonization and growth, yield and P economy of sugarcane cv. COJ 86. Microscopic examination of co-cultivated sugarcane cultures confirmed successful colonization by AM- fungus [G. fasciculatum]. The colonization rate increased from 48% after 10 days to 78% after 30 days of co-cultivation. Root dry weight of in vitro AM infected sugarcane was 2.45g compared to 1.97 g for the control plants. These plants had better survival rate of 89.6% than the non-mycorrhizal plants. Field transfer of these plants showed 100% survival in ex vitro followed by 98.6% in vitro non-AM infected plants. Significant increases in percentage colonization, cane height, sugar content, and yield were recorded in AM infected [both in vitro and ex vitro] plants even at the two P levels representing half and three fourth of the recommended rate [30 kg/ha]. The total plant biomass and cane yield at half P level in AM infected plants were significantly higher than three fourth P levels of non-AM infected plants. At Coimbatore, India, Sundara and Hari [2004] conducted series of field experiments to assess the efficacy of PSB, Gluconacetobacter and AM applications to sugarcane individually and in combination. The microbial treatments were given at 0 and 50% of the recommended P application rate [75kg P2O5/ha] on plant and ratoon crops in two soil types [alfisol and vertisol] with separate controls [0, 50 and 100% P without microbes]. A total of 6 experimental crops were raised. Overall results indicated a three-fold increase in the PSB and mycorrhiza population at 120 days of planting compared to the population at planting. However, the population build-up at later stages was marginal. All the treatments comprising of PSB, AM-fungi or Gluconacetobacter recorded higher population of the respective microorganism as compared to uninoculated control. Highest population of PSB and AM-fungi was recorded in triple inoculation treatments [PSB + Gluconacetobacter + AM-fungus ] at 50% or zero P application. Generally, microbial pairing resulted in higher PSB and mycorrhizal population than any single biofertilizer application. Application of the microbial cultures increased available P status in the soil. Among the microbes used, AM-fungi consistently gave higher cane yields both at zero and 50% P application. Combined applications of microbes did not show any additional benefit though there was an indication of PSB and AM-fungi exhibiting some synergistic effect in one of the experimental crops on vertisol. With the microbial application, reducing the P application rate to 50% of the recommended rate did not affect the cane yield or juice quality thereby suggesting considerable saving of to P.

CONCLUSION The present stage of work on improving P nutrition of sugarcane employing microbes suggest that phosphate solubilizing organisms together with arbuscular mycorrhizal fungi can

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be effective in enhancing P nutrition and hence, P fertilizer input in sugarcane cultivation could be reduced to an extent of 25 to 50% . However, more accurate investigations using diverse varieties, growing environments and efficient microbial species is required before the potential of naturally abundant yet functionally different microbes is realized.

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REFERENCES Allen, MF; Swenson, W; Querejeta, JI; Egerton-Warburton, LM; Treseder, KK. Ecology of mycorrhizae: a conceptual framework for complex interactions among plants and fungi, Ann. Rev. Phytopathol,[ 2003]. 41, 371-303. Alexander, AG. Sugarcane physiology, Elsavier, Amsterdam, [1973]. Bajpai, PD; Sundara Rao, WVB. Phosphate solubilizing bacteria, Soil Sci. Plant Nutr., [1971]. 17, 46-53. Banik, S; Dey, BK. Phosphate solubilizing microorganisms of a lateritic soil. 1 solubilization of inorganic phosphates and production of organic acids by microorganisms, isolated in sucrose calcium phosphate agar plates, Zentralbl Bakt Abt II, [1981]. 136, 478-486. Bardia, MC; Gaur, AC. Rock phosphate dissolution by bacteria, Ind. J. Microbiol, [1972]. 12, 269-271. Begum, IF; Priya, OS. A survey of VAM infection and phosphorus nutrition of Saccharum officinarum. J. Ecotoxicol. Environ. Monit, [2004], 14, 261-66. Blackburn, F. Sugarcane, Longman, London, [1984]. Boddey, RM; Urquiaga, S; Resi, VM; Dobereiner, J. Biological nitrogen fixation associated with sugarcane, Plant Soil, [1991]. 137, 111-117. Brundrett, MC. Coevolution of roots and mycorrhizas of land plants, New Phytol [2002], 154, 275-304. Bucher, M. Functional biology of plant phosphate uptake at root and mycorrhiza interfaces, New Phytol., [2007], 173, 11-26. Cavalcante, VA; Dobereiner, J. A new acid-tolerant nitrogen-fixing bacterium associated with sugarcane, Plant Soil [1988] 108, 23-31. Clements, HF. Sugarcane Crop logging and Crop Control: Principles and Practices. The University Press, Hawaii, Honolulu, 520 pp. [1980]. Cooper, R. Bacterial fertilizers in Soviet Union, Soils and Fertilizers, [1959], 22, 327-333. Duebel, A; Gransee, A ;Merbach, W. Transformation of organic rhizodepositions by rhizosphere bacteria and its influence on the availability of tertiary calcium phosphate, J. Plant Nutr. Soil Sci., [2000],163, 387-392. Deubel. A; Merbach, W. In fluence of microorganismson phosphorus bioavailability in soils. In: Buscot, F; Varma, A.[eds].Soil Biology. Vol 3, Springer-Verlag, Berlin Heielberg [ 2005], pp.177-191. FAOSTAT [2009]. Hari, K. Production of plant growth promoting substances and phosphate solubilization by gluconacetobacter diazotrophicus isolated from sugarcane varieties, J. Soil Biol. Ecol, [2003], 23, 10-16. Harrison, MJ; Buuren, M Lv, A phosphate transporter from the mycorrhizal fungus Glomus versiforme, Nature,[1995], 378, 626-629.

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B. Sundara

Humbert, RP. The Growing of Sugarcane, Elsevier, Amsterdam. 779 pp. [1968]. Husz, GS. Sugarcane cultivation and fertilization, Ruhr.Stickstoff, A.G.Bochum, Germany, [1972]. Gaur, AC. Phosphate solubilizing microorganisms as biofertilizers. Omega Scientific Publishers, New Delhi, 176 pp. [1990]. Gyaneshwar, P; Naresh Kumar, G; Parekh, LJ; Poole, PS. Role of Soil microorganisms in improving P nutrition of plants, Plant Soil, [2002], 245, 83-93. Kelly, RM; Edwards, DG; Thompson, JP; Magarey, RC. Growth response of sugarcane to mycorrhizal spore density and phosphorus rate. Australian Journal of Agricultural Research, [2005], 56, 1405-1413. Khasawneh, FE; Doll, EC. The use of phosphate rock for direct application to soils, Advances in Agronomy, [1978], 30, 159-206. Kucey, RMN; Janzen, HH; Leggett, ME. Microbially mediated increases in plant available phosphorus, Adv. Agron, [1989] 42, 199-225. Kumaraswamy, K; Guruswamy, N; Rajasekaran, N. Efficiency of phosphobacterium biofertilizer on the yield and quality of sugarcane, Indian Sugar, [1992], 41, 841-845. Magarey, RC; Bull, JI; Reghenzani, JR. The influence of vesicular arbuscular mycorrhizae [VAM] on sugarcane growth in the field, Proceedings of the 2005 Conference of the Australian Society of Sugar Cane Technologists’, Bandaberg, Queensland, Brisbane, Australia, 3-6May, [2005], p 82-290. Marwaha, BC. Biofertilizer--a supplementary source of plant nutrient, Fert. News, [1995], 40, 39-50. Martinez, M., Martinez, A. Effects of phosphate-solubilizing bacteria during the rooting period of sugar cane [Saccharum officinarum], Venezuela 51–71 variety, on the grower’s oasis substrate. In: E. Velázquez and C. Rodríguez-Barrueco [eds]. First International Meeting on Microbial Phosphate Solubilization. Springer Netherlands, pp. 317-323. [2007]. Menkina, RA. Bacterial fertilizers and their importance for agricultural plants, Microbiologia, [1963], 33,352-358. Mishustin, EN; Naumova, AN. The use of bacterial fertilizers on sowing vegetable seeds into peat manure nutrient cubes, Microbiologia, [1956], 25, 41-48. Parihar, DK; Archana, S; Ramteke, PW. Phosphate solubilizing activity of endophytic bacteria isolated from sugarcane plant, Proc Natl Acad Sci India, Section B, Biol Sci, [2004], 74, 247-254. Pareek, RP; Gaur, AC. Release of phosphate from tricalcium phosphates by organic acids, Current Science, [1973], 42, 278-279. Radhika Guleri; Gupta, RP; Gosal, SK; Pander, MS; Gosal, SS. In vitro and in situ mycorrhization of micropropagated sugarcane plants and its effect on yield, Ind. J. Microbiol, [2005], 45, 71-73. Ratti, N; Kumar, S; Verma, HN; Gautam, SP. Improvement in bioavailabilityof tricalcium phosphate to Cymbopogon martini var.motia by rhizobacteria, Amf and Azospirillum inoculation, Microbiol. Re.s, [2001], 156,145-149. Raychaudhury, SP. Phosphate and potassic fertilizers and their management. In: Kanwar, JS [ed.] Soil Fertility—Theory and Practice. Indian Council of Agricultural research, New Delhi, pp.371-409, [1976].

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335

Reddy, CN; Bharati, BK; Rajkumar, HG; Sunanda, DN. Infectivity and efficacy of four native vesicular arbscular mycorrhiza fungi on sugarcane [cv. Co419], Mycorrhiza News, [2004], 16, 9-12. Sharma, BL; Singh, RR; Singh, SB. Integrated response of phosphorus solubilizing bacteria [PSB] with pressmud cake on sugarcane in calcareous soils, Co-operatve sugar , [2003], 35, 37-40. Sharpley, AN; McDowell, RW; Kleinman, PJ. .Amounts, forms, and solubility of phosphorus in soils receiving manure, Soil Sci. Soc. Am J, [2004], 68, 2048-2057. Shinde, DB; Bangar, ND. Studies on dual inoculation of nitrogenous and phosphatic bacterial cultures in sugarcane, J. Maharashtra Agric. Univ, [2003], 28, 190-192. Singh, GB; Solomon, S. Sugarcane-Agro-industrial Alternatives, Oxford and IBH, New Delhi, [1995]. Smith, JH; Allison, FE; Soulides, DA. Evaluation of phosphobacterin as soil inoculants, Soil Sci. Soc. of America Proc, [1961], 25, 109-111. Smith, SE; Read, DJ. Mycorrhizal Symbiosis, 2nd edn. London, Academic, [1997]. Subba Rao, NS. Advances in agricultural microbiology. Butterworth Scientific, London, [1982]. Sundara, B. Effect of levels of phosphorus and potassium and their late application on sugarcane. Indian J. Agron. [1985], 30, 124-127. Sundara, B. Phosphorus efficiency of sugarcane varieties in a tropical alfisol. Fertilizer Research, [1994], 39, 83-88. Sundara, B. Sugarcane Cultivation. Vikas Publishing House, New Delhi, [1998]. Sundara, B; Hari, K. Microbially aided phosphorus nutrition of sugarcane. Annual Report, Sugarcane Breeding Institute, Coimbatore, 2003-2004. p 35. Sundara, B; Natarajan, V. Effect of source and time of phosphorus application with and without phosphorus solubilizing bacteria on sugarcane, Proc. Ann Conv Sugar Technol. Assoc. of India, Sept. 26-28, [1997], Goa, 13-20. Sundara, B; Natarajan,V; Hari, K. Influence of phosphorus solubilizing bacteria on the changes in soil available phosphorus and sugarcane and sugar yields. Field Crops Research, [2002], 77, 43-49. Sundara, B ; Hari, K ; Sivaraman, K ; Rajendra Prasad, N. Evaluation and refinement of low cost technology packages for sugarcane. Annual Report, Sugarcane Breeding Institute, Coimbatore 2003-2004 p 37-38. Sundara Rao, WVB; Bajpai, PD; Sharma, JP; Subbaiah, BV. Solubilization of phosphorus solubilizing organisms using P as tracer and the influence of seed bacterization on the uptake by the crop, J. Ind Soc. Soil Sci, [1963], 11, 209-219. Sundara, Rao, WVB; Sinha, MK. Phosphate dissolving organisms in the soil and the rhizosphere, Ind J. Agric. Sci, [1963], 33, 272-278. Urquiaga, S; Botteon, PBL; Boddey, RM. Selection of sugarcane cultivars for associated biological nitrogen fixation using 15N-labelled soil. In. Skinner, FA., Boddey, RM; Fendrik, I. [ed.] Nitrogen Fixation with non- legumes. Kluwer Academic Publishers, Dordrecht, The Netherlands p 311-319. [1989]. Yadav, K; Singh, Tripurari. Effect of Bacillus megaterium on the solubilization of phosphatic fertilizers influencing yield and uptake by sugarcane, Bharatiya Sugar, [1990], 15, 15-23.

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In: Phosphate Solubilising Microbes for Crop Improvement ISBN 978-1-60876-112-8 Editor: M. S. Khan and A. Zaidi © 2009 Nova Science Publishers, Inc.

Chapter 16

PHOSPHATE SOLUBILIZING MICROORGANISMS FOR AUGMENTING CROP NUTRITION Parvaze Ahmad Wani∗ and Geeta Singh Division of Microbiology, Indian Agricultural Research Institute, New Delhi-12; India

ABSTRACT

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Phosphorus is one of the major plant growth-limiting nutrients although it is abundant in soils in both inorganic and organic forms. Phosphorus plays a very important role in several physiological and biochemical activities. Phosphate solubilizing microorganisms are ubiquitous in soils and play an important role in supplying phosphorus to plants in a more environmentally friendly and sustainable manner. Although solubilization of phosphatic compounds by microbes is very common under laboratory conditions, results in the field have been highly variable. Strains of Pseudomonas, Bacillus and Rhizobium are very important phosphate solubilizing bacterial isolates whereas Aspergillus, Penecillum are very important phosphate solubilizing fungi. The principal mechanism for phosphate solubilization is the production of organic acids. Several genes encoding phosphate solubiizing activity have been identified and cloned. The phosphate solubilizing microorganisms have also been characterised for their plant growth promoting and biocontrol activities. The plant growth promoting activities are indole acetic acid, siderophore, hydrogen cyanide, ammonia and auxin production. These activities have been shown to enhance the growth of plants. When such phosphate solubilizers are inoculated to both legume and non legume plants may increase the growth and yield of crops. Understanding the genetic basis of phosphate solubilization could help in transforming more rhizosphere-competent bacteria into PSMs. Further research should also focus on the microbial solubilization of iron [Fe] and aluminum [Al] phosphates, as well as mobilization of the organic phosphate reserves present in the soils.

∗

Corresponding author: [email protected]

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16.1. INTRODUCTION

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Phosphorous [P] is a critical element in natural and agricultural ecosystems throughout the world. This macronutrient element is a key component of cellular compounds and is vital to both plant and animal life [Nyle and Ray, 1999]. Phosphorus plays a significant role in several physiological and biochemical plant activities like photosynthesis, transformation of sugar to starch, and transporting the genetic traits. Moreover, phosphorus causes early ripening in pants, decreasing grain moisture, improving crop quality and is the most sensitive nutrient to soil pH [Malakooti, 2000]. Phosphorus in soils is immobilized or becomes less soluble either by absorption, chemical precipitation, or both. The mobility of this element is very slow [10-15%] in the soil and can not respond to its rapid uptake by plants. This leads to develop a phosphorus depleted zones near the contact area of roots and soil in rhizosphere. Consequently, farmers are advised to apply several times more phosphorus than is required by the plants [Nyle and Ray, 1999]. However, excessive application of phosphorous causes environmental and economic problems. Therefore, the plants consistently need an assisting system which could extend beyond the depletion zones and help to absorb the phosphorus from a wider area by developing an extended root system. Microorganisms are known to solubilize insoluble phosphate through the production of organic acids [e.g., oxalic, citric, butyric, malonic, lactic, succinic, malic, gluconic, acetic, glyconic, fumaric, adipic, and 2ketogluconic acid] and chelating oxo acids from sugars [Asea et al., 1988]. Among the naturally abundant microbes, fungi and bacteria are known as effective organisms for phosphate solubilization [Reyes et al., 1999]. Among the soil bacterial communities, Pseudomonas spp. and Bacillus spp. whereas, Aspergillus and Penicillium among fungi have been recognized as the most important phosphate solubilizers.

16.2. WHY BIOFERTILIZERS ARE USED FOR PLANT P NUTRITION? Theoretical estimates have suggested that the accumulated P in agricultural soils is sufficient to sustain maximum crop yields worldwide for about 100 years [Goldstein et al., 1993]. Phosphate solubilizing microorganisms [PSMs] can help in increasing the availability of accumulated phosphates for plant growth by solubilization of complex or insoluble phosphate [Goldstein, 1986; Kucey et al., 1989; Richardson, 1994; Subba Rao, 1982]. In addition, the phosphate solubilizing micro-organisms involved can enhance plant growth by increasing the efficiency of biological nitrogen fixation, enhancing the availability of other trace elements, such as Fe, zinc, etc. and by production of plant growth promoting substances [Kucey et al., 1989]. Most of the impact with the use of micro-organisms as biofertilizers has been directed towards understanding the biological nitrogen fixation that occurs in the symbiotic system of legume and bacteria of the rhizobiaceae family. In contrast, the fundamental work on microbial P solubilization has been substantially less, though it is known that P is the most limiting factor for N2 fixation by Rhizobium-legume symbiosis. Therefore, to avoid environmental hazards caused by the use of agrochemicals and to provide an inexpensive source of P to plants through the application of natural resource [PSMs], attention is paid to exploit the maximum use of PSM in different agro-ecosystems around the world.

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16.3. ROLE OF PHOSPHATE IN BIOLOGICAL NITROGEN FIXATION Major research has been done on the plant growth promoting rhizobacteria that concentrated on understanding and improving nitrogen fixation which involves the symbiosis between legume and rhizobia resulting in the formation of a specialized structures called nodule, in which the bacterial symbiont fixes N in return for reduced carbon [C] from the plant host [Denarie et al., 1996; Long, 1996; Mylona et al., 1995]. However, it is known that the P availability limits the formation of N2 fixing nodule [MacDermott, 1999]. The positive response to phosphate has been shown by the legumes like, alfalfa [Medicago Sativa] [AlNiemi et al., 1997; Deng et al., 1998], clover [Trifolium pratense], common bean [Phaseolus vulgaris], cow pea [Vigna sinensis] [Cassman et al., 1981] and pigeon pea [Cajanus cajans] [Itoh, 1987]. The phosphate is also the limiting factor for the nitrogen fixing aquatic legumes like, Sesbania rostrata [Ventura and Ladha, 1997], which is an important constituent of the green-manure technology for rice [Oryza sativa] cultivation [Ladha et al., 1992]. The phosphate solubilization by Rhizobium has been reported by only a few researchers [Chabot et al., 1993, 1996, Halder et al., 1991, 1992, Ahmad et al., 2008]. For example, it has been shown that the phosphate-solubilizing Rhizobium leguminosarum increased the growth of maize [Zea mays] and lettuce [Lactuca sativa] when they were inoculated to the plant [Chabot et al., 1996]. The phosphate solubilization by Rhizobium has been attributed to 2-ketogluconic acid, which indicates that the reduction of the pH was responsible for the phosphate solubilizing activity of the bacterium.

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16.4. PHOSPHATE SOLUBILIZERS AND THEIR NATURE The microbial association with the plant has been shown to facilitate the plant growth under P-deficient conditions. It can result either in better uptake of the available P, or making the unavailable phosphate-sources available to the plant. The arbuscular mycorrhizae [AM] belong to the former group and the later category includes various bacteria and fungi capable of solubilizing insoluble mineral phosphate complexes [e.g., calcium phosphate complexes]. AM-fungi are known to be ubiquitous in nature and enhance the P nutrition to through the large surface area, and their high affinity for phosphate [Hayman, 1974, 1983; Mosse, 1980]. Organic acid production by AM-fungi can also solubilize the insoluble mineral phosphates [Lapeyrie, 1988]. It has further been suggested that AM-fungi could also affect the availability of Fe phosphates [Bolan et al., 1987; Cress et al., 1984], but so far no alternative to the original mechanism of Sanders and Tinker [1973] has been accepted. Production of organic acids by AM-fungi though affect the availability of acid-labile insoluble phosphate, but the whole issue of mycorrhiza mediated increase in available P requires extensive assessment. However, ectomycorrhizal fungi for example have been shown to possess P solubilizing activity [Lapeyrie et al., 1991]. They utilize P from inositol phosphates and possess phosphatase activity that mediate the release of P from soil organic matter [Antibus et al., 1991; Koide and Schreiner, 1992]. However, the use of AM-fungi as phosphate biofertilizer is dreadfully hindered due primarily to their inability to grow under in vitro environment. In addition, mycorrhizal infection also depends on the P status of the plant [Abbott et al., 1984]. And, it is reported that the AM- fungi are not able to colonize plant

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roots strongly under P sufficient conditions [Amijee et al., 1989; Koide and Schreiner, 1992]. In certain cases, the growth rates of plants were reduced by AM colonization in the presence of available P [Peng et al., 1993; Son and Smith, 1995].

16.5. MOLECULAR GENETICS AND PHOSPHATE SOLUBILIZATION Mineral phosphates in general are solubilized by organic acids, either by reduction in the pH, or chelating the cat-ions associated with P [Kucey et al., 1989]. Better understanding of the genetic basis of the secretion of organic acids is likely to pave the way for transferring the mps ability to various bacteria that are competent to colonize a particular rhizosphere [Figure 1]. The rhizosphere competence is a major determinant for the success of the inoculant, because in the rhizosphere, C sources are available to the microbes to produce enough organic acid. Additionally, the solubilized P can be better utilized by the plant before it gets precipitated again. Some genes involved in mps in different bacterial species have been isolated [Table 1]. In this context, molecular approaches have been used to understand the genetic basis of gluconic acid production in P-solubilizing bacteria. For example, Escherichia coli is known to possess a gene for apo glucose dehydrogenase [enzyme responsible for conversion of glucose to gluconate], but not for the essential cofactor pyrroloquinoline quinone [PQQ]. The genes involved in PQQ biosynthesis from P-solubilizing Erwinia herbicola were cloned by using its genomic DNA library to select E. coli transformants for a mps phenotype. Using this approach, two genes were obtained from E. herbicola, a gene encoding PQQ synthase [Goldstein and Liu, 1987] and a second gene that is a putative PQQ transporter [Liu et al., 1992].

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Table 1. Cloning of genes involved in mineral phosphate solubilization Microorganism Erwinia herbicola

Gene or Plasmid Mps

Pseudomonas cepacia

gabY

Enterobacter agglomerans Rahnella aquatilis

pKKY

Serratia marcescens

pKG3791

pK1M10

Features

Reference

Produces gluconic acid and solubilizes mineral P in E. coli HB101 Produces gluconic acid and solubilizes mineral P in E. coli JM109 Solubilizes P in E. coli JM109 Does not lower pH Solubilizes P and produces gluconic acid in E. coli DH5α Probably related to PQQ synthesis Produces gluconic acid and solubilizes mineral P

Goldstein and Liu [1987] Babu-Khan et al. [1995] Kim et al. [1997] Kim et al. [1998b]

Krishnaraj and Goldstein [2001]

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Figure 1. Development of genetically modified phosphate-solubilizing micro-organisms. In this figure RCB indicates rhizosphere competent bacteria; mps-mineral phosphate solubilisation; CS-citrate synthase; PEPC-Pep carboxylase; GDH-glucose dehydrogenase and PS-phosphate solubilizing [adapted from Gyaneshwar et al. 2002].

Another gene putatively involved in PQQ transport was similarly cloned [Babu-khan et al., 1995]. This approach led to the isolation of two genes similar to the pqqD and pqqE genes from P-solubilizing Rahnella aquatilis [Kim et al., 1998]. Genes conferring mps phenotype have been cloned in E. coli from a non-P solubilizing Synechocystis PCC 6803. The fact that mps trait can be linked to particular genes can aid in generating novel PSMs by incorporating the mps genes in rhizosphere competent bacteria like, Rhizobium and Pseudomonas. Additionally, a high-affinity P transporter from mycorrhizal fungi, Glomus vermiformis, has been cloned and its expression was found to be localized to the external hyphae of the fungus. Over expression of these high affinity transporters could allow the development of better P scavenging AM-fungal strains. Genetic engineering thus, could help not only in increasing the survival of the inoculant strain by incorporating the ability to utilize certain nutrients better than the rest of the microbial population but also to produce microbes with greater efficiency to solubilise insoluble P. For example, genes for utilization of salicylate were transferred to a growth promoting bacteria, and the recombinant bacterium was able to survive and enhance plant growth better than the wild type.

16.6. FUNCTIONAL DIVERSITY AMONG PHOSPHATE-SOL UBILING ORGANISMS Plant growth promoting rhizobacteria [PGPR] including PSMs can be found in the rhizosphere, at root surfaces and in association with roots, which can improve the extent or quality of plant growth directly and/or indirectly. The direct promotion by PGPR entails

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either providing the plant with plant growth promoting substances that are synthesized by the bacterium or facilitating the uptake of certain plant nutrients from the environment. The indirect promotion of plant growth occurs when PGPR prevent deleterious effects of one or more phytopathogenic microorganisms. The mechanisms by which PSM facilitate plant growth may include the production of plant growth regulators like, indoleacetic acid, gibberellic acid, cytokinins and ethylene [ Wani et al. 2007], asymbiotic and symbiotic N2 fixation [Halder et al. 1990; Ahmad et al. 2008], displaying antagonism against phytopathogenic microorganisms by producing siderophores or antibiotics and cyanide [Wani et al. 2008b) Sakthivel and Gnanamanickam 1987]. Among the phosphate solubilizers, Pseudomonas, Bacillus and Alcaligens piechaudii have shown to synthesize IAA at lower levels [Barazani and Friedman, 1999 Rajkumar et al., 2006]. Of these, most popular bacteria studied and exploited as biocontrol agent includes the species of fluorescent Pseudomonas and Bacillus. Some of the growth promoting substances released by PSMs are briefly explained in Table 2. In addition to these traits, PSM strains must be rhizospheric competent, be able to survive and colonize the rhizosphere. Unfortunately, the interactions between interacting PGPR including PSM with plants have been found as extremely inconsistent. And hence, the results obtained in vitro cannot be reproduced under field environment. The variations in the performance of PSM has been suggested due to changes in the environmental factors including climate, weather conditions, soil characteristics or the composition or activity of the indigenous microbial flora of the soil. However, to achieve the optimum growth in plants using interactive communities of PGPR and plants, it is suggested to find solution as to how the rhizobacteria could provide maximum benefits to plant and how the effects of variable weathers both on functional properties of PSM and their interactions with plants could be reduced. Table 2. Growth promoting substances produced by phosphate-solubilizing microorganisms Organisms Azotobacter, Fluorescent Pseudomonas, and Bacillus Pseudomonas corrugate Bacillus spp.

Pseudomonas, Bacillus Aspergillus sp, Penicillium sp Pinus pinea, P. pinaster and Lactarius deliciosus Bacillus sp. Pseudomonas sp., Bacillus sp. Bacillus subtilis Pseudomonas fluorescens Pseudomonas sp.

Growth regulators IAA, Siderophore, Ammonia, HCN, P-solubilization P- solubilization P-solubilization, IAA, siderophores, ammonia production, HCN, chromium reduction, metal solubilization Siderophores, IAA, P-solubilization P-solubilization ACC, Auxin, siderophore and P- solubilization P-solubilization P- solubilisation, IAA, siderophore IAA and P-solubilization IAA, siderophore and P-solubilization IAA, siderophore and P-solubilization

Reference Ahmad et al., [2008] Trivedi et al., [2008] Wani et al. [2007 b, c]

Rajkumar et al., [2006] Pradhan and Shukla [2006] Barriuso et al., [2005] Canbolat et al., [2006] Rajkumar et al. [2006] Zaidi et al., [2006] Gupta et al., [2005] Gupta et al., [2002]

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16.6.1. Phosphate Solubilizing Microorganisms as Biological Control Soil borne pathogens are well known for their devastating effects on plant health and yield. For successful disease management, it is important to find the most effective and economical ways to protect the plant from various pests or diseases. In recent years, the use of PGPR including PSM as an inducer of systemic resistance in crop plants against different pathogens has been demonstrated under field conditions [Vidhayasekaran and Muthamilan, 1999; Khan et al. 2009]. And consequently, in recent years, management of plant disease using microbial preparation has emerged as an important and environmentally reliable means of controlling/reducing the plant pathogens. Phosphate solubilizers while acting as a biological control agent suppress the populations of pathogens by producing of metabolites. These metabolites may include siderophores that bind Fe making it less available to certain members of the native pathogenic microflora. Certain fluorescent Pseudomonas, particularly P. fluorescens and P. Putida were adapted to colonize plant roots which promoted plant growth under field conditions most likely through siderophore formation. In a study, Cattelan et al. [1999] reported that five of 22 soil bacterial isolates proved positive for solubilization of inorganic phosphates and also inhibited the growth of Sclerotium rolfsii and Sclerotinia sclerotiorum. Fungal growth inhibition was assessed by measuring the mycelial radial growth in plate assays on media that was either amended or unamended with 0.1 mM FeCl2. Of these five isolates, two significantly affected the soybean growth in a P-deficient soil amended with insoluble phosphate. A bacterial strain NJ-101 isolated from agricultural soil and identified as Pseudomonas sp. proved to release 74.6 mg/ml soluble phosphate from inorganic phosphate source, produced 11.4 μg/ml IAA, and siderophores on chrome azurol S [CAS] agar plates and inhibited the growth of Fusarium pathogens [Bano and Musarrat 2004]. In other study, Dey et al. [2004], investigated nine bacterial soil isolates and found that eight strains produced siderophores while five produced IAA, ammonia, and solubilized inorganic phosphate and inhibited soil borne fungal pathogens such as Sclerotium rolfsii. The performance of these rhizobacterial isolates was repeatedly evaluated for three years in pot and field trials. After bacterial inoculation, the content of phosphate in soil, shoot, and kernels significantly in three years trials in both rainy and post-rain seasons. Of these, three Pseudomonas fluorescens isolates demonstrated multiple properties and consistently enhanced growth yield and phosphate uptake of peanut under pothouse and field conditions, decreasing the incidence of pathogens. Earlier studies by the same research group demonstrated that fluorescent Pseudomonas sp. EM85 and Bacillus sp. MR- 11 [2] produced 0.108 and 0.092 mg catechol type of siderophores/mg protein and 3.84 and 3.71 μg IAA/ml, respectively [Pal et al., 2001]. Pseudomonas sp. EM85 solubilized tricalcium phosphate [14.13 mg/100 ml] by the production of gluconic, citric, succinic, and αketobutyric acid whereas, in the culture broth of Bacillus sp. MR-11 [2], gluconic, citric, tartaric, and α-ketobutyric acids were found and the amount of soluble phosphate reached 25.7 mg/100 ml. It was speculated that the phytohormone production by P-solubilizing microorganisms may contribute to their stimulatory effect on plant growth. Later studies have identified the exact indole substances that show auxin activity and quantified their amount produced in liquid media with tryptophan by P-solubilizing Pseudomonas, Bacillus, and Acinetobacter under different environmental parameters [Lehinos 1994]. Recently, Vassilev et al. [2006b] have demonstrated the capacity of living cells of the biocontrol bacterium Bacillus thuringiensis, entrapped in k-carrageenan, to solubilize insoluble inorganic

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phosphate and simultaneously produce IAA in a repeated-batch fermentation process. After five batch-fermentation cycles, an average concentration of 6.9 mg IAA/l was obtained in the presence of 1.5 g rock phosphate/l compared to 4.7 mg IAA/l in the control experiment without rock phosphate. The latter was solubilized with a maximum of soluble phosphate of 115 mg/l after the fourth batch cycle. The addition of tryptophan raised the production of auxin to 20.7 mg/l per batch but bacterial growth and rock phosphate solubilization were lower. When introduced into a soil–plant system, the same bacterial formulation was found to boost plant growth and P-uptake and stimulate the establishment and development of the endomycorrhizal fungus Glomus deserticola co-inoculated into the soil–plant system [Vassilev and Vassileva, 2004]. A similar inoculation scheme containing both B. thuringiensis and AM-fungi represents an attractive, less environmentally damaging alternative to chemical methods for phosphate fertilization and pathogen control. Among the fungal isolates, Trichoderma species are the most commonly studied biocontrol microorganisms, which also exhibit plant-growth-promoting activity [Harman and Bjorkman 1998]. In a study, Altomare et al. [1999] investigated the capability of the plantgrowth promotion and biocontrol potential of T. harzianum T-22 to solubilize in vitro insoluble minerals including rock phosphate [RP]. Organic acids were not detected in the culture filtrates which indicated that acidification was probably not the major mechanism of solubilization as the pH never fell below 5. The fungal-solubilizing activity was attributed both to chelation and to reduction processes, which also play a role in the biocontrol of plant pathogens. A similar biocontrol effect of P-solubilizing filamentous fungi against Fusarium wilt in tomato [Fusarium oxisporum f. sp. lycopersici; Fol] was shown by Khan and Khan [2001, 2002]. Root-dip applications of Bacillus subtilis, P. fluorescens, Aspergillus awamori, A. niger, and Penicillium digitatum caused a significant decline in the rhizosphere population of Fol. Tomato yield was enhanced, being greatest with A. awamori and P. digitatum. Direct soil-plant inoculation with A. niger, A. awamori, and P. digitatum decreased the rhizosphere Fol population by 23–49% while the tomato yield increased by 28–53% in field experiments. The authors propose that organic acids produced by these microorganisms may inhibit fungal infection but other metabolites such as bulbiformin and phenazin could also be involved, particularly in the treatments with B. subtilis and P. fluorescens as also reported by Dalla [1986]. Interestingly, an efficient biotechnological scheme for preparing a material with biocontrol and plant-growth-promoting activities was reported by Vassilev et al. [2005a]. Sugar beet wastes were mineralized by an acid-producing strain of A. niger with a simultaneous solubilization of RP under conditions of solid-state fermentation. The product of this process, used as soil amendment, resulted in 347 and 467% higher [vs unamended control] plant biomass in plant–soil experiments contaminated or not inoculated with Fol, respectively. Disease severity and number of Fol colony-forming units reached the lowest levels, particularly when plants were mycorrhized with G. deserticola. Moreover, the biocontrol activity of A. niger, was attributed partly due to siderophore production. Other PS fungus, Penicillium variabile P16 deserves special mention for its ability to solubilize inorganic phosphates when encapsulated in polysaccharide gels. This fungus P. variabile increased the glucose oxidase [GOD] production in the presence of polysaccharides of plant origin, which were found to serve as activators of defensive systems in this filamentous fungus [Petruccioli et al. 1999]. In fact, GOD is reported to play a significant role in antibiosis in the soil environment: the hydrogen peroxide enzymatically produced is cytotoxic for microorganisms.

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In studies on the biological control by soil microorganisms that participate in phosphate solubilization and plant growth promotion, mycorrhizal fungi merit special attention. AMfungi are accepted as an important part of biocontrol microorganisms [Johansson et al. 2004] as they strengthen resistance to certain wilt and root-rot pathogens by enhancing the nutritional status of the host plant and/or releasing unspecified substances. For this reason, AM-fungi are currently considered as biological-control microorganisms against soil borne diseases. The importance of such studies is validated by the fact that under natural conditions, nearly 90% of all plant species are estimated to form this type of symbiotic fungal infection. Greenhouse and growth-chamber experiments have demonstrated repeatedly that these root symbionts can benefit plants by providing one of the essential nutrients [primarily phosphate]. And hence, the improved phosphate uptake by the mycorrhizal plants is greatly emphasized. The main contribution of AM-fungi to the host is to reach and translocate phosphate through their extracortical hyphae, which can penetrate as much as 9 cm into the soil. In addition, numerous studies have demonstrated the ability of AM-fungi to solubilize otherwise insoluble phosphate sources. The effects of AM-fungi on pathogens are most likely to be indirect and include improved nutrition or altered physiology of the host. Many details are known about the physiological and biochemical changes in plants due to symbiosis [Smith and GianinazziPearson 1988]. Changes in the balance of phytohormones may determine AM effects on plant growth and health due to their regulatory functions. Another factor that determine the biocontrol activity of any organisms is the release of antibiotic by such microbes, antibiosis and competition cannot easily be separated. For instance, inoculation of antibiotic negative mutants in the rhizosphere did not affect the target organism as did the parent antibiotic producer; it is a good indication that antibiosis is a major factor. In various studies the production of antibiotics [e.g., pyrrolnitrin, phycocyanin, 2,4diacetyl phloroglucinol] by microbial inocula is demonstrated, which can cause suppression of pathogens [Glick, 1995; Thomashow and Weller, 1995]. Glick, [1995] however, very strongly believed that the most effective mechanism that a phosphate solubilizers can employ to prevent proliferation of phytopathogens is the synthesis of antibiotics.

16.7. PHOSPHATE SOLUBILIZERS AND THEIR ROLE IN CROP IMPROVEMENT 16.7.1. Inoculation Effects of Phosphate Solubilizers on Cereal Crops Several studies indicate that phosphate-solubilizers may act as natural elicitors for improving the growth and yield of wheat, rice and maize. For example, Javed and Arshad [1997] isolated 38 cultures of rhizobacteria from a rhizosphere soil and assessed their ability to produce IAA in vitro. Seeds of two wheat cultivars were inoculated with these bacterial strains and sown in the field under optimum fertilized conditions [NPK,-150, 75, 50 Kg ha-1]. Grain yields of wheat var. inqlab and LU-265 due to inoculation were increased by 5.3 and 18.5%, respectively, compared with the non-inoculated control. Inoculation with phosphatesolubilizers also significantly increased the number of tillers, straw weight and 1000-grain weight in both cultivars. Plant height was increased only in Lu-26S. In a similar study, Chen et al, [1994] isolated PGPR from the roots and rhizosphere of 57 crops and selected five

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strains for seed inoculation. Inoculation significantly promoted the yield of wheat with an average increase ranging from 6.3 to 15% compared to the non-inoculated control. Pseudomonas is another most promising and widely studied phosphate-solubilizer which has been reported to improve plant growth and yield of crops when used either alone or in combination with other PGPR. However,plant responses are often variable depending upon the bacterial strain, crop variety and experimental site. For example, Khaliq et al., [1996] conducted a field experiment to evaluate the potential of two isolates of Pseudomonas using wheat as a test crop under fertilized conditions. Bacterial inoculation promoted grain and straw yields, tillering, N concentration in grains and total N uptake upto 20, 14, 17, 41 and 65%, respectively, over the non-inoculated control. The effect of Azotobacter in improving the growth and yield of wheat has been observed in several studies. The Azotobacter inoculation in the presence of NPK [@ 150, 75 and 50 kg ha-1, respectively] significantly increased the grain yield [38.5%], straw yield [15.3%], number of tillers [12.5%], spikelets [10.7%] and 1000 grain weight [7.3%] of wheat in comparison to non-inoculated control [Zahir et al. 1996]. A similar increase in rice following Pseudomonas fluorescens inoculation is reported [Sakthivel et al. 1986]. For example, inoculation of Burkholderia vietnamiensis to rice cultivars in two pot and four field trials at different locations of Vietnam, showed an enhancement of 33, 57, 30 and 13% in shoot weight, root weight, leaf area and number of tillers/hill, respectively, compared to non-inoculated control plants. In other study, strain of Rhodobacter capsulatus significantly increased the plant dry weight, number of productive tillers, grain and straw yields of rice var. Giza 176, grown in pot amended with different levels of nitrogen fertilizer compared to non-inoculated plants [Elbadry et al., 1999a]. The results of this study further suggested that N fertilizer could be saved upto 50%. Similarly, a substantial increase in root weight [41%], straw yield [12%], grain yield [11.7-20%] and total biomass [18.7%] following PGPR inoculation over non-inoculated rice is reported [Mehnaz et al. 1998; Sherchand, 2000]. The inoculation of phosphate-solubilizing Pseudomonas to maize, resulted in an increase of 18.9% in grain yield, while it enhanced the cob weight, mass of 1000 grain and straw weight significantly by 20.8, 11.6, 17.2 and 27.1%, respectively, relative to non-inoculated plants [Javed et al., 1998]. In other experiment, seeds inoculated with Azotobacter and Pseudomonas in combination with NPK [@150, 100 and 100 kg ha-1 respectively] significantly increased grain yield [19.8%], cob weight [21.3%], 100 grain weight [9.6%], plant height [8.5%] and N content in both grain and straw [19.8 and 18% respectively], compared to non-inoculated plants [Zahir et al., 1998a].

16.7.2. Interaction of Phosphate Solubilizers with Legume Crops Phosphorus is a major plant nutrient and phosphate-solubilizing microorganisms can benefit the plants either used alone or in combination with other plant growth promoting rhizobacteria [Wani et al., 2007, Khan and Zaidi, 2007]. The plants inoculated with phosphate-solubilizing microorganisms though have shown sufficient growth enhancement and increased P contents, varied considerably in their effectiveness in different agroecosystems [Khan et al. 2007]. For example, a significant increase in mungbean yield was observed with the inoculation of Rhizobium spp. and PS bacteria [Khan et al., 1997, 1998].

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Table 3. Co-inoculation of legumes with phosphate- solubilizing and nitrogen fixing microorganisms Crop

Rhizobia

Alfalfa

R. meliloti

Co-inoculating PS solubilizers Pseudomonas

Chickpea

Mesorhizobium

Pseudomonas

Mesorhizobium

Bacillus

Rhizobium

Pseudomonas, Bacillus Pseudomonas sp.

Clover Common bean Greengram

R. leguminosarum bv.trifolii 24 Rhizobium

A. brasilense

Soybean

Bradyrhizobium sp. [Vigna] B. japonicum

Bacillus P. fluorescens

White clover

R. leguminosarum bv. trifolii

Azospirillum lipoferum

Plant responses to inoculation

References

Significant increase in plant growth, nitrogenase activity, nodule number, total nodule weight and total plant nitrogen Significant increase in nodule weight and shoot biomass when coinoculated with Mesorhizobium and Pseudomonas in sterilized chillum jar conditions. In pot experiments, co-inoculation significantly increased root and shoot biomass. Co-inoculation significantly increased plant dry weight, nodulation, N content, protein content and seed yield, compared to single inoculation. Significantly increased nodule weight, root and shoot biomass and total plant nitrogen Co-inoculation significantly increased shoot and nodule weight in comparison to plants inoculated with R. leguminosarum bv. trifolii. Co-inoculation promoted root hair formation and an increase in secretion of the nod gene induced flavonoids resulting in greater number of nodules. Co-inoculation enhanced nodulation and growth of greengram

Knight and LangstonUnkefer [1988] Sindhu et al. [2002a]

Co-inoculation increased colonization of B. japonicum on soybean roots, nodule number and the acetylene reduction activity [ARA] Enhanced the number of nodules and ARA

Chebotar et al. [2001]

Wani et al. [2007e] Parmar and Dadarwal [1999] Derylo and Skorupska [1993] Burdman et al. [1996]

Sindhu et al. [2002b]

Tchebotar et al., [1998]

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Parvaze Ahmad Wani and Geeta Singh

Moreover, the microbes that are involved in P solubilization as well as better scavenging of soluble P can enhance plant growth by improving the efficiency of BNF, accelerating the availability of other trace elements and by production of phytohormones and antimicrobial compounds. Accordingly, increase in yield of various legumes have been observed following seed or soil inoculation with N2 fixing organisms and PSM [Zaidi and Khan, 2007] or PSM [s] and other PGPR [Mukherjee and Rai, 2000; Zaidi and Khan, 2007]. Similarly, about 37% increase in the grain yield of black gram was reported following the inoculation of Rhizobium and Bacillus megaterium [Prabakaran et al., 1996]. These studies also demonstrated an increase in P uptake by plants. The results of PSM-plant inoculations, however, may not conclusively show a direct role for the PSMs in supplementing soil phosphates for plants because [1] no increase has been found in about 70% of the experiments [2] the increase in crop yields were not compared with crop yields with addition of superphosphates [3] the plant growth promoting activity of PSMs, other than P solubilization, has not been determined and [4] enhancement in P uptake mediated by AM-fungi as a consequence of PSM inoculation was not considered in interpreting the results.

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16.7.3. Co-Inoculation of Phosphate Solubilizers and Nitrogen Fixers The mixed inoculation of phosphate-solubilizers and N2 fixers has received increasing attention in the recent years. Co-inoculation of N2 fixers and P-solubilizers may provide both N and P to plants and consequently improve the overall performance of crops in different agricultural soils. In addition, P-solubilizers are able to produce phytoalexin, antibiotics against pathogens, siderophores and efficiently colonize root surfaces, thereby out-competing pathogens [Parmar and Dadarwal, 1999]. Thus, dual inoculation of N2 fixers and Psolubilizers is likely to stimulate plant growth more profoundly than single inoculation of either organism. For example, Oliveira et al. [1997] tested the effect of three plant growth promoting rhizobial strains for N2 fixation and growth of clover plants under green house conditions and found that though the inoculation of Azospirillum brasilense strain Sp7 and a local rhizobia promoted nodulation and nitrogenase activity but the mixed inocula of N2 fixers and P-solubilizers further enhanced biological and chemical properties including symbiosis, nitrogenase activity, N content and grain yield. The Table 3 provides results observed for several legumes following co-inoculation with P-solubilizers and N2 in greenhouse and field experiments.

CONCLUSION Divergent populations of microbial communities inhabiting soils have enormous potential and are capable of providing essential nutrients including P and several other growth promoting substances to plants. In addition, they can also protect plants from the severe deleterious effects of soil pathogens and consequently improve the growth and yields of various crops. However, due to lack of sufficient information on the functional variations within microbial communities, inadequate understanding of the factors governing the microbial association with plants and the nature and nutritional diversity in exudates released

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by different plant genotypes into rhizosphere, has, to some extent, limited the use of beneficial microbes in agricultural practices. A better and sound understanding of such microbes is likely to reduce the dependence on chemical fertilizers besides their role in maintaining the fertility status of soil.

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REFERENCES Abbott, LK; Robson, AD and De Bore, G [1984]. The effect of phosphorus on the formation of hyphae in soil by the vesicular-arbuscular mycorrhizal fungus Glomus fasciculatum. New Phytol, 97, 437–446. Ahmad, F; Ahmad, I and Khan, MS [2008]. Screening of free–living rhizospheric bacteria for their multiple plant growth promoting activities. Microbiol. Res, 163, 173-181. Al-Niemi, TS; Kahn, ML and McDermott, TR [1997]. P metabolism in the Rhizobium tropici-bean symbiosis. Plant Physiol, 113, 1233– 1242. Amijee, F; Tinker, PB and Stribley, DP [1989]. Effects of phosphorus on the morphology of vesicular-arbuscular mycorrhizal root system of leek [Allium porrum L]. Plant Soil, 119, 334–336. Antibus, RK; Sinsabaugh, RL and Linkins, AE [1991]. Phosphatase activities and phosphorus uptake from inositiol phosphates by ectomycorrhizal fungi. Can. J. Bot, 70, 794–801. Altomare, C; Norvell, WA; Bjorkman, T; Harman, GE [1999]. Solubilization of phosphates and micronutrients by the plant growth promoting and biocontrol fungus Trichoderma harzianum rifai 1295-22. Appl. Environ Microbiol, 65,2926–2933. Asea, PEA; Kucey, RMN and Stewart, JWB [1988]. Inorganic phosphate solubilization by two Penicillium species in solution culture and soil. Soil Biol. Biochem, 20, 459-464. Babu-Khan, S; Yeo, C; Martin, WL; Duron, MR; Rogers, R and Goldstein, A [1995]. Cloning of a mineral phosphate-solubilizing gene from Pseudomonas cepacia. Appl. Environ. Microbiol, 61, 972–978. Bano, N; Musarrat, J [2004]. Characterization of a novel carbofuran degrading Pseudomonas sp. with collateral biocontrol and plant growth promoting potential. FEMS Microbiol Lett, 231, 13–17. Barriuso, J; Pereyra, J; Lucas García, MT; Megías, JA; Gutierrez Mañero, M; and Ramos, B [2005]. Screening for Putative PGPR to Improve Establishment of the Symbiosis Lactarius deliciosus-Pinus sp. Microbial Ecology. 50, 82-89. Bolan, NS; Robson, AD and Barrow, NI [1987]. Effect of vesicular arbuscular mycorrhiza on avalibility of iron phosphates to plants. Plant Soil, 99, 401–410. Burdman, S., Volpin, H., Kigel, J., Kapulnik, Y and Okon, Y. [1996]. Promotion of nod gene inducers and nodulation in common bean [Phaseolus vulgaris] root inoculated with Azospirillum brasilense. Appl. Environ. Microbiol. 62, 3030-3033. Barazani, OZ and Friedman, J [1999]. Is IAA the major root growth factor secreted from plant growth mediating bacteria. J. Chem. Ecol, 25, 2397-2407. Canbolat, MY; Bilen, S; Cakmakci, R; Sahin, F and Aydin, A [2006]. Effect of plant growth promoting bacteria and soil compaction on barley seedling growth, nutrient uptake, soil properties and rhizosphere microflora. Biol. Fertil. Soils, 42, 350-357.

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350

Parvaze Ahmad Wani and Geeta Singh

Cassman, KG; Whiteny, AS and Fox, RL [1981]. Phosphorus requirements of soybean and cow pea as affected bymode of N nutrition. Agron. J., 73, 17–22. Cattelan, AJ; Hartel, PG; Fuhrmann, JJ [1999]. Screening for plant growth promoting rhizobacteria to promote early soybean growth. Soil Sci. Soc. Am. J., 63, 1670–1680. Chabot, R; Antoun, H and Cecas, P [1993]. Stimulation de la croissancedu mais et de la laitue romaine par des microorganisms dissolvant le phosphore inorganique. Can. J. Microbiol, 39, 941–947. Chabot, R; Antoun, H and Cesas, MP [1996]. Growth promotion of maize and lettuce by phosphate solubilizing Rhizobium leguminosarum biovar phaseoli. Plant Soil, 184, 311– 321. Chen, Y; Mei, R; Lu, S; Liu, L; Kloepper, JW [1994]. The use of yield increasing bacteria as PGPR in Chinese agriculture. In: Gupta, UK and Utkhede, T [Eds.]. Management of soil borne diseases. Narosa Publishing House, New Delhi, India. Cress, WA; Johnson, GV and Barton, LL [1984]. The role of endomycorrhizal fungi in iron uptake by Hilaria jamesii. J. Plant Nutr, 40, 547–555. Dalla, GC [1986]. Esperienze di lotta biologicacontrola fusariosi vascolare del garofano [Trials on the biological control of vascular kilt of carnations]. Ann. Inst .Sper. Floric, 17, 3012 Deng, S; Kahn, ML and McDermott, TR [1998]. Characterization and transposon mutagenesis of a non-specific acid phosphatase cloned from Rhizobium meliloti. Arch. Microbiol, 170, 18–26. Derylo, M and Skorupska, A [1993]. Enhancement of symbiotic nitrogen fixation by vitaminsecreting fluorescent Pseudomonas. Plant Soil, 154, 211-217. Denarie, J; Debelle, F and Prome, JC [1996]. Rhizobium lipochitooligosaccharide nodulation factors: Signaling molecules mediating recognition and morphogenesis. Ann. Rev. Biochem., 65, 503–535. Dey, R; Pal, KK; Bhatt, DM; Chauhan, SM [2004]. Growth promotion and yield enhancement of peanut [Arachis hypogaea L.] by application of plant growth-promoting rhizobacteria. Microbiol. Res., 159, 371–394. Elbadry, M; Gamal-Eldin, H and Elbana, K [1999a]. Effects of Rhodobacter capsulatus inoculation in combination with graded levels of nitrogen fertilizer on growth and yield of rice in pots and lysimeter experiments. World. J. Microbiol. Biotechnol, 15, 393-395. Glick, BR; Karaturovic, DM and Newell, PC [1995]. A novel procedure for rapid isolation of plant growth promoting Pseudomonas. Can. J. Microbiol, 41, 533-536. Goldstein, AH; Rogers, RD and Mead, G [1993]. Mining by microbe. BioTechnol, 11, 1250– 1254. Golstein, AH [1986]. Bacterial phosphate solubilization: Historical perspective and future prospects. Am. J. Alt. Agric, 1, 57–65. Goldstein, AH and Liu, ST [1987]. Molecular cloning and regulation of a mineral phosphate solubilizing gene from Erwinia herbicola. Biotechnology, 5, 72–74. Gupta, DK.; Tohoyama, H; Joho, M and Inouhe, M [2002]. Possible roles of phytochelatins and glutathione metabolism in cadmium tolerance in chickpea roots. J. Plant Res, 115, 429-437. Gupta, A; Rai, V; Bagdwal, N and Goel, R [2005]. In situ characterization of mercury resistant growth promoting fluorescent Pseudomonads. Microbiol. Res, 160, 385-388.

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Phosphate Solubilizing Microorganisms for Augmenting Crop Nutrition

351

Gyaneshwar, P; Naresh Kumar PG; , Parekh, LJ; Poole, PS [2002]. Role of soil microorganisms in improving P nutrition of plants. Plant Soil, 45, 83-93. Halder, A. K. Mishra, A. K. Bhattacharyya, P. and Chakrabartty, P. K. [1990]. Solubilization of inorganic phosphate by Rhizobium and Bradyrhizobium. J Gen Appl Microbiol, 36, 8192. Halder, AK; Misra, AK and Chakrabarty, PK [1991]. Solubilization of inorganic phosphates by Bradyrhizobium. Indian J. Exp. Biol, 29, 28–31. Halder, AK; Banerjee, A; Misra, AK and Chakrabarty, PK [1992]. Role of ammonium and nitrate on release of soluble phosphate from hydroxyapatite by Rhizobium and Bradyrhizobium. J. Basic Microbiol, 32, 325–330. Harman, GE; Bjorkman, T [1998]. Potential and existing uses of Trichoderma and Gliocladium for plant disease control and plant growth enhancement. In: Harman, GE; Kubicek, CP [eds.]. Trichoderma and Gliocladium, [vol 2, pp. 229-265]. Taylor and Francis, London, UK. Hayman, DS [1974]. Plant growth responses to vesicular-arbuscular mycorrhiza VI. Effects of light and temperature. New Phytol., 73, 71–80. Hayman, DS [1983]. The physiology of vesicular-arbuscular endomycorrhizal symbiosis. Can. J. Bot, 61, 944–963. Itoh, HS [1987]. Characteristics of phosphorus uptake of chick pea in comparison with pigeon pea, soybean and maize. Soil Sci. Plant Nutr., 33, 417–422. Javed, M; Arshad, M [1997]. Growth promotion of two wheat cultivars by plant growth promoting rhizobacteria. Pak. J. Bot, 29, 243-248. Javed, M; Arshad, M; Ali, K [1998]. Evaluation of rhizobacteria for their growth promoting activity in maize. Pak. J. Soil Sci, 14, 36-42. Johansson, J; Paul, LR; Finlay, RD [2004]. Microbial interactions in the mycorrhizosphere and their significance for sustainable agriculture. FEMS Microbiol. Ecol. 1, 1–13. Khaliq, A; Arshad, M; Khalid, A and Zahir, ZA [1996]. Potential of Azotobacter and Pseudomonas for enhancing wheat [Triticum aestivum L.] yield. Paper presented at 7th international symposium on BNS with non-legumes, Oct. 16-21, 1996, Faisalabad, Pakistan [Abst.], p. 170. Khan, MR and Khan, SM [2001]. Biomanagement of Fusarium wilt of tomato by the soil application of certain phosphate-solubilizing microorganisms. Int. J. Pest Manag. 47, 227–231. Khan, MR and Khan, SM [2002]. Effect of root-dip treatment with certain phosphatesolubilizing microorganisms on the Fusarium wilt of tomato. Biores. Technol., 85, 213– 215. Khan, MS; Zaidi, A; Ahemad, M; Oves, M; Wani, PA. Plant growth promotion by phosphate solubilizing fungi–current perspective. Arch. Agron Soil Sci., [2009], DOI: 10.1080/03650340902806469. Kim, KY; Jordan, D and McDonald, GA [1997]. Solubilization of hydroxyapatite by Enterobacter agglomerans and cloned Escherichia coli in culture medium. Biol. Fert. Soils, 24, 347-352. Kim, KY; Jordan, D; McDonald, GA [1998]. Effect of phosphatesolubilizing bacteria and vesicular-arbuscular mycorrhizae on tomato growth and soil microbial activity. Biol. Fertil Soil, 26,79–87.

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352

Parvaze Ahmad Wani and Geeta Singh

Kim, KY; Jordan, D and Krishnan, HB [1998b]. Expression of genes from Rahnella aquatilis that are necessary for mineral phosphate solubilization in Escherichia coli. FEMS Microb. Lett, 159, 121–127. Knight, TJ and Langston-Unkefer, PJ [1988]. Enhancement of symbiotic nitrogen fixation by a toxin releasing plant pathogen. Science, 242, 951-954. Krishnaraj, PU and Goldstein, AH [2001]. Cloning of a Serratia marcescens DNA fragment that induces quinoprotein glucose dehydrogenase-mediated gluconic acid production in Escherichia coli in the presence of stationary phase Serratia marcescens. FEMS Microbiol. Lett, 205, 215–220. Khan, MS; Zaidi, A and Wani, PA [2007]. Role of phosphate solubilizing microorganisms in sustainable agriculture-A review. Agron. Sustain. Dev, 27, 29-43. Khan, MS and Zaidi, A [2007]. Synergicstic effects of the inoculation with plant growth promoting rhizobacteria and an arbuscular mycorrhizal fungus on the performance of wheat. Turk. J. Agric. For, 31, 355-362. Khan, MS; Aamil, M and Zaidi, A [1997]. Associative effect of Bradyrhizobium sp.[vigna] and phosphate solubilizing bacteria on moongbean [Vigna radiata[L.] wilczek]. Biojornal, 10, 101-106. Khan, MS; Aamil, M and Zaidi, A [1998]. Moongbean response to inoculation with nitrogen fixing and phosphate solubilizing bacteria. In: Deshmukh, AM [ed.]. Biofertilizers and Biopesticides, Technoscience Publications, Jaipur, pp.40-48. Koide, TR and Schreiner, PR [1992]. Regulation of vesicular arbuscular mycorrhizal symbiosis. Ann. Rev. Plant Physiol. Plant Mol. Biol, 43, 557–581. Kucey, RMN; Jenzen, HH and Leggett, ME [1989]. Microbially mediated increases in plant available phosphorus. Adv. Agron, 42, 199–228. Ladha, JK; Parrek, RP and Beker, M [1992]. Stem-nodulating legume Rhizobium symbiosis and its agronomic use in low land rice. Adv. Soil Sci, 20, 148–192. Lapeyrie, F [1988]. Oxalate synthesis from soil bicarbonate by fungus Paxillus involutus. Plant Soil, 110, 3–8. Lapeyrie, F; Rangers, J and Vairelles, D [1991]. Phosphate-solubilizing activity of ectomycorrhizal fungi in vitro. Can J. Bot, 69, 342– 346. Lehinos, V [1994]. Effects of pH and glucose on auxin production of phosphate-solubilizing rhizobacteria in vitro. Microbiol. Res., 149,135–138. Long, SR [1996]. Rhizobium symbiosis: Nod factors in perspective. Plant Cell, 8, 1885–1896. Liu, ST; Lee, LY; Taj, CY; Hung, CH; Chang, YS; Wolfrang, JH; Rogers, R and Goldstein, AH [1992]. Cloning of an Erwinia herbicola gene necessary for gluconic acid production and enhanced mineral phosphate solubilization in Escherichia coli HB101: nucleotide sequence and probable involvement in biosynthesis of the coenzyme Pyrroloquinoline Quinone. J. Bacteriol, 174, 5814–5819. Malakooti, MJ [2000]. Sustainable agriculture and yield increment by optimum fertilizer utilization in Iran. 2nd edition. Agricultural Extension Publications, Iran. Mehnaz, S; Mirza, MS; Hassan, U and Malik, K.A [1998]. Detection of inoculated plant growth promoting rhizobacteria in rhizosphere of rice. In: Malik, KA; Mirza, MS; Ladha, JK [Eds.] Nitrogen fixation with non-legumes. [pp. 75-83], Kluwer Acad. Pub, London, Great Britain.

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Phosphate Solubilizing Microorganisms for Augmenting Crop Nutrition

353

Moose, B [1980]. Vesicular-arbuscular mycorrhiza research for tropical agriculture. Research Bulletin 194, Hawaii Institute of Tropical Agriculture and Human Resources, Honolulu, HI, USA; University of Hawaii. Mylona, P; Pawlowskim K and Bisseling, T [1995]. Symbiotic Nitrogen fixation. Plant Cell, 7, 869–885. McDermott, TR [1999]. Phosphorus assimilation and regulation in rhizobia In: Triplett, EW [Eds.] Nitrogen Fixation in Prokaryotes: Molecular and Cellular Biology. Horizon Scientific Press USA. Mukherjee, PK and Rai, RK. [2000]. Effect of vesicular arbuscular mycorrhizae and phosphate solubilizing bacteria on growth, yield and phosphorus uptake by wheat [Triticum aestivum] and chickpea [Cicer arietinum]. Ind. J. Agron, 45, 602-607. Nyle, CB; Ray, RW [1999]. The nature and properties of soils, 12th edn. Upper Saddle River, NJ: Prentice Hall, pp 540–542. Oliveira, A; Ferreira, EM and Pampulha, ME [1997]. Nitrogen fixation and yield of clover plants co-inoculated with root-colonizing bacteria. Symbiosis, 23, 35-42. Pal, KK; Tilak, VBP; Saxena, AK; Dey, R; Singh, CS [2001]. Suppression of maize root diseases caused by Macrophomina phaseolina, Fusarium moniliforme and Fusarium graminearum by plant growth promoting rhizobacteria. Microbiol Res,156, 209–223. Parmar, N and Dadarwal, KR [1999]. Stimulation of nitrogen fixation and induction of flavonoid like compounds by rhizobacteria. J. Appl. Microbiol, 86, 36-44. Peng, S; Eissentat, DM; Graham, JH; Williams, K and Hodge, NC [1993]. Growth depressions in mycorrhizal citrus at high phosphorus supply: Analysis of carbon costs. Plant Physiol, 101, 1063–1071. Petruccioli M, Federici F, Bucke C, Keshavarz T [1999]. Enhancement of glucose oxidase production by Penicillium variabile P16. Enzyme Microb Technol 24:397–401. Prabakaran, J; Ravi, K.B and Srinivasan, K. [1996]. Response of Vamban-1 blackgram to N2 fixer and P mobilizers in acid soil. In: Microbiology Abstracts, XXXVII Annual Conference of the Microbiologists of India, IIT, Chennai, Dec, 4-6, p. 120. Pradhan, N and Sukla, LB [2006]. Solubilization of inorganic phosphates by fungi isolated from agriculture soil. Afr J. Biotechnol, 5, 850-854. Rajkumar, M; Nagendran, R; Kui JL; Wang, HL and Sung, ZK. [2006]. Influence of plant growth promoting bacteria and Cr [VI] on the growth of Indian mustard. Chemosphere, 62, 741-748. Reyes, I; Bernier, L; Simard, RR and Antoun, H [1999]. Effect of nitrogen source on the solubilization of different inorganic phosphates by an isolate of Penicillium rugulosum and two UV induced mutants. FEMS Micobiol. Ecol., 28, 281-290. Richardson, AE [1994]. Soil micro-organisms and phosphate availability. In: Eds. Pankhurst, CE; Doube, BM; Gupts, VVSR and Grace, PR [Eds.] Soil Biota Management in Sustainable Agriculture. [pp. 50–62], CSIRO, Melbourne, Australia. Sakthivel, N; Sivamani, E; Unnamlai, N and Gnanamanickam, SS [1986]. Plant growth promoting rhizobacteria in enhancing plant growth and suppressing plant pathogens. Cur. Sci, 55, 22-25. Sakthivel, N. and Gnanamanickam, S. S. [1987]. Evaluation of Pseudomonas fluorescens for suppression of sheath-rot disease and for enhancement of grain yields in rice [Oryza sativa L]. Appl. Environ. Microbiol., 53, 2056–2059.

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354

Parvaze Ahmad Wani and Geeta Singh

Sanders, FE and Tinker, PB [1973]. Phosphate flow into mycorrhizal roots. Pest. Sci, 4, 385– 395. Sherchand, K [2000]. Responses of effective microorganisms and other nutrients to rice and wheat under field conditions at Khumaltar. Nepal. Em. World J. 1, 40-44. Subba Rao, NS [1982]. In: Subba Rao, NS [Ed.] Advances in Agricultural Microbiology, [pp. 229–305], Oxford and IBH Publ. Co. Sindhu, SS; Suneja, S; Goel, AK; Parmar, N and Dadarwal, KR [2002a]. Plant growth promoting effects of Pseudomonas sp. on co-inoculation with Mesorhizobium sp. cicer strain under sterile and wilt sick soil conditions. Appl. Soil Ecol, 19, 117-120. Sindhu, SS; Gupta, SK.; Suneja, S and Dadarwal, KR [2002b]. Enhancement of greengram nodulation and growth by Bacillus sp. Biologia Plantarum, 45, 117-120. Smith, SE; Gianinazzi-Pearson, V [1988]. Physiological interactions between symbionts in vesicular-arbuscular mycorrhizal plants. Annu. Rev. Plant Physiol. Plant Mol. Biol, 39, 211–244. Son, CL and Smith, SE [1995]. Mycorrhizal growth responses: interaction between photon irradiance and phosphorus nutrition. New Phytol, 108, 305–314. Tchebotar, VK.; Kang, UG; Asis, CA. Jr. and Akao, S [1998]. The use of GUS-reporter gene to study the effect of Azospirillium-Rhizobium coinoculation on nodulation of white clover. Biol. Fertil. Soils, 27, 349-352. Thomashow, LS and Weller, DM [1995]. Concept in the use of introduced bacteria for biological disease control. In: Stacey, G; Keen, N [Eds.] Plant microbe interaction, [vol. 1, pp. 187-235], Chapman and Hall, New York, USA. Trivedi, P and Sa, T [2008]. Pseudomonas corrugata [NRRL B-30409] mutants increased phosphate solubilization, organic acid production, and plant growth at lower temperatures. Curr. Microbiol, 56,140–144. Vassilev, N and Vassileva, M [2004]. Multifunctional properties of a plant growth promoting bacterium entrapped in k-carrageenan. In: Pedraz, JL; Orive ,G; Poncelet, D [Eds.] XII international workshop on bio-encapsulation, [pp 162-166], Vitoria, Univ Pais Vasco, 24–26 September 2004. Vassilev, N; Nikolaeva, I and Vassileva, M [2005a]. Biocontrol properties of microbiallytreated sugar beet wastes in presence of rock phosphate. J Biotechnol, 118, S1:S177. Vassilev, N; Nikolaeva, I and Vassileva, M [2006b]. Phosphate ore solubilization and simultaneous indole-3-acetic acid production by a gel-entrapped bacterium in fermentation conditions. Chem. Eng. Commun. [in press]. Ventura, W and Ladha, JK [1997]. Phosphorus requirements when used as biofertilizers for long term rice cultivation. Soil Sci. Soc. Am. J, 61, 1240–1244. Vidhayasekaran, P and Muthamilan, M [1999]. Evaluation of powder formulation of Pseudomonas fluorescence Pf1 for control of rice sheath blight. Biocontrol Sci. Technol, 9, 67-74. Wani, PA; Khan, MS and Zaidi, A (2008b) Chromium reducing and plant growth promoting Mesorhizobium improves chickpea growth in chromium amended soil. Biotechnol. Lett, 30:159–163. Wani, PA; Khan, MS and Zaidi, A (2007c). Effect of metal tolerant plant growth promoting Rhizobium on the performance of pea grown in metal amended soil. Arch. Environ. Conatm. Toxicol. doi: 10.1007/00244-9097-y.

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Wani, PA; Khan, MS and Zaidi, A [2007e]. Synergistic effects of the inoculation with nitrogen fixing and phosphate-solubilizing rhizobacteria on the performance of field grown chickpea. J. Plant Nutr. Soil Sci, 170, 283–287. Zaidi, S; Usmani, S; Singh, BR and Musarrat, J [2006]. Significance of Bacillus subtilis strain SJ- 101 as a bio- inoculant for concurrent plant growth promotion and nickel accumulation in Brassica juncea. Chemosphere, 64, 991-997. Zaidi, A and Khan, MS [2007]. Stimulatory effects of dual inoculation with phosphate solubilizing microorganisms and arbuscular mycorrhizal fungus on chickpea. Aus. J. Expt. Agri. 47, 1016-1022. Zahir, ZA; Arshad, M and Hussain, A [1996]. Response of wheat [Triticum aestivum L.] to Azotobacter inoculation under fertilized condition. Sarhad J. Agri, 7, 133-138. Zahir, ZA; Akram, M; Arshad, M and Khalid, A [1988a]. Improving maize yield by inoculation with plant growth promoting rhizobacteria. Pak. J. Soil Sci, 15, 7-11.

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In: Phosphate Solubilising Microbes for Crop Improvement ISBN 978-1-60876-112-8 Editor: M. S. Khan and A. Zaidi © 2009 Nova Science Publishers, Inc.

Chapter 17

PHOSPHATE SOLUBILIZING MICROORGANISMS: PROSPECTS, PROMISES AND PROBLEMS Aftab Afzal∗ and Asghari Bano Department of Plant Sciences Quaid-I-Azam University Islamabad, Pakistan

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ABSTRACT Phosphorus [P] is an essential plant nutrient and forms an important constituent of macromolecules but is one of the most limiting mineral nutrients in the agricultural system. To increase the availability of P to plants, large amounts of chemical P fertilizer are applied to soil. A large proportion of fertilizer P after application, however, is quickly transformed into the insoluble form. Therefore, very little amount of the applied P is available for uptake by plants warranting continuous application of external P necessary. This chapter gives a brief account of P fixation phenomenon, reasons of P unavailability to plants, role of chemical fertilizers in P availability, current status of phosphatesolubilizing microbes, interaction of phosphate solubilizing bacteria with other plant growth promoting rhizobacteria for bioavailability of P, and crop improvement by phosphate-solubilizing microbes. Use of molecular techniques in relation to phosphatesolubilizing organisms is also reviewed and discussed. Economics of using phosphatesolubilizing microorganisms as inoculants and factors affecting the efficiency of such organisms especially in field conditions is also briefly discussed in this chapter.

17.1. IMPORTANCE OF PHOSPHORUS Phosphorus is an essential inorganic nutrient for all living organisms. It is required as a structural component in nucleic acids and phospholipids, as an element in intermediates in carbon metabolism, and a wide range of enzymes. After nitrogen [N], P is quantitatively the most important inorganic nutrient for plant growth, and often limits primary productivity in natural systems as well as cropping systems, unless supplied as fertilizer [Vance et al., 2003].

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The P is absorbed mainly during the vegetative growth and, thereafter, most of the absorbed P is re-translocated into various organs of plants including fruits and seeds. P-deficient plants exhibit retarded growth [reduced cell and leaf expansion, respiration and photosynthesis], and often a dark green color [higher chlorophyll concentration] and reddish coloration [enhanced anthocyanin formation]. It has been reported that the level of P supply during reproductive stages regulates the partitioning of photosynthates between the source leaves and the reproductive organs, an effect essential for N2-fixing legumes [Marschner, 1993]. This nutrient is absorbed by plants from the soil solution as monovalent [H2PO4] and divalent [HPO4] orthophosphate anions, each representing 50% of total P in solution at a nearly neutral pH [pH 6–7]. At pH 4–6, H2PO4 is about 100% while at pH 8, H2PO4 and HPO4 represents 20% and 80% of total P, respectively [Black, 1968].

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17.2. PHOSPHORUS FIXATION AND ITS UNAVAILABILITY TO PLANTS Phosphorus is a non-renewable resource and the global P reserves are consistently decreasing. It is generally believed that current P reserves are likely to be halved by 2040 or by 2060 [Steen, 1998]. Whilst global P reserves on one hand is depleting, P levels in many agricultural soils on the other hand is building up, because 80–90% of P applied as fertilizer is adsorbed by soil particles, rendering it unavailable for plants that lack specific adaptation to access adsorbed P [Gerke et al., 1994; Jones, 1998]. Of the different forms of P, organic P moves freely with soil water in much the same way as do the nitrate and hence, can be leached out. In contrast, inorganic P binds strongly to clays and oxide surfaces in acid soils, and is precipitated as relatively insoluble calcium phosphates in alkaline soils. These fixation processes maintain the P concentration at a low level in the soil solution. Although P content in an average soil is 0.05%, only 0.1% of the total P is available to the plants because of its fixation and low solubility. For instance, superphosphate applied to the alkaline calcareous soils gets converted into insoluble calcium phosphate [49-59%], iron and aluminum phosphate [14-19%], while water soluble fraction ranges between 5-9% only [Ahmad et al., 1992]. The plant tissues recover only 11-19% of the applied phosphorus [Sharif, 1985]. The average phosphorus fixation of the added phosphorus in clay, clay loam, loam, sandy loam and loamy sand soils is 71, 62, 56, 29 and 29%, respectively after one month of incubation [Chaudhry and Qureshi, 1982].

17.3. ROLE OF CHEMICAL FERTILIZERS IN P AVAILABILITY AND CURRENT STATUS OF P RESERVES Rock phosphate is a general term for rock that contains a high concentration of phosphate minerals, which commonly belong to the apatite group. Phosphate rock minerals are the only significant global resources of phosphorus (Table 1). The United States is the world's leading producer and consumer of phosphate rock, which is used to manufacture phosphate fertilizer

∗

Correspondence to: [email protected], [email protected]

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and industrial products. China, the United States and Morocco are the world's largest miners of phosphate rock, each producing about a quarter of the total world production. Table 1. World Rock Phosphate Resources Country

Reserve Base (In 000 tonnes)

Australia Brazil Canada China Egypt India Israel Jordon Morocco and Western Sahara Russia Senegal South Africa Syria Togo Tunisia USA Other Countries World Total

1200000 370000 200000 13000000 760000 160000 800000 1700000 21000000 1000000 160000 2500000 800000 60000 600000 4000000 2000000 50000000

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Source : Mineral Commodity Summaries, 2004.

Other countries with significant production include Brazil, Russia, Jordan and Tunisia. Phosphate rock is mined, beneficiated, and either solubilized to produce wet-process phosphoric acid, or smelted to produce elemental phosphoric acid or smelted to produce elemental phosphorus. Phosphoric acid is reacted with phosphate rock to produce the fertilizer triple superphosphate or with anhydrous ammonia to produce the ammonium phosphate fertilizers. Phosphorus from rock phosphate is also used in animal feed supplements, food preservatives, anti-corrosion agents, cosmetics, fungicides, ceramics, water treatment and metallurgy. Many kinds of rock contain mineral components containing phosphate or other phosphorus compounds in small amounts. However, RP are of limited solubility, so that the availability of phosphorus is limited as compared with other forms of phosphate. The two main sources for phosphate are guano, formed from bird droppings, and rocks containing concentrations of the calcium phosphate mineral, apatite. The total world reserves of rock phosphate and apatite are estimated at 47,000 million tonnes. The primary source of phosphate in fertilizers is the mineral apatite, which is primarily tri-calcium phosphate [Ca3 [PO4]2]. It is the major constituent of phosphate rock [RP], the basic raw material used in the production of phosphatic fertilizers. The phosphate containing rocks are found in special geological deposits and some phosphate containing iron ores or other P

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compounds. Depending upon the dominance of F, Cl or OH in the apatite crystal structure, it is known as fluorapatite, chlorapatite or hydroxyapatite, respectively. Weathering processes over long periods of time result in the accumulation of primary apatites or apatite-containing bones, teeth, etc. of animals of earlier geological periods. Many such deposits occur near the earth’s surface, from where they are obtained by opencast mining and utilized either directly or as fertilizer.

17.3.1. Selection of the Appropriate P Fertilizer

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The choice of P fertilizer for agronomic purposes depends on soil factors, climate conditions, crop characteristics, economics and secondary effects of fertilizers. Despite numerous comprehensive studies made worldwide, no universally applicable recommendations about the use of such fertilizers are available. However, some suggestions in this regard may help the agrarian communities who frequently apply chemical fertilizers for enhancing the productivity of crops around the word. For example, water soluble P [WSP] fertilizers have been found best for slightly acid to neutral and alkaline P-deficient soils, particularly for short-duration crops which require available phosphate immediately. However, a high degree of water solubility can be a disadvantage in soils with strong P sorption where phosphate ions are transformed rapidly into less available forms. Phosphate forms with only moderate water solubility provide the best results in moderate to slightly acid soils. Slow-acting RP requires sufficient amounts of soil acidity and biological activity for its conversion into easily available P forms. The special advantage associated with the use of RP is its lower cost and a lower solubility, which decreases the rate at which the P is adsorbed in soils rich in active Fe or Al compounds [FAO, 2006].

17.3.2. Phosphorus and Environmental Issue The P that can contribute to the enrichment of water bodies, and hence lead to eutrophication, is a combination of the P that is attached to soil particles less than 0.45 µm in size that are transported during soil movement. The risk of P losses to the environment through surface runoff is greatest on sloppy lands, and where the fertilizer is surface applied and then followed by rainfall or irrigation. Most governments have set limits on the concentration of P in waters. In the United States of America, the Environmental Protection Agency [EPA] has recommended a limit of 0.05 mg/l total P for controlling eutrophication in streams that enter lakes and 0.1 mg/l for total P in flowing streams. It has not been possible to prescribe safe P concentrations in runoff leaving a field because of the considerable P transfers that occur between the field and the waterway. Phosphate leaching is only a problem on soils that are well supplied or oversupplied with P, especially where they have inadequate capacity to immobilize P. Maintenance of good soil cover is the best protection against such losses. Subsurface leaching of P can take place where: [i] P is in soluble organic form, as in manure; [ii] the capacity of the soil for binding inorganic P has been exceeded and [iii] a preferential flow of water through channels and cracks in the soil prevents contact with the adsorption sites in the soil [Laegreid et al., 1999]. With good nutrient management, the phosphate losses to the environment can be kept low and within a tolerable range [FAO,

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2006a]. It is proposed that if phosphate [residual or applied] is made available to plants through its solubilization by PSMs, it is expected that less P would be available for leaching or surface runoff and ultimately reduce eutrophication.

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17.4. CURRENT STATUS OF PHOSPHATE SOLUBILIZING MICROBES Soil- plant- microbe interaction has received greater attention in recent times. A variety of microorganisms are known to inhabit soils and play important roles in plant growth and development. The concept of using soil microorganisms to improve mobilization of poorly available forms of soil P is however, not new. Phosphate solubilizing microorganisms have been used as biofertilizer since 1950’s [Kudashev 1956; Krasilnikov 1957] and since then are being applied as inoculants. For example, in the former Soviet Union a commercial biofertilizer named phosphobacterin was first prepared by using Bacillus megaterium var. phosphaticum adsorbed on kaolin and also widely used in East European countries and India. Later on, a carrier based preparation commonly called IARI microphos was developed by the Indian Agricultural Research Institute, New Delhi, India using efficient phosphate-dissolving strains of Pseudomonas striata, Bacillus polymyxa and Aspergillus awamori, A. niger and Penicillium digitatum [Gaur, 1983] packed in a wood charcoal and soil mixture. These cultures were tested in multilocal field trials and were found highly effective. In addition, phytate-mineralizing bacteria have shown diverse biotechnological applications [Igual et al., 2001; Konietzny and Greiner 2004]. And hence, the inoculation of plants by enzyme producing phosphobacteria has been shown to improve P uptake by plants [Rodriguez et al., 2006]. These bacterial effects occur in the rhizosphere, and because some phosphobacteria may render more P soluble than is required for their growth and metabolism, the surplus P is available for uptake by plants [Mehta and Nautiyal, 2001]. These bacteria have been isolated from varying ecological niches e.g., plain areas, high altitudes of Baltistan viz. Khaplu3200masl, Skardu-2400masl [Mateen 2008] and from Khewra salt mine [ Naz 2008], from deserts and even from volcanic soils [Jorquera, 2008]. Some of the potential phosphatesolubilizing microbes are listed in Table 2.

17.4.1. Mechanism of Action of PSM Several soil bacteria, particularly phosphobacteria, posses the ability to solubilize insoluble inorganic P and make it available to plants. The solubilization effect is generally due to the production of organic acids by these organisms. The organic acids produced by phospho-microbes includes monocarboxylic acid [acetic, formic], monocarboxylic hydroxy [lactic, glucenic, glycolic], monocarboxylic, ketoglucenic, decarboxylic [oxalic, succinic], dicarboxylic hydroxy [malic, maleic] and tricarboxylic hydroxy [citric] acids in order to solubilize inorganic phosphate compounds [Lal, 2002]. They are also known to produce amino acids, vitamins and growth promoting substances like, indole acetic acid [IAA] and gibberellic acid [GA] which facilitate the growth of plants. According to Pradhan and Sukla [2005] drop in pH during growth of P solubilizing fungi was more prominent in absence of TCP in the liquid medium suggesting that absence of soluble P in media induced the acid

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production. Thus, the process of acidification, chelation and exchange reactions are involved in the conversion of insoluble P into soluble form [Abd-Alla, 1994; Whitelaw, 2000; Goldstein, 1986]. Therefore, such microorganisms may not compensate only for higher cost of industrial production of fertilizers but also mobilize the fertilizers added to soil. Table 3 shows phosphate-solubilizing capacity of selected P-solubilizers. Table 2. Examples of important P-solubilizing microbes

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Genera

Selected Reference

Bacteria

Fungi

Bacillus

Aspergillus

B. brevis, B. cereus, B. circulans, B. firmus, B. licheniformis, B. megaterium, B. mesentricus, B. mycoides, B. polymyxa, B. pumilis, B. pulvifaciens and B. subtilis

A. niger, A. flavus, A. nidulans, A. awamori, A. carbonum, A. fumigatus, A. terreus and A. wentii

Taha et al. [1969], Barea et al. [1976], Banik and Day [1981], Venkateshwarlu et al., [1984], Sattar and Gaur [1985], Ali et al., [1989], Gaind and Gaur [1999], Rajarathinam et al. [1995], Bhattacharya et al. [1998], Kole and Hajra [1997], Kole and Hajra [1998]. Subba and Bajpai [1965], Chhonkar and Subba [1967], Prerna [1997].

Paeciliomyces fusisporus, Penicillium digitatum, P. simplicissimum, P. aurantiogriseum, Sclerotium rolfisii and species of Cephalosporium, Alternaria, Cylindrocladium, Fusarium and Rhizoctonia

Kole and Hajra [1997], Bardiya and Gaur [1974], Nair and Rao [1977], Jisha. [1997], Pal [2000], Gupta [1998]. Varsha-Narsian [1994]

yeasts, Torula thermophila, Saccharomyces cerevisiae and Rhodotorula minuta

Gupta [1998] Mishra [1985] Datta [1982]

Pseudomonas Pseudomonas striata, P. cissicola, P. fluorescens, P. pinophillum, P. putida, P. syringae, P. aeruginosa, P. putrefaciens and P. stutzeri

Escherichia freundii, E. intermedia, Serratia phosphaticum and species of Achromobacter, Brevibacterium, Corynebacterium, Erwinia, Micrococus, Sarcina and Xanthomonas Cyanobacteria, viz. Anabaena sp., Calothrix brauni, Nostoc sp., Scytonema sp. and Tolypothrix eylonica

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Table 3. Phosphate solubilizing capacity of selected P- solubilizers PSM Bacterial strains CPS-2, CPS-3 and Ca-18 Bacillus megaterium Trichoderma viride [TV 97] Trichoderma virens [PDBCTVs 12] Pseudomonas sp. Trichoderma virens [PDBCTVs 13] Aspergillus sp. Penicillium sp.

Solubilization Index [on solid media] 21 to 83 mm

Solubilizing Capacity [in liquid media] 65 to 130.5 μg/mL

Reference

[12.43 µg·mL–1]. [9.03 µg·mL–1], [9.0 µg·mL–1],

Rudresh [2005] Rudresh [2005] Rudresh [2005]

[8.83 µg·mL–1]

Afzal [ 2008] Rudresh [2005]

4.1

480 μg/ml 275 μg/ml

Gull et al. [2005]

Pradhan and Sukla . [2006] Pradhan and Sukla . [2006]

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Broadly, the process of solubilization and growth promotion by P-dissolving microbes involves- [i] organic acid production which lowers the pH in the rhizosphere and help to dissociate the bound forms of phosphate leading to P availabilty to plants [ii] humic and fulvic acids along with carboxylic, phenolic groups can release P from compost etc. [iii] phosphatases enzyme released by plants and microbes render P available [iv] proton extrusion during NH4+ and release of CO2 during respiration which is converted to carbonic acid subsequently decreasing pH which help in P solubilization [v] phytohormone production like IAA, GA and cytokinins which increases root elongation for better P acquisition [vi] bacteriocin production which provide protection to plants against soil and seed borne pathogens and [vii] degradation of pesticides; thus helping in reducing harmful impact of chemicals on soil microbes.

17.5. CROP IMPROVEMENT BY PSM The yield components of wheat increased as a result of soil inoculation with the phosphate-dissolving isolates [Wahid and Mehana, 2000]. Seed inoculation of wheat varieties with phosphate-solubilizing and phytohormone-producing Azotobacter chroococcum showed a better effect over the control [Kumar et al. 2001]. PSM [mixture of Pseudomonas and Bacillus sp.] inoculation increased number of tillers/m2, grain and biological yield compared to control [Afzal et al., 2005]. Similarly wheat inoculated with mixed inocula [B. megaterium or A. lipoferum 137] exhibited high shoot dry weight, compared to the control [El-Komy, 2005]. Soni and Aery [2004] concluded that farmyard manure [FYM] and biogas slurry [BGS] along with phosphate-solubilizing bacteria and high grade RP showed a substantial increase in leaf area, shoot root dry weight, seed number plant-1 and seed weight plant-1 of wheat over the control. A possible solution to minimize the problem of wheat production in calcareous soils is to increase N and P assimilation with bioinoculants using phosphatesolubilizing micro-organisms. The combination of arbuscular mycorrhizal [AM] fungus Glomus species [G1 and G2] and Pseudomonas putida increased dry matter accumulation in

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roots and shoots of wheat plants grown in soil fertilized with 50% P2O5 compared with the control wheat fertilized with 100% N and P2O5 [Valdivia et al., 1999]. Similarly, P fertilizer saving in wheat cultivation due to inoculation with phosphate-solubilizing microorganisms has also been documented by Suri et al. [2006]. Increased dry matter and grain yield of many agronomic crops following mixtures of P-solubilizers and P sources have also been reported by many workers [Mukherjee and Rai, 1999; Agrawal and Sharma, 2005; Qureshi and Narayanasamy, 2005; Sharma and Prasad, 2003; Egamberdiyeva et al., 2004]. When rock phosphate was inoculated with phosphate-solubilizing bacteria [Pseudomonas striata], it increased the grain yield by 0.9-1.8 t/ha and straw yield by 0.8-2.1 t/ha of the rice-wheat system [Sharma, 2003]. Inoculation with phosphate-solubilizing microorganisms resulted in significant increases in plant height, dry matter, nodulation and grain yield in soybean [Paratey and Wani, 2005]. Dual inoculation of soybean with phosphate-solubilizer [Sordaria fimicola] along with sulfur oxidizer [Aspergillus terreus] and phosphorus sources showed significant increase in nodulation, their dry weight, pod and dry matter yield [Rasal et al., 2004]. Less P fertilizer with P solubilizers and increased seed yield have been augmented by Dubey and Sunil-Agarwal [1999] and Dubey [2000] in soybean and by Kumpawat [2006] in cluster bean.

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17.5.1. Nutrient Improvement through PSM PSM inoculation increased P content in the seed, compared to control [Afzal et al., 2005]. Sharma and Prasad [2003] reported that Mussoorie rock phosphate [MRP] inoculated with PSB, increased P uptake of both rice and wheat. When the rock phosphate was inoculated with phosphate-solubilizing bacteria [Pseudomonas striata], it increased N uptake [by 18-38 kg/ha], P uptake [by 2.7-6.6 kg/ha], and K uptake [by 16-41 kg/ha] of the rice-wheat system [Sharma, 2003]. Improved nutrient uptake in wheat following inoculation with phosphate solubilizers have been documented by Valdivia et al. [1999], Tabar et al. [2002] and Wakelin et al. [2004]. Agrawal and Sharma, [2005] reported that application of 60 kg single super phosphate/ha in combination with A. chroococcum and PSB inoculation resulted in the highest N and P uptake. Amongst the phosphate solublizers, A. awamori showed the most significant effect in improving available P, followed by P. striata. The number of viable counts of phosphate- solubilizers in the rhizosphere soil after harvesting was improved due to P sources and seed inoculation with phosphate-solubilizers over control [Qureshi et al., 2005]. Phosphate-dissolvers significantly reduced pH and increased available P in the soil treated with either RP or super phosphate [Wahid and Mehana, 2000]. In a pot experiment, the inoculation of winter wheat with mixed culture of Pseudomonas fluorescens and P. putida increased the rate of nitrogen fixation in the rhizosphere. The effect of inoculation on the yield depended on the amount of mineral N added to the soil. Application of bacteria made it possible to reduce the rate of N fertilizer by threefold without any loss in the yield. The inoculation increased the uptake of N, P, K, Ca, Mg, Fe, Zn, Mn, and Cu by wheat [Shabaev and Smolin, 2000]. Increased P and calcium contents of shoots of soybeans with the use of low grade RP in conjunction with phosphate-solubilizers and FYM have been reported [Qureshi et al. 2005]. The efficiency of phosphate-solubilizers in solubilizing rock sources and influencing soybean yield followed the order: A. awamori > Pseudomonas striata > Bacillus polymyxa. Less fertilizer use with co-inoculation of N-fixers and P-solubilizers was

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superior with regard to N, P and K uptake [seed + straw] of soybean [Rasal et al., 2004; Dubey 2001a]. The status of residual N in the soil was increased with phosphate-solubilizing bacteria [Pseudomonas striata] compared to initial available N. The results indicated that P application alone as well as with P. striata had marked influence on the residual P status [Dubey, 2001b].

17.5.2. Co-Inoculation Effects of PSM with Other Microbes

Grain Weight (g/pot)

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The co-inoculation of Rhizobium and PSB along with P2O5 was found as the best combination for improving residual N and P in soil and root/shoot weight but PSB with P2O5 was the best combination for increasing yield attributes of wheat [Figure 1]. The results of soybean experiments indicated the effectiveness of dual inoculation in presence of P2O5 for nutrient uptake, yield and yield traits [Figure 2, 3]. Further, it was also investigated that colony forming units [cfu] of Rhizobium and PSB were enhanced by dual inoculation in presence of P2O5 [Afzal, 2008]. It is inferred that with co-inoculation in presence of P2O5, almost 50% P fertilizer could be saved which is economical for the farmer and is environment friendly as well. In other study, 42 % increase in wheat yield due to inoculation of PSB along with P2O5 and 12-38 % in soybean yield is reported. Furthermore, residual soil P was increased by 11-19% after wheat cultivation and 16-50% after soybean cultivation when inoculated plots were compared with un-inoculated ones [Afzal, 2008]. Plant nutrient status was also improved following co-inoculation of PSB and Rhizobium [Table 4]. It is suggested that further experiments related to dual inoculation of beneficial microorganisms should be conducted to help farmers for increasing per hectare yield of important crops as well as for a safer environment.

4 3.5 3 2.5 2 1.5 1 0.5 0

3.73 2.7

2.63 1.57

1.5

l tro on C

5 O P2

hi R

um bi zo

2.7

2.9

1.33

5 B 5 2O B O 2O P PS P S + P2 P + + B + m iu PS um B i b S b o + z o P z hi m hi R iu R ob z hi R

Treatments

Figure 1. Effect of Rhizobium and PSB on grain yield [g/pot] of Wheat; values are mean of two years trial [ adapted from Afzal, 2008].

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8 7 6 5 4 3 2 1 0

7.35

6.94 5.95

5.33

4.71

4.32

3.61

2O 5

5

5

+

P

P2 O

PS B

+ B R hi zo bi um

+

PS

R hi zo bi um

+

+

P2 O

PS B

B

P2 O

PS R hi zo bi um

5

R hi zo bi um

2.46

C on tro l

Grain Yield (g/Pot)

366

Treatments

3000 2199

2500 2000

2251 1953

2220

2319

2407

2453

1532

1500 1000 500 PS R B hi zo bi um + PS R hi B zo bi um + P2 O 5 P R S hi B zo + bi P2 um O 5 + PS B + P 2O 5

R hi zo bi um

P2 O

5

0 C on tro l

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Grain Yield (kg/ha)

Figure 2. Effect of Rhizobium and PSB on grain yield [g/pot] of soybean; values are mean of two years pot trial [adapted from Afzal, 2008].

Treatments

Figure 3. Effect of Rhizobium and PSB on grain yield of soybean; values are mean of two years field trials [adapted from Afzal, 2008].

Khokhar et al. [2006] reported increase in grain yield of maize ranging from 27-50% due to co-inoculation of Rhizobium, Azospirillum and PSB [Pseudomonas]. Seed inoculation using Rhizobium with PSB increased dry matter accumulation, grain yield and protein content of chickpea, dry fodder yield of succeeding maize and total N and P uptake by the cropping system over no inoculation and inoculation with Rhizobium alone. Rhizobium with and without PSB also increased soil N content over control. However, soil P content and bacterial counts remained unaffected by sole application of Rhizobium but improved with Rhizobium when used with PSB over no inoculation [Jat, 2006].

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Table 4. Effect of phosphate solubilizing bacteria and Rhizobium on seed P and N content [%] of wheat and soybean Treatments

Wheat

Soybean [Pot study]

Soybean [Field Study]

Seed P [%]

Seed N [%]

Seed P [%]

Seed N [%]

Seed P [%]

Seed N [%]

Control

0.27bc

2.27c

0.67c

4.6d

0.52b

4.2e

P2O5

0.29bc

2.40bc

0.76b

5.1c

0.59a

4.3e

Rhizobium

0.25c

2.70a

0.71bc

5.7b

0.60a

5.3b

PSB

0.38b

2.40bc

0.75b

5.4b

0.58a

5.2bc

Rhizobium + PSB

0.32bc

2.37bc

0.81a

5.5b

0.57a

6.0a

Rhizobium + P2O5

0.52a

2.50b

0.72bc

5.6b

0.62a

4.8d

PSB + P2O5

0.45b

2.43b

0.84a

5.7b

0.63a

5.1c

Rhizobium + PSB + P2O5

0.49a

2.40bc

0.87a

6.1a

0.63a

6.1a

LSD Value

0.111

0.355

0.0554

0.2215

0.055

0.156

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Adapted from Afzal [2008]; Mean values followed by different letters are significantly [P≤0.05] not different.

Rudresh et al. [2004] reported that combined inoculation of Rhizobium and PSB [Bacillus megaterium] and a biocontrol fungus Trichoderma sp. increased seed germination, nutrient uptake, plant height and yield of chickpea. Yasmeen [2003] and Mateen [2008] reported that three types of plant growth regulators [i.e., auxins, gibberellins and cytokinins] were produced by PSB. The rhizobial strain with P-solubilizing ability showed a more beneficial effect on plant growth and nutrient uptake than the strain without this ability, although both strains had similar effectiveness for N2-fixation in symbiosis with lentil. Synergistic relationships were observed between AM- fungi and some rhizobial strains that related to the compatible pairing of these two microsymbionts. The P-uptake efficiency was increased when P fertilizer was applied along with AM- fungi and/or P-solubilizing rhizobial strains [Zarei et al., 2006].

17.6. USE OF GENETIC ENGINEERING IN P-SOLUBILIZING RESEARCH Introduction or over-expression of genes involved in soil P-solubilization in natural rhizosphere bacteria is an attractive approach to improve the capacity of microorganisms to enhance P solubilization. Here, we present recent advances in the manipulation of genes related to microbial P-solubilization and its relationship with the use of rhizobacteria as improved inoculants.

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17.6.1. Organic Phosphate Solubilization There are two forms of P in soil, organic and inorganic phosphates. Phosphorous can be released form organic compounds in soil by means of three groups of enzymes: phosphatases, phytases, and phosphonatases. The major role apparently corresponds to acid phosphatases and phytases. Several acid phosphatase genes from Gram negative bacteria have been isolated and characterized [Rossolini et al., 1998]. These cloned genes represent an important source of material for the genetic transfer of this trait to other plant growth promoting rhizobacterial strains. Among rhizobacteria, we isolated a gene from Burkholderia cepacia that exhibited phosphatase activity. This gene codes for an outer membrane protein that enhances its synthesis in the absence of soluble P in the medium and was suggested to be involved in P transport to the cell. The heterologous expression of these genes in agriculturally-important bacterial strains is the next step in this approach. We transferred the napA phosphatase gene from the soil bacteria Morganella morganii to Burkholderia cepacia IS-16, a strain used as an inoculant, using a broad-host range vector [pRK293]. An increase in the extracellular phosphatase activity of the recombinant strain was achieved. Insertion of the transferred genes into the bacterial chromosome is advantageous for stability and ecological safety. In our lab, a plasmid for the stable chromosomal insertion of the phoC phosphatase gene from Morganella morganii was constructed and we are currently attempting to insert this gene into Azospirillum spp. and Burkholderia cepacia strains [Rodríguez et al., 2006].

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17.6.2. Inorganic Phosphate Solubilization A few genes involved in mineral phosphate solubilization [mps] from different species, such as Erwinia herbicola, Pseudomonas cepacia, Enterobacter agglomerans, and Rhanella aquatilis have been isolated. These genes conferred the ability to hydrolyze insoluble P substrates to Escherichia coli and in some cases, they have been found to be involved in the synthesis of the coenzyme pyrroloquinoline quinone [PQQ]. The PQQ synthetase gene from E. herbicola [Goldstein and Liu 1987] was subcloned in our lab using a broad-host range vector [pKT230]. The recombinant plasmid [pL230] was expressed in E. coli, and thereafter transferred to the PGPB B. cepacia and P. aeruginosa strains, using tri-parental conjugation. Several of the exconjugants recovered in the selection medium showed a larger-sized clearing halo in medium with tricalcium phosphate as the sole P source. This indicates the heterologous expression of this gene in the recombinant strains, which gave rise to improved mps ability of these PGPB [Rodríguez et al., 2006]. Recently, using specific primer sets and PCR amplification, the presence of two genes involved in P- solubilization [gdh and pqqE] were detected in PSB [Perez et al., 2007]. It is suggested that current database for genes is too small to provide adequate coverage [Lim et al., 2007]. Reports on how genetic engineering could help to improve the P-solubilizing efficiency of naturally abundant yet functionally different microbes are still scarce. However, the preliminary success in the genetic manipulation of P-solubilizing microbes provides a promising and exciting perspective for obtaining PGPB strains with enhanced P solubilization ability, and thus, a more efficient use of microbes as agricultural inoculants.

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17.7. ECONOMICS OF USING PSM INOCULANTS The economics of biofertilizers or microbial inoculants can be calculated either by costing the biologically fixed N or P [residual or freshly applied] solubilized in terms of the cost of fertilizer N or P that produces a similar yield increase, or by deducting the cost of inoculant plus its application cost from the value of extra yield produced. Residual benefit from the N fixed as a result of inoculation is not easy to compute except in terms of the value of extra crop produced [FAO, 2006]. Phosphate solubilizing microorganisms including species of Aspergillus, Bacillus, Escherichia, Arthrobacter and Pseudomonas have been reported to add 30–35 kg P2O5 ha–1 to soils [Guar et al., 2004].

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17.8. PROBLEMS OF PSM INOCULANTS UNDER FIELD CONDITIONS The success of microbial inoculants under field environment requires- [i] an efficient strain able to realize the claimed effect [ii] the technology to produce an efficient inoculant with a good shelf-life [iii] a convenient method of inoculation delivery and [iv] a good survival of the inoculated microorganisms in order to obtain a prolonged effect. Although, uncountable number of microbial strains are processed and characterized for P-solubilizing activity at laboratory level in controlled conditions and are tested under field conditions by scientists throughout the world. At farmers end, the success of such inoculants is however, not consistent due to great variations in field conditions around the world. Such differences in the performance of PS microbes observed under lab and land condition could probably be due to- [i] use of irrelevant/incompetent strains for specific crops and specific biological and physical factors such as soil osmotic potential, soil temperature, soil nutrient status and indigenous microflora in soil [ii] use of non-sterile system of production which allows various contaminants to grow along the specific culture and [iii] lack of high-tech production infrastructure, skilled manpower, storage and transport of the viable products and good quality control enforcement system. The reasons for erratic response of biofertilizers at farmers end includes- [i] poor inoculant quality [ii] lack of awareness among farmers [iii] non-availability of inoculants in ready to use form and [iv] wide variations in agro-climatic situations, soil types, nutrient status of soil and native microbial populations. Although quality control of inoculants was a matter of concern till early seventies in some developed countries like Canada, France, Australia and Brazil but rigorous enforcement of quality control system has changed the entire scenario dramatically.

CONCLUSION The use of phosphate solubilizing microorganisms as biofertilizer is likely not only to reduce the application of more expensive phosphatic fertilizers but is also environment friendly. Besides providing P to plants in a more environmentally friendly and sustainable manner, phosphate solubilizing microorganisms could also enhance plant growth by increasing the efficiency of biological nitrogen fixation [BNF] and the availability of trace elements [Fe, Zn etc] and through production of plant growth promoting substances [Kucey et

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al., 1989]. Although, solubilization of P compounds by microbes is very common under laboratory conditions, results in the field have been highly variable which in turn has hampered the large-scale use of PSMs in sustainable agriculture. Though, many reasons have been suggested for this variation in their performance, but none of them have been conclusive. Despite their proven ability in augmenting plant growth, the detailed mechanisms as to how P-solubilizing microbes improve P pool of soils and uptake of P by plants are not well explained at biochemical and molecular level. Recent work in our laboratory has shown that the conditions employed to isolate PSMs obviously do not reflect soil conditions and that PSMs capable of effectively releasing P from soil are not as highly abundant as was suggested in earlier studies. Moreover, mineral phosphate-solubilizing ability of microbes has been found linked to specific genes, which are reported to be present even in non Psolubilizing bacteria. Therefore, understanding the genetic basis of P-solubilization could possibly help not only in developing a better phosphate solubilizing inoculant but also transforming more rhizosphere-competent non P-solubilizing bacteria into PSMs. Upon inoculation, such improved PS bacterial strains or trans-conjugants may boost the agricultural productivity in different agro-ecosystems.

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REFERENCES Abd Alla M.H. [1994]. Phosphatases and the utilization of organic phosphorus by Rhizobium leguminosarum biovar viceae. Lett. Appl. Microbiol. 18 : 294–296. Afzal,A [2008]. Role of Rhizobium and PSM in growth and yield of wheat [Triticum aestivum L.] and soybean [Glycine max L.]. PhD Thesis. Quaid –I-Azam University Islamabad, Pakistan. Afzal, A; M. Ashraf; S. A. Asad and M. Farooq. [2005]. Effect of phosphate solubilizing microorganisms on phosphorus uptake, yield and yield traits of wheat [Triticum aestivum L.] in rainfed area. Int. J. Agri. Biol. 7[2]: 207-209. Agrawal, M. M. and C.P. Sharma. [2005]. Effect of PSB and Azotobacter on the availability of phosphorus in wheat [Triticum vulgare L.]. Farm Sci. J. 14[2]: 76-78. Ahmad, N; M.T. Saleem and I.T. Twyford. [1992]. Phosphorus Research in Pakistan, pp: 59–92. A Review: Symposium on the role of phosphorus in crop production. Government of Pakistan Planing and Development Division, NDFC, Islamabad. Ali, M. E; A. M. Massoud and I. A. I. El-Xhander. [1989]. Effect of different isolates of PSB on soil pH and available soil P. Proceedings of the Conference Agricultural Development and Research, Ain Shams University, Cairo, Egypt. Banik, S. and B. K. Dey [1981]. Solubilization of inorganic phosphate and production of organic acids by microorganisms isolated in sucrose tricalcium phosphate agar plates. Zbl. Bakteriol.136, 478–486. Bardiya, M. C. and A. C. Gaur [1974]. Isolation and screening of microorganisms dissolving low grade rockphosphate. Folia Microbiol., 1974, 19, 386–389. Barea, J. M., E. Navarro, and E. Montoya [1976]. Production of plant growth regulation by rhizosphere phosphate solubilizing bacteria. J. Appl. Bacteriol., 40[2], 129–134.

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Phosphate Solubilizing Microorganisms

371

Bhattacharya, P; T. K. Ghosh and R. K. Jain [1998]. Evaluation of native phosphate solubilizing microorganisms from Vidarbha soils. J. Maharastra Agric. Univ. Pub. 22[2], 252–253. Black, C. A. [1968]. Soil-plant relationships. 2nd Ed. John Wiley and Sons, Inc., New York. Chaudhry, T.M. and S.M. Qureshi [1982]. Agricultural Chemistry Section.Review of Work 1926-80. ARI., Tandojam, Government of Sindh. Chhonkar, P. K. and N. S. Subba Rao [1967]. Phosphate solubilization by fungi associated with legume root nodules. Can. J. Microbiol., 13, 743–753. Datta, M; S. Banik and R. K. Gupta [1982]. Studies on the efficacy of a phytohormone producing, phosphate solubilizing Bacillus firmus in augmenting paddy yield in acid soils of Nagaland. Plant Soil 69, 365–373. Dubey, S. K. [2001a]. Biomass and nitrogen accumulation pattern in soybean [Glycine max L.] as influenced by phosphate solubilizing bacteria in vertisols. Indian J. Plant Physiol. 6[4]: 441-443. Dubey, S. K. [2001b]. Balance sheet of N and P as influenced by phosphate solubilizing bacteria and phosphate fertilization in rainfed soybean [Glycine max L.] grown on vertisols. Annals Agric.Res. 22[1]: 162-164. Dubey, S. K. and A. Sunil. [1999]. Effect of phosphate-solubilizing micro-organisms as single and composite inoculant on rainfed soybean [Glycine max L.] in Vertisol. Indian J. Agric. Sci. 69[8]: 611-613. Dubey, S. K.[2000]. Effectiveness of rock phosphate and superphosphate amended with phosphate solubilizing microorganisms in soybean grown on Vertisols. J. Indian Soc. Soil Sci. 48[1]: 71-75. El-Komy, H. M. A. [2005]. Coimmobilization of Azospirillum lipoferum and Bacillus megaterium for successful phosphorus and nitrogen nutrition of wheat plants. Food Tech. Biotech. 43[1]: 19-27. Egamberdiyeva, D; D. Juraeva; S. Poberejskaya; O. Myachina; P. Teryuhova; L. Seydalieva and A. Aliev. [2004]. Improvement of wheat and cotton growth and nutrient uptake by phosphate solubilizing bacteria. Proceedings-of-the-26th-Southern-Conservation-TillageConference-for-Sustainable-Agriculture,-Raleigh,-North-Carolina,-USA, 8-9-June, 2004. 58-66. FAO. [2006]. Plant nutrition for food security. A guide for integrated nutrient management. FAO Fertilizer and Plant Nutrition Bulletins. Pp 99-102. FAO. [2006a]. Plant nutrition for food security. A guide for integrated nutrient management. FAO Fertilizer and Plant Nutrition Bulletins. Pp 199-200. Gaind, S. and A. C. Gaur. [1999]. Microbial phosphate solubilization as influenced by sodium chloride. Indian J. Exptl. Biol., 37, 209–210. Gaur, A.C. [1983]. Biofertilizers and crop productivity. Adv Soil Sci, 1, 127-178. Ed. K.V. Paliwal. Books and Periodicals, New Delhi, India. Gaur, R; N. Shani; Kawaljeet, Johri; B. N., Rossi, P. and M., Aragno. [2004]. Diacetyl phloroglucinol-producing Pseudomonas do not influence AM fungi in wheat rhizosphere. Curr. Sci., 86, 453–457. Gerke, J; W. Ro¨mer and A. Jungk [1994]. The excretion of citric and malic acid by proteoid roots of Lupinus albus L.; effects on soil solution concentrations of phosphate, iron, and aluminumin the proteoid rhizosphere in samples of an oxisol and a luvisol. Zeitschrift fu¨r Pflanzenerna¨hrung und Bodenkunde 157: 289–294.

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372

Aftab Afzal and Asghari Bano

Goldstein A. and S.T. Liu. [1987]. Molecular cloning and regulation of a mineral phosphate solubilizing gene from Erwinia herbicola. Biotechnology 5, 72-74. Goldstein, A.H. [1986]. Bacterial solubilization of mineral phosphates: historical perspectives and future prospects. Am. J. Altern. Agric 1 : 51-57. Gull, M; F. Y. Hafeez; M. Saleem and K. A. Malik [2005] Phosphorus uptake and growth promotion of chickpea by co-inoculation of mineral phosphate solubilizing bacteria and a mixed rhizobial culture. Aust. J. Exp. Agri. 44[6] 623 – 628. Gupta, R. P; M. K. Vyas and M. S. Pandher [1998]. Role of phosphorus solubilizing microorganisms in P-economy and crop yield. In Soil–Plant–Microbe Interaction in Relation to Nutrient Management [ed. Kaushik, B. D.], Venus Printers and Publishers, New Delhi, pp. 95–101. Igual J.M; A. Valverde; E. Cervantes and E.Velásquez [2001]. Phosphate solubilizing bacteria as inoculants for agriculture: use of updated molecular techniques in their study. Agronomie 21: 561–568. Jat, R. S. [2006]. Direct and residual effect of vermicompost, biofertilizers and phosphorus on soil nutrient dynamics and productivity of chickpea-fodder-maize Sequence. J. Sustainable Agri. 28[1]: 41–54. Jisha, M. S. [1997]. Optimization of factors for efficient solubilization of mineral phosphates. Ph.D. Thesis, P.G. School, IARI, New Delhi. Jones, D. L. [1998]. Organic acids in the rhizosphere - a critical review. Plant Soil 25-44. Jorquera, M.A. and M.T. Hernandez. [2008]. Isolation of culturable phosphobacteria with both phytate-mineralization and phosphate-solubilizing activity from the rhizosphere of plants grown in a volcanic soil. Biol. Fertil. Soils. Khokhar, S.N; M.A.Khan; A.Afzal and R.Ahmed. [2006]. Interaction of diazotrophs with phosphorus solubilizing bacteria: their effect on seed germination, growth and grain yield of maize, under natural conditions. Int.J.Biol.Biotech.,3[4]: 773-777. Kole, S. C. and J. N. Hajra [1997]. Isolation and evaluation of tricalcium and rock phosphate solubilizing microorganisms from acidic terai and lateritic soils of West Bengal. J. Interacademicia 1[3], 126–128. Kole, S. C. and J. N. Hajra [1998]. Occurrence and acidity of tricalcium phosphate and rock phosphate solubilizing microorganisms in mechanical compost plants of Calcutta and an alluvial soil of West Bengal. Environ. Ecol. 16[2], 344–349. Konietzny U. and R.Greiner [2004]. Bacterial phytase: potential application, in vivo function and regulation of its synthesis. Braz. J. Microbiol. 35:11–18. Krasilinikov, N. A. [1957]. On the role of soil micro-organism in plant nutnition. Microbiologiya 26: 659-672. Kucey, R. M. N; H. H. Janzen and M. E. Leggett. [1989]. Microbially mediated increases in plant-available phosphorus. Advan. Agron. 42: 199-228. Kudashev, I. S. [1956]. The effect of phosphbacterin on the yield and protein content in grains of autumn wheat, maize and soybean. Doki. Akad. Skh. Nauk. 8: 20-23. Kumar, V; R. Kumar and N. Narula [2001]. Establishment of phosphate-solubilizing strains of Azotobacter chroococcum in the rhizosphere and their effect on wheat cultivars under green house conditions. Microbiol. Res. 156, 87-93. Kumpawat, B. S. [2006]. Effect of phosphorus levels and phosphate-solubilizing bacteria on clusterbean [Cyamopsis tetragonoloba] and its residual effect on wheat [Triticum aestivum L.] under limited water supply. Crop-Research-Hisar 31[1]: 14-16.

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Phosphate Solubilizing Microorganisms

373

Laegreid, M; O.C. Bockman and O. Kaarstad [1999]. Agriculture, fertilizers and the environment. Cambridge, UK, CABI Publishing, University Press. 294 pp. Lal, L. [2002]. Phosphatic biofertilizers. Agrotech, Publ. Academy, Udaipur, India, 224p. Lim B.L; P.Yeung; C. Cheng and J. E. Hill [2007]. Distribution and diversity of phytatemineralizing bacteria. ISME J 1: 321-330. Marschner, H. [1993]. Mineral Nutrition of Higher Plants. London, Academic Press Ltd., Harcourt Brace and Co. Publishers. Mateen, A. [2008]. Isolation and characterization of phosphate solubilizing bacteria from two altitudes of Baltistan. M.Phil Thesis. Dept. of Plant Science, Quaid-I-Azam University Islamabad,Pakistan. Mehta, S. and C.S. Nautiyal. [2001]. An efficient method for qualitative screening of phosphate-solubilizing bacteria. Curr. Microbiol. 43:51–56. Mishra, M. M. [1985]. Solubilization of insoluble inorganic phosphate by soil microorganisms – A review. Agric. Rev. 6, 23. Mukherjee, P. K. and R. K. Rai. [1999]. Sensitivity of P uptake to change in root growth and soil volume as influenced by vesicular arbuscular mycorrhizae [VAM], phosphate solubilizing bacteria [PSB] and P levels in wheat and chickpea. Annals Agric. Res. 20[4]: 528-530. Nair, S. K. and N. S. S. Rao [1977]. Distribution and activities of phosphate solubilizing microorganisms in the rhizosphere of coconut and cacao under mixed cropping. Plantation Crops. 5, 67–70. Naz, I. [2008]. Physiological and molecular characterization and phytohormone production of Rhizobium and Azospirillum from rhizosphere and roots of halophytes from Khewra salt range and attock. M.Phil Thesis. Dept. of Plant Science, Quaid-I-Azam University Islamabad, Pakistan. Pal, K. K; K. V. B. R. Tilak; A. K. Saxena; R. Dey and C. S. Singh [2000]. Enhancement of phosphate solubilization and siderophore production by Tn5 mutagenesis of a biocontrol rhizobacterium Pseudomonas spp. Em 85. J. Microb. World 2, 9–15. Paratey, P. R. and P. V. Wani. [2005]. Response of soybean [cv. JS-335] to phosphate solubilizing biofertilizers. Legume Res. 28[4]: 268-271. Pérez, E; M, Sulbarán; M.M. Ball and L.A.Yarzábal [2007]. Isolation and characterization of mineral phosphate-solubilizing bacteria naturally colonizing a limonitic crust in the south-eastern Venezuela region. Soil Biol. Biochem. 39:2905–2914. Pradhan, N. and L.B. Sukla. [2006]. Solubilization of inorganic phosphates by fungi isolated from agriculture soil. Afr. J. Biotech. 5, 850-854. Prerna, A; K. K. Kapoor and P. Akhaury [1997]. Solubilization of insoluble phosphate by fungi isolated from compost and soil. Environ. Ecol., 15[3], 524–527. Qureshi, A. A. and G. Narayanasamy. [2005]. Residual effect of phosphate rocks on the dry matter yield of and P uptake of soybean. J. Indian Soc. Soil Sci. 53[1]: 132-134. Qureshi, A. A; G. Narayanasam;, P. K. Chhonkar and V. R. Balasundaram. [2005]. Direct and residual effect of phosphate rocks in presence of phosphate solubilizers and FYM on the available P, organic carbon and viable counts of phosphate solubilizers in soil after soybean, mustard and wheat crops. J. Indian Soc. Soil. Sci. 53[1]: 97-100. Rajarathinam, K; T. Balamurugan; R. Kulasekarapandian; S. Veerasami and M. Jayabalan [1995]. Isolation and screening of phosphate solubilizers from soil of Kamarajar district [Tamil Nadu]. J. Ecotoxicol. Environ. Monit., 5[2], 155–157.

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Aftab Afzal and Asghari Bano

Rasal, P. H., B. B. Sangale and K. B. Pawar. [2004]. Effects of phosphate solubilizing and sulphur oxidizing microorganisms on yield and phosphorus uptake of soybean. J. Maharashtra Agric. Uni. 29[1]: 51-53. Rodriguez, H; R. Fraga; T. Gonzalez and Y. Bashan [2006]. Genetic of phosphate solubilization and its potencial applications for improving plant growth-promoting bacteria. Plant Soil 287:15–21. Rossolini, G.M; S. Shipa; M.L. Riccio; F. Berlutti; L.E. Macaskie and M.C. Thaller [1998]. Bacterial non-specific acid phosphatases: physiology, evolution, and use as tools in microbial biotechnology. Cell. Mol. Life Sci. 54, 833-850. Rudresh, DL., M.K. Shivaprakash, MK; Prasad, RD. (2005) Effect of combined application of Rhizobium, phosphate solubilizing bacterium and Trichoderma spp. on growth, nutrient uptake and yield of chickpea (Cicer aritenium L.). Appl Soil Ecol, 28, 139-146. Rudresh, D.L; M.K. Shivaprakash, and R.D. Prasad. [2005]. Tricalcium phosphate solubilizing abilities of Trichoderma spp. in relation to P uptake and growth and yield parameters of chickpea [Cicer arietinum L.]. Can. J. Microbiol./Rev. Can. Microbiol. 51, 217-222. Sattar, M. A. and A. C. Gaur [1985]. Characterization of phosphate dissolving microorganisms isolated from some Bangladesh soil samples. Bangl. J. Microbiol., 2, 22– 28. Shabaev, V. P. and V. Y. Smolin. [2000]. The response of winter wheat to inoculation with Pseudomonas bacteria on grey forest soil. Pochvovedeni [4]: 497-504. Sharif, M., 1985. Improvement of P fertilizer efficiency. Proc. Inter. Seminar on Fertilizer Use Efficiency. pp: 106–16. Nov., 4-6, 1985, Lahore. Sharma, S. N. [2003]. Effects of phosphate-solubilizing bacteria on the efficiency of Mussoorie rock phosphate in rice [Oryza sativa L.]-wheat [Triticum aestivum L.] cropping system. Indian J. Agric. Sci. 73[9]: 478-48. Sharma, S. N and R. Prasad. [2003]. Yield and P uptake by rice and wheat grown in a sequence as influenced by phosphate fertilization with diammonium phosphate and Mussoorie rock phosphate with or without crop residues and phosphate solubilizing bacteria. J. Agric.Sci. 141[3/4]: 359-369. Soni, P. and N. C. Aery. [2004]. Agronomic effectiveness of high grade rock phosphate with organic sources and phosphate solubilizing bacteria on wheat [Triticum aestivum L.]. Phosphate rich organic manure: an alternate to phosphatic fertilizers. Udaipur, India: Himanshu Publications pp: 168-172. Steen I. [1998]. Phosphorus availability in the 21st century: management of a non-renewable resource, Phosphorus Potassium, 217, 25–31. Subba Rao, N. S. and P. D. Bajpai [1965]. Fungi on the surface of root nodules and phosphate solubilization. Experientia, 21, 386–387. Sundara Rao, W. V. B. and M. K. Sinha [1998]. Phosphate dissolving organisms in soil and rhizosphere. Indian J. Agric. Sci., 33, 272-278. Suri, V. K; G. Chander; A. K. Choudhary and T. S. Verma. [2006]. Co-inoculation of VAmycorrhizae [VAM] and phosphate solubilizing bacteria [PSB] in enhancing phosphorus supply to wheat in a Typic Hapludalf. Crop Res. Hisar. 31[3]: 357-361. Tabar, A. R; N. S. Rastin; H. A. Alikhani and M. Mohammadi. [2002]. Effects of native Pseudomonas fluorescens strains on the nutrient uptake by wheat. Iranian J. Agric. Sci. 33[4]: 771-780.

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Phosphate Solubilizing Microorganisms

375

Taha, S. M; S. A. Z. Mohmoud; A. A. El-Damati and A.M. Abd-El- Hafez [1969]. Activity of phosphate dissolving bacteria in Egyptian soil. Plant Soil 31, 149–160. Thompson, J.A. [1999]. An outsider’s view of the legume inoculant production scenario in India. In Use and development of Microbial inoculants, Eds. T.Singh, A.K.Yadav and S. Raychaudhari,publ. North Eastern Regional Conference on biofertilizers, 22-23 January 1999. pp. 53-80. Vance C.P; C. Uhde-Stone and D.L.Allan [2003]. Phosphorus acquisition and use: critical adaptations by plants for securing a nonrenewable resource. New Phytologist 157: 423– 447. Valdivia, U. B; Y. J. M. Sanchez; C. E. Pena and B. J. M. Fernandez. [1999]. Effect of the inoculation of wheat with Glomus spp. and Pseudomonas putida. RevistaLatinoamericana-de-Microbiologia 41[4]: 231-237. Varsha-Narsian, J. Thakkar and H. H. Patel [1994]. Inorganic phosphate solubilization by some yeast. Indian J. Microbiol., 35[2], 113–118. Venkateshwarlu, B; A. V. Rao and P.Raina [1984]. Evaluation of phosphorus solubilization by microorganisms isolated from aridsoil. J. Indian Soc. Soil Sci., 32, 273–277. Wahid, O. A. A. and T. A. Mehana. [2000]. Impact of phosphate-solubilizing fungi on the yield and phosphorus-uptake by wheat and faba bean plants. Microbiol. Res. 155[3]: 221227. Wakelin, S. A; R. A. Warren; P. R. Harvey and M. H. Ryder. [2004]. Phosphate solubilization by Penicillium spp. closely associated with wheat roots. Biol. Fert. Soils 40[1]: 36-43. Whitelaw, M.A. [2000]. Growth promotion of plants inoculated with phosphate solubilizing fungi. Edited by Donald L. Sparks. Advances in Agronomy, Academic press 69: 99-151. Yasmeen, S. [2003]. Combined effect of phosphorus solubilizing microbes, Rhizobium and PGPR on physiology of soybean [Glycine max.L.]. M.Phil Thesis. Dept. of Plant Science, Quaid-I-Azam University Islamabad, Pakistan. Zarei M.; N. Saleh-Rastin ; H A. Alikhani and N. Aliasgharzadeh .[2006]. Responses of Lentil to Co-Inoculation with Phosphate-Solubilizing Rhizobial Strains and Arbuscular Mycorrhizal Fungi. J. Plant Nutri. 29, 1509 – 1522.

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In: Phosphate Solubilising Microbes for Crop Improvement ISBN 978-1-60876-112-8 Editor: M. S. Khan and A. Zaidi © 2009 Nova Science Publishers, Inc.

Chapter 18

GENETIC MANIPULATIONS OF METAL ACCUMULATION AND HEAVY METAL TOLERANCE: IMPROVING PLANTS FOR ENVIRONMENTAL REMEDIATION M. Nedkovska∗ and N. Gorinova AgroBioInstitute, Department of Phytoremediation, Dragan Tzankov Blvd. N8, Sofia 1164, Bulgaria

ABSTRACT Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Pollution of the environments by the toxic metals is a global threat that has increased substantially in the recent times. The metals discharged from various sources including industries, sewage sludges and agro-chemicals, accumulate in soil and in turn, adversely affect the microbial diversity, physico-chemical properties of soils, leading to the loss of soil fertility and yields of crops. Such toxic metals in general, can not be destructed biologically to more or less toxic products and remain in the environment. Conventional approaches employed to detoxify metal polluted sites generates huge amounts of toxic products and are expensive. On the other hand, the biologically mediated approach, like, phytoremediation, that involves the use of plants to remove, contain, or render harmless environmental pollutants, is an exciting low cost in situ technology with immense market potential. This review presents the results of studies on the recent developments in the field of genetic manipulations of the plants for improving the metal phytoremediation along with application of transgenic plants in metal poisoned soils which is likely to open up new possibilities for remediation of metal polluted soils.

∗

Corresponding author: [email protected]

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18.1. INTRODUCTION 18.1.1. Phytoremediation

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Conventional remediation technologies for the clean up of heavy metals from polluted soil depends on physicochemical engineering and electrochemical approaches. In contrast, phytoremediation, the use of plant-based approaches to remove, contain, or render harmless environmental pollutants, is an emerging technology with immense market potential [Berken et al., 2002]. There are several different types of phytoremediation- [i] phytoextraction: plants take up contaminants from the soil and accumulate them in their tissues, which can then be harvested and removed from the site [Kumar et al., 1995]. For example, hyperaccumulator plants, which naturally grow on heavy metal and metalloid rich soils, facilitate the continuous phytoextraction [Baker and Brooks, 1999] [ii] phytostabilization: plants immobilize contaminants chemically and physically at the site thereby preventing their movement to surrounding areas [Berti and Cunningham, 2000] [iii] phytofiltration; plant roots absorb metals from water and aqueous waste streams [Dushenkov and Kapulnik, 2000] [iv] phytodetoxification: plants change the chemical species of the contaminant to a less toxic form [Lytle et al., 1998] and [v] phytovotalization: makes use of plants and their associated microbes to metabolize some contaminants such as, selenium [Terry et al., 2000]. Phytoremediation takes advantage of the natural ability of plants to extract heavy metals from soil, using energy from sunlight [Doty, 2008]. Its primary advantage is that it is approximately 10 times less expensive than conventional strategies [Chapell, 1998]. Other advantages of phytoremediation include the possibility of useful products such as wood, pulp, or bio-energy that could help finance the clean-up [Doty, 2008]. Plants also supply nutrients for rhizospheric microorganisms that may also aid in remediation of the pollutants. The advantages and limitations of phytoremediation technique are listed in Table 1. Table 1. Advantages and disadvantages of phytoremediation Advantages Less costly than mechanical methods Passive, solar-driven High public acceptance Retain topsoil Less secondary waste generation

Disadvantages Limited to shallow contaminants Phytotoxicity effects of contaminants Slower than mechanical methods Unknown effects of biodegradation products Contaminants may enter the food chain

Adapted from Chapell [1998].

18.2. APPROACHES FOR MAKING THE METAL PHYTOREMEDIATION MORE EFFICIENT The efficiency approaches includefor remediation of maximize biomass

of metal phytoremediation can be improved in several ways. Such [i] screening and identifying the most suitable plant species or varieties metal and [ii] agronomic practices to optimize selected species to production and metal uptake. For instance, planting density and

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fertilization can be optimized to enhance plant productivity [Chaney et al., 2000] and amendments such as, organic acids or synthetic chelators may be added to the soil to enhance metal uptake [Salt et al., 1998; Blaylock and Huang, 2000]. Different plant species may also be combined for maximal phytoremediation efficiency [Horne, 2000]. Farm management practices such as, fertilization and, plant clipping may also affect metal uptake by influencing microbial density and composition in the root zone. The selected species or variety can be bred further for desired property, either through classic [conventional] breeding or via genetic engineering.

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18.2.1. Strategies for Genetic Enhancement of Phytoremediation Phytoremediation by natural plant species [genetically unaltered] is limited by environmental as well as by biological factors. Hyperaccumulating plants can accumulate heavy metals to high concentrations in their tissues, but phytoremediation using hyperaccumulators is slower than physical and chemical processes because of their extremely slow growth rate and low biomass [Cunningham et al., 1997]. These limitations could be overcome by conventional plant breeding practices. Combining the genome of a tolerant, slow-growing hyperaccumulator, with that of a much less tolerant, but fast-growing nonaccumulator, by developing somatic hybrids could be one method of increasing phytoremediation efficiency [Brewer et al. 1999]. However, conventional approaches may take decades. Genetic engineering has potential to produce plant populations with superior traits for phytoremediation in a relatively short time. Using genetic engineering, genes can be transferred from organisms [that cannot be crossed, such as, inserting genes from microbes] into plants and enables one to control gene expression. For example, by choosing an appropriate promoter gene expression can be made to be constitutive or tissue specific [Karenlampi et al., 2000]. Genetic engineering could be used to generate plants with a higher tolerance to heavy metals, greater efficiency in extracting heavy metals and other pollutants from soil, or greater abilities for the accumulation and detoxification of such pollutants [Berken et al., 2002]. A direct method for enhancing the effectiveness of phytoremediation is to overexpress in transgenic plants the genes involved in metabolism, or transport of specific pollutants [Cherian and Oliveira, 2005]. The introduction of these genes can be readily achieved for many plant species using Agrobacterium tumefaciens-mediated plant transformation. Many genes are involved in metal uptake, translocation and sequestration and transfer of any of these genes into candidate plants is a possible strategy for genetic engineering of plants for improvement of phytoremediation traits [Eapen and D, Souza, 2005]. Depending on the strategy, transgenic plants can be engineered to accumulate high concentrations of metals in harvestable parts. Transfer or over-expression of genes will in turn, lead to enhanced metal uptake, translocation, sequestration or intracellular targeting. While bio-engineering of plants for synthesis of metal chelators is likely to improve the capability of plant for metal uptake [Pilon-Smits and Pilon, 2002; Clemens et al. 2002]. An important pathway by which plants detoxify heavy metals is through sequestration with heavy metal-binding proteins called phytochelatins [PCs] or their precursor, glutathione. Manipulating the expression of enzymes involved in glutathione and phytochelatin synthesis may thus, be a good approach for enhancing heavy metal tolerance in plants [Zhu et al.,

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1999]. To identify limiting factors for heavy metal accumulation and tolerance, and to develop transgenic plants with an increased capacity to accumulate and/or tolerate heavy metals, the Escherichia coli gshII gene encoding glutathione synthase [GS] was overexpressed in the cytosol of Indian mustard [Brassica juncea L.] [Zhu et al., 1999]. The transgenic GS plants accumulated significantly more cadmium [Cd] than the wild type: shoot Cd concentrations were up to 25% higher and total Cd accumulation per shoot was up to three fold higher. The transgenic GS plants showed enhanced tolerance to Cd at the seedling and mature plant correlated with gshII expression level of glutathione, phytochelatin, thiol, sulfur [S], and calcium [Ca] than wild type plants. The authors conclude that in the presence of cadmium, GS enzyme was rate limiting for the biosynthesis of glutathione and phytochelatins, and that overexpression of GS offers a promising strategy for the production of plants with superior heavy metal phytoremediation capacity. The GS plants offer great promise for enhancing the efficiency of cadmium phyto-extraction from polluted soils and waste water. These plants may also show increased tolerance to and accumulation of other heavy metals, because PCs are thought to play a role in tolerance of a range of heavy metals especially nonessential heavy metals such as, mercury and lead [Goldsbrough, 1998]. Overexpression of phytochelatin synthase is postulated to give plants a genetically enhanced tolerance to heavy metals [Li et al. 2004]. Kim et al. [2005] established transgenic Arabidopsis and tobacco [Nicotiana tabacum] plants that over-expressed the barley [Hordeum vulgare] nicotinamine synthase [NAS] gene, and showed that increased nicotianamine [NA] biosynthesis confers enhanced tolerance to high levels of metals, particularly Ni, to plants. These results indicated that NA plays a critical role in metal detoxification and, therefore, can be a powerfool tool for use in phytoremediation [Kim et al. 2005] [Figure 1 and Figure 2]. Over-expression of cysteine synthase [O-acetyl-L-serine [thiol] lyase] which catalyzes the final step of cysteine biosynthesis caused the increase of glutathione [GSH] and cysteine contents in tobacco [Noji et al., 2001]. Moreover, the transgenic Arabidopsis thaliana plants over-expressing cysteine synthase in cytosol showed the tolerance to cadmium treatment [Dominguez-Solis, et al. 2001].

Figure 1. Transgenic Arabidopsis expressing NAS gene [HvNAS1] are tolerant to excess metals [adapted from Kim et al. 2005].

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Figure 2. Transgenic tobacco plants expressing NAS gene [HvNAS1] are tolerant to 200 μM NiSO4 [adapted from Kim et al. 2005].

These results suggest that the biosynthesis of cysteine participates in the detoxification of heavy metals and thus enhanced formation of cysteine may lead to resistance to heavy metals [Dominguez-Solis, et al. 2001]. Furthermore, in the transgenic tobacco plants F1 overexpressing cysteine synthase cDNA in cytosol, chloroplasts were significantly more tolerant than wild type plants in agar medium containing Cd, Se, and Ni. The F1 transgenic plants could enhance accumulation of Cd in shoot suggesting that the transgenic plants overexpressing cysteine synthase both in cytosol and chloroplasts could be applied for removing cadmium from contaminated soils using phytoremediation technique [Kawashima, et al. 2004]. In other study, transgenic tobacco, Nicotiana glauca, containing a gene encoding a phytochelatin synthase from wheat [Triticum aestivum], accumulated more metals [Cd, Pb, Cu, Zn, Ni, and B] when grown in mine soil compared with non-transgenic plants [Martinez et al. 2006]. Under hydroponic conditions, the transgenics accumulated 24-fold more cadmium in roots and three-fold more in foliage, and 36-fold more lead in roots and nine-fold more in foliage, compared with wild-type plants. Interestingly, the transgenic tobacco plants grew better than nontransgenic control plants in all the mining soils tested, and had much more biomass than the natural hypeaccumulator Thlaspi caerulescens [Martinez et al. 2006]. One strategy for improving tolerance to cadmium is altered cellular metabolism leading to accumulation of particular solutes, including amino acids that stabilize proteins or stress proteins that protect or modify plants to reduce the content of undesired heavy metals [Chen and Murata, 2002]. A pioneering strategy was revealed in transgenic tobacco plants expressing a mouse metallothionein gene leading to the expression of the heavy metalchelating protein metallothionein and enhanced Cd tolerance [Maiti et al. 1989]. Cadmiumtreated metallothionein [mouse metallothionein gene-mMTF1] overexpressing transgenic tobacco plants exhibited higher riubulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco] and phosphoenylpyruvate carboxylase [PEPC] activities, smaller proline and carbohydrates

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content in the leaves relative to the non-transgenic plants, as shown in Figure 3 [Nedkovska, et al. 2004].

Figure 3. Changes in the activity of Rubisco in the leaves of non-transgenic [⎯] and transgenic [----] tobacco plants, treated with increasing CdCl2 concentrations [adapted from Nedkovska et al., 2004].

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In a follow up study, tobacco plants transformed with plant metallothionein gene [MTHis] accumulated significantly more cadmium both in roots and leaves suggesting that the overproduction of metallothioneins in both organs of plants were significantly correlated in terms of enhanced tolerance and accumulation of cadmium, as illustrated in Figure 4 [Gorinova, et al., 2007].

Figure 4. Cadmium accumulation in leaves [left] and roots [right] of transgenic and non-transformed tobacco plants [adapted from Gorinova et al., 2007].

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18.2.2. Genetic Manipulation of Metal Transporters, Alteration of Metabolic Pathway and Oxidative Stress Mechanisms

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For the development of efficient transgenic plants for phytoremediation purposes, genes can be transferred from hyperaccumulators or metal accumulating plants to conventional plants. Some of the possible areas of genetic manipulation include-

18.2.2.1. Metal Transporters It is known that this manipulation could alter metal tolerance/accumulation in plants. For example, transfer of Zn transporter- ZAT gene from Thlaspi goesingense to Arabidopsis thaliana resulted in two fold higher Zn accumulations in roots [Van der Zaal et al., 1999]. Likewise, transfer of yeast protein [YCF1], a member of ABC transporter family involved in transfer of cadmium into vacuoles by conjugation with glutathione, when transferred to Arabidopsis thaliana was shown to over-express and resulted in transgenic plants with enhanced lead and cadmium tolerance [Song et al., 2003]. In other study, Gaxiola et al. [2002] described experiments using genetic manipulations to alter the ion transport across the vacuolar membrane of Arabidopsis and yeast and suggested that it is possible to alter ion transport broadly by altering the proton gradient. And hence, the availability of mutants and the ability to transfer genes between these two organisms are likely to set the stage for a powerful method of analyzing a process as complex as ion transport. The ability to alter ion transport has both theoretical and practical applications. The ability to manipulate the proteins-pumps, transporters, and ion channel-responsible for the movement of ions across the vacuolar membrane will further enhance our understanding of the role of this organelle in the growth and development of plants. The capability of engineering the level and behavior of these pumps offers the possibility of increasing the tolerance of the plants of agricultural importance capable of growing in soils of high salinity and restricted water availability, as well as plant biofilters capable of detoxifying industrial waste sites containing ions toxic to humans [Gaxiola et al. 2002]. Moreover, new metabolic pathway can be introduced into plants for hyperaccumulation or phytovotalization as in case of MerA and MerB genes, introduced into plants which resulted in plants being several fold tolerant to mercury [Hg] and volatilized elemental mercury [Bizily et al., 2000]. Alteration of oxidative stress related enzymes may further altered metal tolerance. For example, enhanced aluminum tolerance by over-expression of glutathione-S-transferase and peroxidase is reported [Ezaki et al., 2000]. 18.2.2.2. Alteration in Roots and Biomass by Genetic Engineering In phytoremediation studies, it is important for plants to produce highly branched root systems with large surface area so that an efficient uptake of toxic metals could become possible. Eapen and Souza [2005] showed that Agrobacterium rhizogenes could enhance the root biomass in some hyperaccumulator plants. The hairy roots induced in some of the hyperaccumulators were shown to have high efficiency for rhizofiltration of radionucleides and heavy metals [Nedelkoska and Duran, 2000]. Biomass of known hyperaccumulators can be altered by introduction of genes affecting phytohormones synthesis and consequently leading to an enhanced biomass. Recently, biosynthetic pathways for most of the plant hormone classes and genes encoding many of the enzymes have been identified and cloned. These advances offer new opportunities to manipulate hormone content and regulate their

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biosynthesis [Hedden and Phillips, 2000]. As an example, increased gibberellins biosynthesis in transgenic trees was shown to promote growth and biomass production [Eriksson et al., 2000].

18.2.3. Examples of Transgenic Plants

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Each metal has specific mechanism for uptake, translocation and sequestration. Manipulation of metal transporters and vacuolar targeting of metal will have fruitful applications in the development of plants for phytoremediation. The regulatory control and use of tissue specific promoters offer great promise to develop plants for removal of elemental pollutants and radionucleides. Moreover, hyperaccumulators are loaded with acids and acid anions which have a function in metal storage or plant internal metal transport. Transgenic plants could be developed to secrete metal selective ligands into the rhizosphere which could specifically solubilize elements for phytoremediation. Understanding hyperaccumulators is likely to help in transfer of genes from hyperaccumulators to candidate plants and building a library of well characterized genes that function in mineral acquisition and storage will help in developing novel plants with improved hyperaccumulating traits. Based on these facts, it is essential to design suitable strategies for developing transgenic plants specific to each metal. However, a very few strategies have been used for development of transgenic plants [Eapen and Souza, 2005]. In this section, an attempt is made to highlight some of the strategies adopted for developing transgenic plants used in phytoremediation of toxic metals.

18.2.3.1. Phytoreduction of Mercury Mercury and mercurial compounds are hazardous to all biological organisms. Bacteria have evolved mechanisms for colonizing mercury contaminated environments and an operon of mercury resistance [mer] genes encoding for transporters and enzymes for biochemical detoxification have been identified [Summer, 1986]. Genetically engineered plants, like, Arabidopsis thaliana, Nicotiana tabacum and Liriodendron tulipifera with merA and merB genes have been produced [Bizily et al., 2000]. Such genetically altered transgenic plants grew well in the presence of toxic levels of mercury. In other study, three modified merA constructs were used for transformation of yellow poplar [Populus canadensis] proembryogenic masses, each having different amounts of altered coding sequences [Rugh et al., 2000]. These transgenic poplar trees when grown in soil with 40 ppm of Hg [II], produced higher biomass. 18.2.3.2. Tolerance to Selenium Selenium is another major environmental pollutant whose doses above its requirement [normal concentration] can result in toxic effects. The assimilation of sulfate and selenate is activated by ATP sulfurylase. Transgenic plants which over-expressed ATP sulfurylase gene [APS] had four fold higher APS enzymatic activity and accumulated three times more Se per plant than the wild type. The APS transgenic plants were more tolerant to Se, growing at higher rates than wild type [Pilon-Smith et al., 1999]. To obtain better insights into the effect of Se on metabolism and mechanisms of plants involved in Se tolerance, Hoewyk et al.

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[2008] studied the transcriptome of Arabidopsis plants grown with and without selenate and identified Se-responsive genes. These genes may help create plants that can better tolerate and accumulate Se, which may enhance the effectiveness of Se phytoremediation or serve as Sefortified food [Hoewyk et al. 2008].

18.2.3.3. Tolerance to Arsenic Arsenic is extremely toxic metalloid pollutant which is hazardous to human health. Dhankler et al. [2002] developed transgenic Arabidopsis plants, which could transport oxyanion arsenate to aboveground, reduced arsenite and sequester it in thiol peptide complexes. These transgenic plants accumulated 4 to 17-fold greater fresh shoot weight and accumulated two to three-fold more arsenic per gram of tissue than wild plants. 18.2.3.4. Iron Uptake Usually, soils contain insoluble Fe [III] oxides and hydroxides. A FRO2 gene encoding a ferric chelate reductase isolated from Fe deficient roots of Arabidopsis thaliana was capable of restoring ferric chelate reductase activity in an Arabidopsis mutant deficient in this enzyme [Robinson et al., 1999]. Two Fe [III] reductase FRE1 and FRE2 genes have been isolated from S. cerevisiae and transferred to tobacco [Mok and Machteld, 1998].

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18.3. REQUIREMENTS FOR DEVELOPING GENETICALLY ENGINEERED PLANTS USED FOR PHYTOREMEDIATION Candidate plants for genetic engineering for phytoremediation should possess the following properties, like- [i] be able to produce a high biomass over a short or long duration, such as trees [ii] be suitable to conveniently undergo genetic transformation. In this context, some of high biomass hyperaccumulators for which regeneration protocol is already developed include Indian mustard [Brassica juncea], sunflower [Helianthus annus], tomato [Lycopersicon esculentum], and yellow poplar [Liriodendron tulipifera L.]. Many of candidate plants for genetic engineering for phytoremediation are crop plants and when these plants are used for phytoremediation, they should not be consumed by humans and animals. However, for engineering any plants, at least three different approaches to enhance metal uptake can be envisioned, like- [i] enhancing the number of uptake sites [ii] alteration of specificity of uptake system to reduce competition by unwanted cations and [iii] increasing intracellular binding sites [Eapen and D, Souza, 2005].

18.3.1. Potential Genes for Improving Phytoremediation Phytoremediation is a physiological process and many toxic elements are taken up by plants by default pathway along with essential elements. Efforts are made to understand the genetics and biochemical processes involved in metal uptake, transport and storage by hyperaccumulating plants which in turn is expected to provide a greater insight into the process of metal detoxification by plants [Clemens et al. 2002; Pollard et al. 2002]. Principal phytoremediation strategies for metals are stabilization and accumulation. To further enhance

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the efficiency of metal phytoremediation, the following strategies may be used-[i] new suitable plant species may be identified [ii] selected species may be further bred for the desired property, via classical breeding or genetic engineering and [iii] agronomic practices may be optimized [Bennet et al., 2003]. Plant metabolomic engineering offers possibilities for introducing into plants new and/or altered pathways for biosynthesis of amino acids and their derived metabolites for tolerance to abiotic stresses [Sanjaya et al. 2008]. Remarkable progress made in transferring foreign genes into tomato has increased resistance to biotic and abiotic stresses [Chan et al. 2005]. Recently, Sanjaya et al. [2008] studied the molecular mechanisms of tryptophan and cadmium defense in transgenic Arabidopsis and tomato plants. Tryptophan is essential amino acids in plants; it plays a major role in the regulation of plant development and defense responses. These authors demonstrated the potential of tryptophan to confer cadmium tolerance in transgenic Arabidopsis and tomato plants. Though, this role of tryptophan was unknown, but this finding opens up new horizons in metabolic engineering for the further investigation on tryptophan with respect to metal physiology in plants [Figure 5].

Figure 5. Schematic view of metabolic engineering of tryptophan [Trp] biosynthesis pathway in plants with increased cadmium tolerance and nutritional quality [adapted from Sanjaya et al., 2008].

One of the abiotic stresses in agriculture arises from low iron [Fe] availability due to high soil pH and about 30% of arable land are too alkaline and hence, unfavorable for crop production [Takahashi et al., 2001]. Takahashi et al. [2001] reported that genetically Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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engineered rice [Oryza sativa] plants tolerated low iron availability in an alkaline soil carrying the barley [Hordeum vulgare] naat genes, which expresses crucial enzymes involved in the biosynthesis of phytosiderophores, natural iron chelators capable of solubilize iron in the soil. These genes were introduced into rice using Agrobacterium-mediated transformation. The two transgenes were expressed in response to low iron nutritional status in both the shoots and roots of rice transformants. Transgenic rice plants expressing the two genes had a higher nicotinamine aminotransferase activity and secreted larger amount of phytosiderophores than nontransformants under iron-deficient conditions. The transgenic rice showed an enhanced tolerance to low iron availability and had 4.1 times greater grain yields than that of the nontransformant rice in an alkaline soil [Takahashi et al., 2001]. In other studies, the expression of several iron-deficiency-responsive genes from tobacco and barley plants was evaluated in each host plant grown in iron deficient but cadmium treated soils. These conditions significantly enhanced NtIRT1 and HvIDS2 gene expression in roots, whereas other genes expression was similar in shoots and roots. Some expression under cadmium exposure differed from that under both [iron deficient and cadmium treated] conditions. It is possible that both the genuine Fe-deficiency-responsive mechanism and an unidentified mechanism, which can be directly regulated by cadmium, contribute to gene expression to maintain metal homeostasis within the plant [Hodoshima et al., 2007]. A novel stress-induced aldose/aldehyde reductase gene [ALR] was identified from the alfalfa somaticembryo-derived library. The data suggested that elevated ALR protein synthesis can ensure overall stress tolerance against a wide range of stress factors such as cadmium and low temperatures [Hegedüs et al. 2004]. The comparative transcriptional analysis of the Cd response of the Zn/Cd- hyperaccumulator Thlaspi caerulescens and the non-accumulator Arabidopsis indicates that there are specific responses to Cd exposure in each species and emphasizes the role of genes involved in lignin, gluthatione and sulphate metabolism [Van de Mortel et al. 2008]. Some technical factors however, limit the use of engineered plants. For instance, there are only a few plant systems of metal resistance and/or sequestration that are sufficiently characterized. However, metal resistance and accumulation are common in microorganisms [Silver, 1996]. Isolating genes from such metal tolerant bacterial species and introducing it into a plant could be an option to enhance detoxification of metals by plants. But such genetic transfer from prokaryotes to eukaryotic plant systems is complicated by the fact that the resistance is normally encoded by a large plasmid containing an operon with many genes involved in the resistance mechanism. In this context, Rugh et al. [1998] has been able to transfer only a single gene from such an operon.

18.3.2. Somatic Cell Hybridization It is worthwhile attempting somatic hybridization between high biomass candidate plants and low biomass metal hyperaccumulators to develop hybrids with biomass and hyperaccumulation capabilities. For example, somatic cell hybrids were produced between Brassica juncea, a high biomass lead [Pb] accumulator species and Thlaspi caerulescens, a known zinc and nickel hyperaccumulator. This hybrid showed increased resistance to Pb, Ni and Zn and the total amount of lead phytoextracted was much greater because of the amount of the biomass produced [Dushenkov et al., 2002]. In other study, Brewer et al. [1999]

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generated somatic hybrid between Brassica napus and the Zn hyperaccumulator Thlaspi caerulescens. Regenerated hybrids were able to maintain between 100% and 30% of biomass production in an agar based nutrient medium with 1mM added Zn2+, when compared to control plants grown at a low concentration of 30 μM added Zn2+. A concentration of 1mM added Zn2+ reduced biomass production of the Brassica napus parent to less than 10% of control biomass production. Hybrids survived up to four months and some of them flowered.

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18.4. DETOXIFICATION OF TRACE ELEMENTS In phytoremediation, tolerance to elevated concentrations of trace elements either in the rooting medium or in plant tissues is desirable. A number of transgenic approaches were aimed at reducing the concentration of free metal cation in the cytoplasm [Berken et al., 2002]. Three major strategies can be followed- [i] chemical transformation and/or volatilization [ii] overproduction of metal chelators or compounds which bind the trace element and [iii] efflux from the cytoplasm, through transport into the vacuole [Berken et al., 2002]. A number of transgenic plants have been engineered to contain large amounts of recombinant proteins with a possible role in chelation, assimilation or membrane transport of trace elements. And to estimate the quality of trace element accumulation or votalization rates, such transgenic plants were grown in hydroponics or agar-based media. However, no conclusive data are so far available on the performance of these transgenic plants on solid substrates or under field conditions, where trace element bioavailability is substantially lower. It is therefore, difficult to predict the effects of the expression of a transgene at the level of whole plant. Understanding the metal homeostasis in plants will be vital for developing successful phytoremediation technologies. The attempt to genetically engineer plants with improved phytoremediation efficiency has largely been centered on transformation of the nuclear genome. An alternative and novel approach could be to engineer the chloroplast genomes of higher plants. This approach offers several advantages over nuclear transformation like- [1] very high levels of transgene expression [De Cosa et al., 2001] [2] uniparental plastid gene inheritance [in most crop plants] that prevent pollen transmission of foreign DNA [Daniell and Parkinson, 2003] [3] the absence of gene silencing [Lee et al., 2003] and positioning effect [ Daniell et al. 2001] [4] the ability to express multiple genes in a single transformation event [Daniell and Dhingra, 2002] [5] the ability to express bacterial genes without codon optimization [De Cosa et al., 2001] [6] integration via a homologous recombination process that facilitates targeted transgene integration [Daniell et al., 2002] and [7] sequestration of foreign proteins in the organelle, which prevent adverse interactions with cytoplasmic environment [Lee et al., 2003]. By applying chloroplast genome approach, Ruiz et al. [2003] for the first time reported that chloroplast genome engineered plants had enhanced phytoremediation ability. For this, a native bacterial operon was used for expression in plants without codon optimization. Using this approach, they integrated a native operon containing the merA and MerB genes, coding for mercuric ion reductase and organomercurial lyase, into tobacco Nicotiana tabacum chloroplast genomes. The results show that the chloroplast transgenic plants were more resistant than wild type to the highly toxic organomercurial compound.

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18.5. TRANSGENIC-FIELD TESTING AND RISK ASSESSMENT

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Large-scale field testing of transgenic plants is required to assess their capability under conventional agronomic practices. Currently, no transgenics has so far been commercially used for phytoremediation purposes in derelict soils. However, three transgenic Indian mustard lines were tested under field conditions for their ability to remove selenium [Se] from Se- and boron-contaminated saline sediment. The transgenic lines over expressed genes encoding the enzymes adenosine triphosphate sulfurylase [APS], gamma-glutamyl-cysteine synthetase [ECS] and glutathione synthetase [GS], respectively. The APS, ECS, and GS transgenic plants accumulated 4.3, 2.8, and 2.3-fold more Se in their leaves than wild type, respectively. The GS plants significantly tolerated the contaminated soil better than wild type, attaining an aboveground biomass/area almost 80% of that of GS plants grown on clean soil, compared to 50% for wild type plants. This was probably the first report suggesting that genetically engineered plants can successfully reduce the toxicity of heavy metals under metal poisoned field environments [Banuelos and Terry, 2005]. Recently, Banuelos et al. [2007] tested two new transgenic Indian mustard Brassica juncea L. lines having over expressing genes encoding the enzymes selenocysteine lyase [cpSL] and selenocysteine methyltransferase [SMT] under field conditions for their ability to accumulate selenium [Se] from Se- and boron-contaminated saline sediment. The findings of this study demonstrated that cpSL and SMT transgenic lines had significantly greater Se phytoremediation potential than wild type Indian mustard. Generally, the application of genetically engineered plants under field environment poses some threat to wild life and subsequently to humans. While, risks associated with mercury or selenium volatilizing plants pose negligible threat to the environment [Meagher et al., 2000; Lin et al., 2000]. Moreover, the risk of escape of the genes from the transgenic plants is also negligible. The risk of metal ingestion by wild life can be prevented by suitable fencing off the area and use of non-palatable species [Skarzhinskaya et al., 2003].

CONCLUSION AND FUTURE PERSPECTIVES Phytoremediation is indeed an emerging area with great potential and promises. Further development of phytoremediation requires integrated multidisciplinary approaches including scientists from plant biology, genetic engineering, soil chemistry, soil microbiology, as well as agricultural and environmental engineering groups. The understanding of the biochemical processes involved in plant heavy metal uptake, transport, accumulation and tolerance will help in systematic improvements in phytoremediation using molecular approaches. It has been shown in a number of studies that plant trace element metabolism can be genetically manipulated, leading to plants with altered metal tolerance, accumulation and/or biotransformation capacity [Liu et al., 2005]. The remedial capacity of plants can be significantly improved by genetic manipulation and plant transformation technologies [Cherian and Oliveira, 2005]. The knowledge gained from such studies in conjunction with biotechnology has helped to improve, substantially, the phytoremediation capability of plants. New transgenic plants have been developed with improved capacity for metal uptake, transport, and accumulation, as well as for detoxification of heavy metals. However, in order

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to attain further benefits, following questions needs to be addressed- [i] how manipulation of metal transporters and their cellular targeting to specific cell types such as, vacuoles, allow for safe compartmentation of heavy metals in locations do not disturb other cellular functions? [ii] how the risk of transgene escape could be avoided while manipulating the chloroplast genome? [iii] how the entry of metals into food chain could be restricted once they enter into soils? [Li et al. 2004] [iv] how the colonization and interaction between metal tolerant microbes and transgenic plants be improved? [v] How transgenic research could be helpful in addressing the problem of mixed contamination occurring in many of the polluted sites. However, a multigene approach involving the simultaneous transfer of several genes into suitable candidate plants may help to remove contaminants of mixed or complex nature [Cherian and Oliveira, 2005] and [vi] extensive field trials for transgenic plants possessed with metal toxicity reducing ability are required before it becomes commercially viable and acceptable by the farming communities. In days to come, plant genetic engineering for improved phytoremediation could benefit from the data of genomic and postgenomic projects including proteomics. Advances in bioinformatics and the development of more sophisticated expression systems may be used to create efficient, publicly acceptable plants that can be used for wide-scale phytoremediation projects [Leduc and Terry, 2006]

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REFERENCE Baker A.J., Brooks R.R. [1989] Terrestrial higher plants which hyperaccumulate letallic elements – a review of their distribution, ecology and phytochemistry, Biorecovery 1, 81126. Banuelos G., Terry N. [2005] Field trial of transgenic Indian mustard plants shows enhanced phytoremediation of selenium-contaminated sediment, Environ. Sci. and Technol. 39, 1771-1777. Banuelos G.,Leduc D., Pilon-Smits E. A., Terry N. [2007] Transgenic Indian mustard overexpressing selenocysteine lyase or selenocysteine methyltransferase exhibit enhanced potential for selenium phytoremediation under field conditions, Environ. Sci. Technol.41, 599-605. Bennet L.E., Burkhead J.L., Hale K.L., Terry, N., Pilon M., Pilon-Smits E.A.H. [2003] Analysis of transgenic Indian mustard plants for phytoremediation of metal contaminated mine tailings, J. Environ. Qual. 32, 432 - 440. Berken A., Mulholland M.M., LeDuc D.L., Terry N. [2002] Genetic engineering of plants to enhance Selenium phytoremediation, Critical Rev. in Pl. Science, 21[6], 567-582. Berti W.R., Cunningham S.D. [2000] Phytostabilization of metals. In: Raskin I., Ensley B.D. [eds] Phytoremediation of toxic metals, using plants to clean up the environment, WileyInterscience, New York, pp. 71-88. Bizily S.P., Rugh C.L., Meagher R.B. [2000] Phytodetoxification of hazardous organomercurials by genetically engineered plants, Nat. Biotechnol. 18, 213-217. Blaylock M., Huang J. [2000] Phytoextraction of metals. In: Phytoremediation of contaminated soil and water. CRC Press LLC, pp. 53-70.

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Brewer E.P., Saunders J.A., Angle J.S., Chaney R.L., McIntosh M.S. [1999] Somatic hybridization between the zinc hyperaccumulator Thlaspi caerulescens and Brassica napus. Theor. Appl. Genet. 99, 761-771. Chan Y.L., Prassad V., Sanjaya, Chen K.H., Liu P.C., Cheng C.P., Chan M.T. [2005] Transgenic tomato plants expressing an Arabidopsis thionin [Thi2.1]driven by fruitinactive promoter battle against phytopathogenic attack, Planta 221, 386-393. Chaney R., Li Y., Brown S., Homer F., Malik M., Angle J., Baker A., Reeves R., Chin M. [2000] Improving metal hyperaccumulator wild plants to develop commercial phytoextraction systems: approaches and progress. In: Phytoremediation of contaminated soil and water. CRC Press LLC, pp. 129-152. Chapell, J. [1998] Phytoremediation of TCE in groundwater using Populus. US Environmental Protection Agency. http://clu-in.org/products/intern/phytotce.htm Chen T.H., Murata N. [2002] Enhancment of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes. Current Opinion In Plant Biology 5, 250-257. Cherian S., Oliveira M.M. [2005] Transgenic plants in phytoremediation: recent advances and new possibilities. Environ. Science and Technol. 39, 9377-9390. Clemens S., Palmgren M.G., Kranmer U. [2002] A long way ahead: understanding and engineering plant metal accumulation. Trends Plant Sci. 7, 309-314. Cunningham S.D., Shann L.R., Crowley D.E., Anderson T.A. [1997] Phytoremediation of contaminated water and soil. In: Kruger E.L., Anderson t.A., Coats J.R. [eds] Phytoremediation of soil and water, ACA Symposium Ser.N 664, American Cemical Society, Washington, DC. Daniell H., Lee S.B., Panchal T., Weibe P.O. [2001] Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic chloroplasts, J. Mol. Biol. 311, 1001-1009. Daniell H., Dhingra A. [2002] Multigene engineering: dawn of an exciting new era in biotechnology. Curr. Opin. Biotechnol. 13, 136-171. Daniell H., Khan M.S., Allison L. [2002] Milestones in chloroplast genetic engineering: an environmental friendly era in biotechnology, Trends Plant Sci. 7, 84-91. Daniell H., Parkinson L. [2003] Jumping genes and containment, Nat. Biotechnol 21, 374375. Dhankler O.P., Li Y., Rosen B.P., Shi J., Salt D., Senecoff J.F. [2002] Engineering tolerance and hyperaccumulation of arsenic in plants by combining arsenate reductase and γglutamyl cysteine synthase expression, Nat. Biotechnol. 20, 1140 – 1145. De Cosa B., Moar W., Lee S.B., Miller M., Daniell H. [2001] Hyper-expression of the Bt Cry2Aa2 operon in chloroplast leads to formation of insecticidal crystals, Nat. Biotechnol 19, 71-74. Dominguez-Solis J.R., Gutierez-AlcalaG., Romero L.C., Gotor C. [2001] The cytosolic Oacetylserine [thiol] lyase gene is regulated by heavy metals and can function in cadmium tolerance, J. Biol. Chem. 276, 9297-9302. Doty, SH. [2008] Enhancing phytoremediation through the use of transgenics and endophytes, New Phytologist [on line], 1-16. Dushenkov S., Kapulnik Y. [2000] Phytofiltration of metals, In: Raskin I., Ensley B.D. [eds] Phytoremediation of toxic metals, using plants to clean up the environment, WileyInterscience, New York, pp. 89-106.

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M. Nedkovska and N. Gorinova

Dushenkov S., Skarzhinskya M., Glimelius K., Gleba D., Raskin I. [2002] Bioengineering of phytoremediation plant by means of somatic hybridization, Int. J. Phytoremediation 4, 117-126. Eapen S., D,Souza S.F. [2005] Prospects of genetic engineering of plants for phytoremediation of toxic metals. Biotechnology Advances, 23, 97-114. Eriksson M.E., Israelson M., Olson O., Moritz T. [2000] Increased giberellin biosynthesis in transgenic trees promotes growth, biomass production and xylem fiber length, Nat. Biotechnol. 18, 84-788. Ezaki B., Gardner R.C., Ezaki Y., Matsumoto H. [2000] Expression of Aluminium induced genes in transgenic Arabidopsis plants can ameliorate Al stress and/or oxidative stress, Plant Physiol. 122, 657-665. Gaxiola R.A., Fink G.R., Hirschi K.D. [2002] Genetic manipulation of vacuolar proton pumps and transporters, Plant Physiol. 129, 967-973. Goldsbrough P. [1998] Metal tolerance in plants: the role of phytochelatins and metallothioneins, in: Terry N., Banuelos G. [Eds.], Phytoremediation of trace elements. Ann Arbor Press, MI. Gorinova N., Nedkovska M., Todorovska E., Simova-Stoilova L., Stoyanova Z., Georgieva K., Demirevska-Kepova K., Atanassov A., Herzig R., [2007] Improved phytoaccumulation of cadmium by genetically modified tobacco plants [Nicotiana tabacum L.]. Physiological and biochemical response of the transformants to cadmium toxicity, Environ. Pollut. 145, 161-170. Hedden P., Phillips A.L. [2000] Manipulation of hormone biosynthesis genes in transgenic plants, Curr. Opin. Biotechnol. 1, 130-137. Hegedüs A., Erdei S., Janda T., Tóth E., Horváth G., Dudits D. [2004] Transgenic tobacco plants overproducing alfalfa aldose/aldehyde reductase show higher tolerance to low temperature and cadmium stress, Plant Sci. 166, 1329-1333. Hodoshima H., Enomoto Y., Shoj K., Shimada H., Goto F., Yoshihara T. [2007] Differential regulation of cadmium-inducible expression of iron-defficiency-responsive genes in tobacco and barley, Physiol. Plantarum 129, 622-634. Hoewyk van D., Takahashi H., Inoue E., Hess A., Tamaoki M., Pilon-Smits E. [2008] Transcriptome analyses give insights into selenium-stress responses and selenium tolerance mehanisms in Arabidopsis, Physiol. Plantarum 132, 236-253. Horne A. [2000] Phytoremediation by constructed wetlands. In: Terry N., Bonuelos G. [eds.], Phytoremediation of contaminated soil and water, pp. 13-14, Lewis, Boca Raton, Florida. Karenlampi S., Schat H., Vangronsveld J, Verkleij J., Van der Lelie D., Mergeay M., Tervahauta A. [2000] Genetic engineering in the improvement of plants for phytoremediation of metal polluted soils, Environ. Pollut. 107, 225-231. Kim S., Takahashi M., Higuchi K., Tsunoda K., Nakanishi H., Yoshimura E., Mori S., Nishizawa N. [2005] Increased nicotianamine biosynthesis confers enhanced tolerance of high levels of metals, in particular Nickel, to plants, Plant Cell Physiol. 46, 1809-1818. Kumar N.P., Dushenkov V., Motto H., Raskin I. [1995] Phytoextraction; the use of plants to remove heavy metals from soils. Environ. Sci. Technol. 29, 1232-1238. Leduc D., Terry N. [2006] Genetic engineering stress tolerant plants for phytoremediation, in: Rai, A.R., Takabe T. [eds], Abiotic Stress tolerance in plants, Springer, Printed in Netherlands, pp. 123-133.

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Genetic Manipulations of Metal Accumulation and Heavy Metal Tolerance

393

Lee S.B., Known S., Park S., Jeong M., Han S., Byun M., Daniell H. [2003] Accumulation of trehalose within transgenic chloroplasts confers drought tolerance, Mol. Breed. 11, 1-13. Li Y., Dhankler O.P., Carreira L., lee D., Chen A., Schroeder J.I., Balish R.S., Meagher R.B. [2004] Overexpression of phytochelatin synthase in Arabidopsis leads to enhanced arsenic tolerance and cadmium hypersensitivity, Plant Cell Physiol. 45[12], 1787-1797. Lin Z.Q., Schmenauer R.S., Cervinka V., Zayed A.,. Lee A., Terry N. [2000] Selenium volatilization from the soil-Salicornia bigelovii for treatment system for the remediation of contaminated water and soil in the San Joaquin valley, J. Environ. Qual. 29, 10481056. Liu Y., Zhu Y.G., Chen B.D., Christie P., Li X.L. [2005] Yield and arsenate uptake of Arbuscular mycorrhizal tomato colonized by Glomus mosseal BEG167 in as spiked soil under glasshouse conditions, Environ. Int. 31, 867 - 873. Lytle C.M., Lytle F.W., Yang N., Qian J.H., Hausen D., Zayed A. Terry N. [1998] Phytoconversion of hexavalent chromium [Cr6+] to trivalent chromium [Cr3+] by wetland plants. Environ. Sci. Technol. 32, 3087-3083. Maiti I.B., Wagner G.J., Yeargan R., Hunt A.G. [1989] Inheritance and expression of the mouse metallothionein gene in tobacco: impact on Cd tolerance and tissue Cd distribution in seedlings. Pl. Physiology 91, 1020-1024. Martinez M., Bernal P., Almela C., Velez D., Garcia-Agustin P., Serrano R., Navaro-Avino J. [2006] An engineered plant that accumulates higher levels of heavy metals than Thlaspi caerulescens, with yields of 100 times more biomass in mine soils, Chemosphere 64, 478-485. Meagher R.B., Rugh C.L., Kandasamy M.K., Gragson G., Wang N.J. [2000] Engineered phytoremediation of mercury pollution in soil and water using bacterial genes, in: Terry N., Baňuelos G. [Eds.], Phytoremediation of Contaminated Soil and Water. Boca Raton, Lewis, F1, pp. 201-221. Mok D.W., Machteld C.M. [1998] Expression of the yeast FRE genes in transgenic tobacco, Plant Physiol. 118, 51-58. Nedelkoska T.J., Doran P.M. [2000] Hyperaccumulation of cadmium by hairy roots of Thlaspi caerulescens, Biotechnol. Bioeng. 67, 607-615. Nedkovska M., Gorinova N.., Markovska Y., Atanassov A. [2004] Changes in the activities of carboxylating enzymes, organic acids and carbohydrates content in cadmium-treated metallothionein overexpressing transgenic tobacco plants, Biotechnol. and Biotechnol. Eq. 18, 101-106. Noji M., Saito M., Nakamura M., Aono M., Saji H., Saito K. [2001] Cysteine synthase overexpression in tobacco confers tolerance to sulphur-containing environment pollutants, Plant Physiol. 126, 973-980. Pilon-Smith E., Hwang S., Mel C., Zhy Y., Tai J., Bravo R. [1999] Overexpression of ATP sulfurylase in Indian mustard leads to increased selenate uptake, reduction and tolerance, Plant Physiol. 119, 123-132. Pilon-Smith E., Pilon M. [2002] Phytoremediation of metals using transgenic plants, Crit. Rev. Plant Sci. 21, 439-456. Pollard A.J., Powell K.D., Harper F.A., Smith J.A.C. [2002] The genetic basis of metal hyperaccumulation in plants. Crit. Rev. Plant Sci. 21, [6], 539-566. Robinson N.J., Procter C.M., Connoly E.L, Guerinot M.L. [1999] A ferric chelate reductase for uptake from soils, Nature 397, 694-697.

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M. Nedkovska and N. Gorinova

Rugh C.L., Senecoff J.F., Meagher R.B., Merkle S.A. [1998] Development of transgenic yellow poplar for mercury phytoremediation, Nat. Biotechnol. 16, 925-928. Rugh C.L., Bizily S.P., Meagher R.B. [2000] Phytoremediation of environmental mercury pollution, in: Raskin I., Ensley B. [Eds.], Phytoremediation of toxic metals using plants to clean up the environment, New York, Wiley, pp. 231 – 245. Ruiz O.N., Hussein H.S., Terry N., Daniell H. [2003] Phytoremediation of organomercurial compounds via chloroplast genetic engineering, Plant Physiol. 132[3], 1344-1352. Salt D., Smith R., Raskin I. [1998] Phytoremediation. Ann. Rev. Plant Physiol. Plant Mol. Biol. 49, 643-668. Sanjaya, Hsiao P., Su R., Ko S., Tong C., Yang R. [2008] Overexpression of Arabidopsis thaliana tryptophan synthase beta1 [AtTSB1] in Arabidopsis and tomato confers tolerance to cadmium stress, Plant, Cell and Environment [online], 1-12. Silver S. [1996] Bacterial resistance to toxic metal ions – a review. Gen. 179, 9-19. Skarzhinskaya M., Svab S., Maliga P. [2003] Plastid transformation in Lesquerella fendleri, an oilseed Brassicacea, Transgenic Res.12, 115-122. Song W.Y., Sohn E.J., Martinoia E., Lee Y.J., Yang Y.Y., Jasinski M. [2003] Engineering tolerance and accumulation of lead and cadmium in transgenic plants, Nat. Biotechnol. 21, 914 – 919. Summer A.O. [1986] Organization, expression and evolution of genes for mercury resistance, Annu. Rev. Microbiol. 40, 607-634. Takahashi M., Nakanishi H., Kawasaki S., Nishizawa K., Mori S. [2001] Enhanced tolerance of rice low iron availability in alkaline soils using barley nicotinamine aminotransferase genes, Nat. Biotechnol. 19, 466-469. Terry N., Zayed A.M., de Souza M.P., Tarun A.S. [2000] Selenium in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51, 401-432. Van der Zaal B.J., Neuteboom L.W., Pinas J.E., Chardonnen A.N., Schat H., Verkleij J.A [1999] Overexpression of a novel Arabidopsis gene related to putative Zn transporter genes from animals can lead to enhanced Zn resistance and accumulation, Plant Physiol. 119, 1047-1055. Van de Mortel J., Schat H., Moerland P., Themat van L., Ent der Van S., Blankestijn H., Ghandilyan A., Tsiatsiani S., Aarts M. [2008] Expression differences for genes involved in lignin, glutathione and sulphate metabolism in response to cadmium in Arabidopsis thaliana and the related Zn/Cd-hyperaccumulator Thlaspi caerulescens, Plant Cell Environ. 31, 301-324. Zhu Y.L., Pilon-Smith E., Jonanin L., Terry N. [1999] Overexpression of glutathione synthase in Indian mustard enhances Cd accumulation and tolerance, Plant Physiol.119, 73-80.

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Chapter 19

BIOLOGICAL CONTROL OF PLANT NEMATODES WITH PHOSPHATE- SOLUBILIZING MICROORGANISMS Mujeebur Rahman Khan∗1, Shahana Altaf2, Fayaz A. Mohidin3, Uzma Khan4 and Arshad Anwer5 1,4,5

Department of Plant Protection, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh 202 002, India 2 UCSF Medical Center, University of California, San Francisco, USA 3 Department of Botany, University of Kashmir, Srinagar, India

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Plant nematodes are important pests of agricultural crops and cause singnificant crop damage throughout the world. Management of nematode may be achieved by applying phosphate-solubilizing microorganisms. Experiments conducted mostly under pot conditions have revealed that soil application or seed treatment with the fungi such as, Aspergillus niger, Penicillium digitatum, P. anatolicum, Paecilomyces lilacinus, Trichoderma spp. etc. reduced the crop damage, reproduction and soil population of some important ecto- and endop-parasitic nematodes. Culture filtrates of these fungi inhibited the hatching of eggs and induced mortality to nematode larvae in vitro. The use of phosphate-solubilizing bacteria in nematode management has been extensively employed, and the results have demonstrated that application of effecient strains of Bacillus subtilis, B. polymyxa, Pseudomonas fluorescens, P. stutzeri, P. striata etc. effectively controlled the nematodes and profoundly improved the crop yields. Overall performance of phosphate-solubilizing microorganisms against plant nematodes have been found to a level that ensures their commercial exploitation. This necessitates further in-depth studies under field condition in different agro-climates. Development of efficient immobilizing systems which could support the longer survival of the PSMs and also their multiplication during storage and marketing is likely to prove critical in commercializing such microbes in crop protection.

∗

Correspondence to: [email protected]

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19.1. INTRODUCTION Plant parasitic nematodes are considered important pathogens of agricultural crops. Nematodes cause damage to plants by injuring and feeding on the root hairs, epidermal cells, cortical and/or stealer cells [Khan, 2008]. A large number of nematodes, like, Tylenchus, Hoplolaimus, Rotylenchus, Helicotylenchus, Tylenchorhynchus, Belonolaimus, Trichodorus and Longidorus are ectoparasites feeding on root surface. However, a considerable number of nematodes enter fully inside the host root and are called endoparasites, such as, root-knot nematodes [Meloidogyne spp.], cyst forming nematodes [Heterodera spp.] and root-lesion nematode [Pratylenchus spp.]. Whereas some nematodes such as, citrus nematode [Tylenchulus semipenetrans] and reniform nematode [Rotylenchulus reniformis] are semiendoparasites as they partially enter the host tissue. The most common effect of nematode parasitism is debilitation of the plant with a symptom of stunting of plant growth and mild yellowing of foliage that resembles with nutritional deficiency. As a result, instead of namaticide, fertilizer is applied by farmers which prove ineffective and noneconomic. Nonspecific or general symptoms of nematode infestation appear as patches of plants irregularly distributed in a field showing stunted growth, sparse and dull green or pale yellow foliage [Luc et al., 2005]. The infested plants show incipient wilting despite adequate soil moisture during sunny days but recover at night. Further, roots so weakened and damaged by nematodes are easily invaded by many bacteria and fungi, leading to accelerated root decay. This secondary damage also does not draw immediate attention and an incurable stage is soon reached leading to severe yield losses [Khan and Reddy, 1993] both quantitatively and qualitatively under subtropical and tropical conditions [Sasser, 1989]. For example, Molya of wheat [Heterodera avenae], ufra of rice [Ditylenchus angustus], root rot of maize [Pratylenchus zeae], root-knot of cereals, pulses [Meloidogyne spp.] etc. are some of the diseases which cause tremendous economic loss to all kind of crops [Luc et al., 2005]. Nematodes may cause about 7-12% yield loss to various crops. The yield losses vary greatly depending on inoculum level and host species. The severe infection may result to as much as 80-90% yield decline in an individual field and sometimes plants fail to give yield of economic value. However, nematodes do not always cause hidden damage. When fields are heavily infested, characteristic symptoms appear on roots or shoots. Specific symptoms include root-lesions, root-rot, root pruning, root-galls, cessation of root growth etc. [Khan, 2008] [Figure 1]. Some nematodes also cause characteristic symptoms on above ground parts. For example, Anguina tritici causes seed galls and as a result, normal grains of wheat, barley and oat turn brown to black and irregular in shape. Similarly leaf tips of rice become white due to infection of Aphelenchoides besseyi. In addition to direct damage, nematodes often aid or aggravate the diseases caused by fungi, bacteria and viruses or may break resistance of cultivars to pathogens. Hairy root of roses caused by Agrobacterium rhizogenes is of minor importance, but in the presence of Pratylenchus vulnus the disease becomes severe [Sitaramaiah and Pathak, 1993]. The fusarium wilt resistant cultivars of cotton become susceptible in the presence of root-knot nematodes [Atkinson, 1892]. The crop damage, however, depends largely on the plant species or cultivar, nematode species, level of soil infestation and the prevailing environmental conditions. Plant nematodes may also act as vector for bacteria, fungi and viruses. For instance, Anguina tritici carries Clavibacter tritici and Dilophospora alopecuri to shoot meristem of wheat [Khan and Dasgupta, 1993].

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Figure 1. Symptoms of nematode infection in plants, root necrosis caused by Pratylenchus sp. [A] root galls by Meloidogyne spp. [B] cysts by Globodera sp. [C] and seed galls by Anguina tritici [D].

Nematode transmitted polyhedral viruses [NEPO], which are ringspot viruses for example, tobacco ring spot virus, is transmitted by Xiphinema and Longidorus species. Trichodorus and Paratrichodorus species act as vector for certain tobra viruses such as tobacco rattle and pea early browning viruses [Taylor and Brown, 1997]. Despite a significant impact on agriculture, nematodes have not been recognized as major pest of crops, probably because of the damage caused by nematodes is less obvious than that caused by fungi or other pathogens. Crop losses due to nematodes are greater in the developing countries than the developed countries. It is probably due to unplanned agricultural practices, unawareness of the farmers about nematodes and non availability of nematicides. In the developed countries where management practices are properly implemented, the nematode damages are relatively low; still causing considerable yield losses. Foe example annual monetary loss due to nematodes has been estimated over $ 6.0 billion in USA alone [Agrios, 2005]. In the high input agricultural practices, the control of pests and pathogens using biological preparation seems quite difficult due to wide application of chemical pesticides. However, there are numerous instances where one or the other antagonistic organism has reduced the populations of plant pathogens including plant nematodes. For example, suppression of cereal-cyst nematode, Heterodera avenae found in certain soils [suppressive] under monocultures of susceptible cereals in northern Europe is the best example of natural control of any plant-parasitic nematode [Kerry, 1982]. The suppression is attributed to the population build up of parasitic fungi Pochonia chlamydosporia [Verticillium chlamydosporium] as continuous monoculturing leads to parasitization of the developing females resulting in 95-97% reduction in nematode population [Kerry et al., 1982].There are

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also some other microorganisms that have demonstrated considerable potential antagonism against plant nematodes [Stirling, 1993; Khan, 2005]. Biological control of nematodes may be achieved with two kinds of microorganisms. Classical parasites or predators (e.g., Paecilimyces lilacinus, Dactylaria candida, Pasteuria penetrans) have been used in the nematode control since old times (Khan and Khan, 1995). In recent years, interest has also been developed in using plant growth promoting microorganisms. Of these, phosphate-solubilizing microorganisms [PSM] are most important and may prove efficient biocontrol agents of plant nematodes. The PSMs may suppress rhizosheric nematode populations by promoting host growth, inducing systemic resistance and/or producing nematoxic metabolites [Kirkpatric et al., 1964], like, bulbiformin [Brannen, 1995], phenazin [Toohey, 1965] and pyoleutorin [Howell and Stepinovic, 1980]. Nematode management employing phosphate-solubilizing fungi or bacteria has advantage over classical biocontrol agents as the former provide essential nutrients to plants in addition to their nematode inhibiting ability. Aspergillus niger, Penicillium spp., Trichoderma, Bacillus subtilis, B. polymyxa, Pseudomonas fluorescens, P. stutzeri, P. striata, nematophagous fungus Arthrobotrys oligospora etc. are some of the promising phosphate-solubilizers inhabiting agricultural soils [Gaur, 1990, Rao, 1990; Rudresh et al., 2005; Duponnois et al., 2006; Pandy et al., 2008] and possess promising potential of nematode antagonism. In the following section, the role of these fungi and bacteria in nematode management is discussed.

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19.2. PHOSPHATE-SOLUBILIZING FUNGI IN NEMATODE MANAGEMENT Numerous phosphate-solubilizing fungi in addition to providing essential nutrients [Thomas et al. 2005] including solubilizing insoluble P [Turan 2006] and consequently making it available to plants [Mittal et al., 2008], are also known to suppress plant nematodes [Table 1] [Khan et al., 2000; Oyekanmi, et al., 2008]. Phosphate-solubilizing fungi may directly affect the various developmental stages of nematodes viz., eggs, larvae and adults [Pocasangre et al., 2007, Pant and Pandey, 2001; Oyekanmi, et al., 2008; Sharon et al., 2009]. The phosphate-solubilizing fungi may suppress plant parasitic nematodes through the following mechanisms of action.

19.2.1. Antibiosis Antibiosis is the phenomenon of suppression of one organism by the other due to release of some toxic substances/metabolites in the environment. Antibiosis provides a competitive saprophytic advantage to phosphate-solubilizing fungi including species of Trichoderma, Aspergillus and Penicillium [Lipping et al., 2008]. Low molecular weight compounds or antibiotics [both volatile and non volatile] produced by Trichoderma species and Aspergillus spp. impede colonization of harmful microorganisms including nematodes in the root zone [Eapen and Venugopal 1995]. Harzianic acid, alamethicins, tricholin, peptaibols, antibiotics, 6-penthyl-α-pyrone, massoilactone, viridin, gliovirin, glisoprenins, heptelidic acid, oxalic

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acid and enzymes are some of the chemicals produced by Trichoderma and Aspergillus species [Mankau, 1969, a, b; Benitez et al. 2004; El-Hasan et al., 2007]. Table 1. Effect of different phosphate solubilizing fungi on plant parasitic nematode infesting agricultural crops Phosphate solubilizing fungi Aspergillus niger A. niger A. niger Paecilomyces lilacinus P. lilacinus P. lilacinus P. lilacinus P. lilacinus

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P. lilacinus Penicillium anatoticum T. harzianum, P. lilacinus T. harzianum and P. fluorescens T. harzianum T. harzianum Trichoderma asperellum-203 and Trichoderma atroviride T. pseudokoningii, T. viride, P. lilacinus, A.niger, G.mosseae , P. fluorescens and P. putida T. harzianum, P. lilacinus T. atroviride T. harzianum [T014] and Pseudomonas fluorescens [PS 07] P. chlamydosporia and T. harzianum T. harzianum and P. chlamydosporia

Nematode managed Meloidogyne incognita M. incognita M. incognita Root-knot nematode, Meloidogyne spp. Meloidogyne spp. Meloidogyne javanica R. reniformis Meloidogyne species

Host plant Tomato Okra Tomato -

Reference Singh et al. [1991] Sharma et al., [2005] Khan and Anwer [ 2007] Pal and Gardener [2006] Schenek [2004] Hewelett et al., [1988] Lysek [1966] Jatala [1986]

Meloidogyne species Globodera rostochinensis M. incognita M. javanica

Tomato Tobacco Ttomato Numerous crops Okra. Potato Chickpea Tomato

M. arenaria, Meloidogyne spp. M. javanica

Corn Cardamom -

Pant and Pandey [2002] Siddiqui and Shaukat [2004] Windham et al [1989] IISR [1995] Sharon et al. [2009]

M. incognita

Soybean

Oyekanmi et al. [2008]

Meloidogyne javanica R. similis

Okra Banana

M. incognita

Gladiolus

Zareen et al. [2001] Felde et al., [2006] Pocasangre et al., [2007] Khan and Mustafa [2005]

Globodera rostochiensis and G. pallid H. cajani

Potato

Saifullah [1996a, b]

Pigeonpea.

Siddiqui and Mahmood [1996]

Khan and Ejaz [1997] Jatala [1986]

19.2.2. Stimulation of Host Defense Response The ability of phosphate-slubilizing fungi including strains of Trichoderma, Aspergillus niger, and Penicillium digitatum to protect plants against root pathogens has long been attributed to its antagonistic effect against the invading pathogen [Chet et al., 1997; Vassilev et al., 2006]. Strains of Trichoderma may induce hypersensitive response [HR], systemic

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acquired resistance [SAR] and induced systemic resistance [ISR] in plants [Harman et al., 2004]. Enzymes such as, phenyl alanine ammonia lyase [PAL] and chalcone synthase [CHS] are produced that trigger the biosynthesis of phytoalexins, chitinases and glucanases which may oppose nematode feeding. Trichoderma spp. may induce host resistance by producing metabolites which may act as elicitors of plant resistance. Such metabolites may also induce expression of genes responsible for synthesis of phytoalexins, PR proteins or other compounds involved in increasing resistance against plant pathogens. It has been reported that T. harzianum induced resistance in bean [Meyer et al., 1998], cucumber [Koike et al., 2001] and cotton [Hanson and Howell, 2004].

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19.2.3. Plant Growth Promotion by Phosphate-Solubilizing Fungi Root colonization by phosphate-solubilizing fungi frequently enhance root growth and development, crop productivity, resistance to abiotic stresses and the uptake and use of nutrients [Mehmet et al., 2005; Shin et al., 2006; Morales et al., 2007; Wakelin et al., 2007]. Crop productivity in fields can increase up to 300% after the addition of T. hamatum or T. koningii or A. niger. The experiments carried out in green houses with seed treatment with Trichoderma spores have shown significantly greater yield [Chet et al., 1997]. Equal degree of yield enhancement was observed when plant seeds were separated from Trichoderma by a cellophane membrane. This indicates that Trichoderma produces growth factors that enhanced the rate of seed germination, plant growth and yield [Benitez et al., 1998]. Trichoderma strains and A. niger that produce cytokinin like molecules, e.g. zeatyn and gibberellin GA3 or GA3 have been recently detected. The controlled production of these compounds could improve biofertilization. Thus, the plant growth promotion may be due to production of plant hormones or increased uptake of nutrients by the plant [Chet et al., 1993]; control of one or more sub potential pathogens [Baker, 1986] and/or strengthening plant’s own defense mechanism [Zimand et al., 1996]. The most important genera of phosphatesolubilizing fungi and their mode of action against plant pathogenic fungi or nematode is discussed in detail in the following paragraphs.

19.2.3.1. Aspergillus Niger Van Teigh in Nematode Management Systematic Position Division Sub division Class Order Family Genus

Ascomycota Pezizomycotina Eurotiomycetes Eurotiales Trichocomaceae Aspergillus

Aspergillus niger is a versatile and abundant microorganism present in almost all soil types and climates. The fungus is an important group of phosphate-solubilizer [Varenyam et al., 2007] and possesses great potential to antagonize plant nematodes. Several studies have been conducted to ascertain the biocontrol potential of A. niger against plant parasitic

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nematodes [Goswami et al., 1994; Walia, 1994; Khan and Tarannum, 1999; Khan and Kounsar, 2000; Hasan, 2004; Khan et al., 2006]. Sharma et al. [2005] evaluated the effect of joint application of Kalisena [a commercial formulation of A. niger AN 27 SD] and T. harzianum as seed treatment [1% w/w] on okra cv. Pusa Kranti against M. incognita alone and in combination with carbofuran applied @0.5 kg/ha. The combined treatments with Kalisena and T. harzianum and carbofuran were more effective and reduced root-knot galling by 20-25% in comparison to sole application. Comparatively, T. harzianum was better than the Kalisena in reducing the root-knot damage in combined application but plant growth was better in sole treatment of bioagents than the combined application of carbofuran. Mittal et al. [2005] reported that degree of antagonism by A. niger against plant parasitic nematodes vary with the strain/isolate of the fungus. Khan et.al. [2006] collected 20 isolates of A. niger from different crops and locations and examined their effectiveness against M. incognita in vitro and in vivo conditions. Three isolates were found more aggressive and produced ammonia, hydrogen cyanide, siderophores and solubilised P in vitro. Culture filtrates of these isolates greatly suppressed the hatching and mortality of M. incognita. A pot trial conducted to test the effects on the root-knot disease on eggplant showed significant decrease in the gall formation and egg mass production especially by A. niger isolates AnC2, AnR3 and AnM3 [Khan et al., 2006]. Khan and Anwer [2007] also reported that different isolates of A. niger aggregates suppressed the galling, egg mass production and decreased the soil population of M. incognita. Effect of A. niger on the fungus nematode disease complex of pulses and vegetables has been examined by researchers. Singh et.al. [1991] conducted a pot experiment to study the effect of M. incognita alone and in combination with A. niger and R. solani on tomato cv. Perfection. Damage was greatest in plants inoculated with M. incognita and R. solani together followed by the fungus and nematode alone respectively. Damage to the plant was less when A. niger was inoculated with the nematode. In another investigation Rekha and Sexena [1999] reported that A. niger, E. purphurascum and P. vermiculatum effectively diluted the adverse effect of both the pathogens and showed an increase in germination when incorporated in soil together with M. incognita or R. solani either separately or in combination. During the last couple of years some field trials have been conducted to control disease complexes. For instance, Haseeb and Kumar [2005] studied efficacy of A. niger, P. lilacinus, T. harzianum, T. virens and P. fluorescens on M. incognita and F. solani disease complex of brinjal and found all the treatments effectively reduced fungus infection and root galls. Anwer and Khan [2005] isolated 40 isolates of A. niger from different crops and localities and evaluated thier effectiveness against root-knot – root rot [M. incognita-R. solani] disease complex of eggplant cv Pusa Kranti and found that application of A. niger isolates checked the galling and reproduction of nematode, decreased the root rot index and improved the growth variables. Among various isolates, isolate AnC2 was found highly effective.

19.2.3.2 Penicillium Thoms. in Nematode Management Systematic Position Division Sub division Class Order

Ascomycota Pezizomycotina Eurotiomycetes Eurotiales

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Trichocomaceae Penicillium

Researchers through out the world have recognized the role of Penicillium spp. in the biologically mediated soil nutrition. Commonly occurring Penicillium spp. in agricultural soils are P. digitatum, P. lilaciumum, P. anatolicum etc. [Lal, 2002].Workers in India and abroad have also found nematode suppressing effects of Penicillium spp. Inoculation with Penicillium anatolicum reduced the Globodera rostochinensis population in Panama without parasitizing the female nematodes [Jatala 1986]. It appears to produce a series of compounds that can alter the permeability of the eggshell to cause free movement of noxious compounds, which it has produced or were present in the soil, into the eggs. This in turn may alter the egg physiology when it occurs in early stages of embryonic development, causing abortive embryonic development and complete vacuolation of eggs within a short period of time. Exposure of G. pallida cysts to these compounds from certain culture filtrates caused reduction in hatching of the eggs due to the effects mentioned above [Jatala et al., 1985; O’Hara 1985]. Apparently when these compounds are liberated in the vicinity of the developing females of G. rostochinensis and G. pallida, they cause deformation of females, which become evident as the females mature to form cysts [Jatala 1986].

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19.2.3.3. Trichoderma Persoon in Nematode Management Systematic Position Division Sub division Class Order Family Sub family Genus

Amastigomycota Deuteromycotina Hyphomycetes Moniliales Moniliaceae Gliosporae Trichoderma

Besides being a potent antifungal agent Trichoderma has also been used as an effective tool in nematode management and have shown a variable degree of parasitism and mode of action [Pocasangre et al., 2007, Pant and Pandey, 2001; Oyekanmi, et al., 2008; Sharon et al., 2009]. Paecilomyces lilacinus is an obligate parasite of root-knot and other nematodes [Pal, 2006]. Eapen and Venugopal [1995] reported that isolates of Trichoderma species have antagonistic potential against a variety of fungi and nematodes. A serine protease gene [28 kDa] with trypsin activity was isolated from Trichoderma strain 2413. The enzyme reduced the number of hatched eggs of root knot nematodes and acted synergistically with other proteins. Strains of Trichoderma species and bacteria Burkholderia cepacia are known to produce extracellular compounds that inhibit egg hatching and motility of M. incognita juveniles [J2]. The number of hatched eggs of the root knot nematode, Meloidogyne incognita was significantly reduced after incubation with pure PRA1 [trypsin like protease] preparations T. harzianum CECT 2413 [Suarez et al., 2004]. Pant et al. [2002] studied the effect of using T. harzianum and neem cake alone and in combination to manage M. incognita in chickpea cv. Type-3. Greatest reduction in the root knot nematode was recorded with the

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application of neem cake and T. harzianum together, followed by neem cake and T. harzianum alone. The green house studies reveals that, individual application of these microbes as seed treatment followed by root drenching considerably suppress root-knot nematode on bell pepper compared with untreated plants [Meyer et al., 2001]. Pant and Pandey [2001] recorded highest degree of suppression in M. incognita population by T. harzianum, P. lilacinus and A. niger applied @ 5000 spores/pot. Significant reduction in M. incognita was also observed when T. harziaum was applied along with the neem cake [Pant and Pandey, 2002]. Siddiqui and Shaukat [2004] reported that combined application of T. harzianum and P. fluorescens in unsterilized soil [sandy loam] caused greatly reduced population densities of M. javanica in tomato. T. harzianum when applied before corn seeding in pots having M. arenaria infested soil there was increase in fresh weight [shoot and root] with a decrease in the number of eggs per gram root system in comparison to untreated control [Windham et al., 1989]. Suppression of root-knot nematodes and improvement in plant growth of cardamom in nurseries has been reported with T. harzianum [IISR, 1995]. Eapen and Venugopal [1995] have shown that isolates of Trichoderma spp. have a broad spectrum of biocontrol activity against a number of pathogenic fungi and nematodes. The culture filtrate of Trichoderma spp. also showed nematicidal properties [IISR, 1995]. Sharon et al. [2009] recorded enhanced parasitism of Trichoderma asperellum-203 and Trichoderma atroviride on nematode egg masses, eggs and juveniles was enhanced when antibodies [monoclonal and polyclonal] were incorporated into in vitro parasitism bioassays [Sharon et al., 2009]. Improved parasitism could be due to bilateral binding of the antibodies to the nematodes and conidia, enabling better conidial attachment to the nematodes. Enhanced germination of antibody-bound conidia further improved parasitism. Differences were observed among antibodies in their effects on fungal parasitism and their interaction with Trichoderma species. Antibody binding to juveniles affected their movement and viability, especially gelatinous matrix-originated juveniles. The fucose-specific lectin Ulex europaeus-I enhanced conidial attachment to nematode life-stages. In vitro application of Trichoderma isolates [MT-20 and S2] parasitized R. similis [zum Felde, 2002; Carñizares Monteros 2003]. Pocasangre et al. [2007] evaluated the antagonistic potential of two strains of endophytic fungus, T. atroviride, towards the burrowing nematode, Radopholus similis, in plantain under field conditions. The two strains were nematode suppressive. Evaluation for nematode damage was carried out every three months after planting in the field. Single inoculation of plants with isolate MT-20 controlled R. similis better than two applications of nematicides invitro. The results indicate possibility of replacing nematicides with endophytes in banana. The greenhouse and laboratory experiments revealed that T. harzianum strain BI [102– 108 spores/ml] decreased infection by M. javanica. The strain was able to penetrate the matrix leading to a significant reduction in egg hatching. The fungus also increased the activity of peroxidase, polyphenol oxidase and phenylalanine ammonia lyase significantly in inoculated plants [Sahebani and Hadavi, 2008]. P. lilacinus reduce galling, egg mass production, soil population and root densities compared to control and other treatments [Zareen et al., 2001]. Cannayane and Jonathan [2008] recorded 90% mortality of M. incognita juveniles and 80% reduction in egg hatching on application with culture filtrate [75%] of T. viride. Bacillus subtilis, T. viride, P. chlamydosporia and P. fluorescens were also found to be inhibitory to R. similis and P. coffeae at the same concentration. Paecilomyces lilacinus isolates colonized M.

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incognita eggs and egg masses, leading to egg necrosis. Arthrobotrys oligospora trapped M. incognita juveniles by forming hyphal networks.

19.2.3.4. Paecilomyces Lilacinus [Thoms.] Samson in Nematode Management

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Taxonomic Position Division Class Order Family Genus

Ascomycota Deuteromycetes Moniliales Moniliaceae Paecelomyces

Paecilomyces species are soil-inhabiting fungus and capable of solubilizing phosphorus in soil [Gaur, 1990]. The fungus particularly, P. lilacinus is, however, known as an aggressive parasite of eggs and adults of nematodes [Jatala et al.,1979] It has been isolated from many cyst and root-knot nematodes and from soil in many locations [Sterling, 1991] Several successful field trials using P. lilacinus against pest nematodes were conducted in Peru [Sterling, 1991]. International Meloidogyne project, led to more field trials on a variety of crops in different agroclimatic regions [Jatala, 1986]. Subsequent tests on potted plants and field plots have shown the fungus is effective in management of numerous plant parasitic nematodes throughout the world in different crop plants [Jatala, 1985; Khan et al., 1997]. Its effectiveness is comparable with several chemical nematicides used. The application of MeloCon WG, a commercial product of P. lilacinus strain 251 on tomato [Burpee "Orange Pixie" hybrid] and cucumber [Ferry-Morse"Marketmore 76"] in Hawaii protected tomato plants against nematode damage [Schenck, 2004]. Field trials, glasshouse trials and in vitro testing of P. lilacinus continue to date and a number of isolates have been isolated from soil, nematodes and occasionally from insects. Isolates vary in their pathogenicity to plant-parasitic nematodes. Some isolates are aggressive parasites while other, though morphologically indistinguishable, are less or non-pathogenic. Sometimes isolates which looked promising in vitro or in glasshouse trials have failed to provide control in the field [Khan et al., 2006]. The application of P. lilacinus alone on in combination with neem leaves suppressed the root-knot nematode on okra. The reproductive rate decreased by 24 and 46% leading to 14.8% increase in yield over control. Ashraf and Khan [2008] evaluated the efficacy of fruit wastes of apple [Malus pumila], banana [Musa paradisiaca], papaya [Carica papaya],pomegranate [Punica granatum] and sweet orange [Citrus sinesis] @ 20g/plant and P. lilacinus @ 2g [mycelium+spores]/plant, alone and in combination against reniform nematode, Rotylenchulus reniformis on chickpea under glasshouse conditions. The individual applications of fruit wastes of sweet orange and fungal biocontrol agent P. lilacinus significantly reduced the nematode multiplication, ultimately leading to increase in plant growth. The best protection of chickpea against R. reniformis was recorded on integration of P. lilacinus with fruit wastes of papaya followed by apple and pomegranate.

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19.3. PHOSPHATE-SOLUBILIZING BACTERIA IN NEMATODE MANAGEMENT There are numerous bacteria which play important role in the mineral solubilization in soil, important species belonging to genera Azotobacter, Beijerinkia, Bacillus, Pseudomonas etc. These bacteria are also known to suppress plant nematodes [Khan et al., 2005b]. Among the phosphate- solubilizing bacteria, Pseudomonas and Bacillus are widely used in nematode management [Table 2]. These bacteria not only suppress the nematode pathogenesis but also promote the plant growth by solubilizing the minerals in soil [Campbell, 1989; Wei et al., 1996; Sikora, 1988; Khan and Khan, 1998; Khan and Tarannum, 1999; Khan et al., 2001; Siddiqui and Shaukat, 2004].

19.3.1. Mechanism of Nematode Suppression by the Phosphate-Solubilizing Bacteria

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The phosphate solubilizing bacteria may suppress nematode pathogenesis and decrease their soil populations through one or all of the mechenisms explained below.

19.3.1.1. Rhizosphere Competence and Colonization Rhizosphere competence describes the relative root colonizing ability of a rhizobacteria [Weller and Thomashow, 1994]. The bacteria may get attached to roots with the help of pili as in case of Pseudomonas fluorescens on wheat roots [Vesper, 1987] or its colonization may involve development and multiplication on root surface, however, endophytic colonization of root is also known and the degree of colonization depends on bacterial strain and plant type. Endophytic growth of Bacillus polymyxa Pw-ZR and Pseudomonas fluorescens Sm3-RN in roots has been recorded on spruce [Shishido et al., 1999], Bacillus strain L324-92R12 and P. fluorescens 2-79RN10 on wheat [Kim et al., 1997] and other strains on pea [Benhamou et al., 1996a, b; M’ Piga et al., 1997]. P. fluorescens CHAO grow endophytically on tobacco [Troxler et al., 1997] and P. fluorescens WCS417r on tomato [Duijff et al., 1997]. 19.3.1.2. Antibiosis In addition to metal chelaters and enzymes numerous antifungal metabolites are produced by bacteria that act against nematodes both in vitro and in vivo. These include bacillomycin [Peypoux et al., 1980, Chevanet et al., 1985], iturin [Delcambe et al., 1975; Peypoux et al., 1978; Phister et al. 2004; Mahadtanapuk et al., 2007], surfactin, mycosubtilin [Peypoux et al., 1986], bacilysin [Roger et al., 1965; Loeffler et al., 1986; Phister et al. 2004] fengymycin [Roger et al., 1965; Loeffler et al., 1986], mycobacillin [Majumdar and Bose, 1970; Mannanov and Sattarova, 2001], ammonia, butyrolactones, 2,4-diacetylphloroglucinol, HCN, kanosamine, oligomycin A, oomycin A, phenazine-1-carboxylic acid, pyoluterin, pyrrolnitrin, viscosinamide, xanthobaccin and zwittermycin A etc. [Milner et al., 1996; Keel and Defago, 1997; Whipps, 1997a; Nielson et al., 1998; Kang et al., 1998; Kim et al., 1999; Thrane et al., 1999; Nakayama et al., 1999].

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Table 2. Effect of different phosphate-solubilizing bacteria on plant parasitic nematode infesting agricultural crops Phosphate solubilizing bacteria Azospirillum lipoferum Azotobacter chroococcum Bacillus subtilis

Nematode managed M. incognita M. incognita M. incognita, M. arenaria and Rotylenchulus reniformis. M. incognita Heterodera zeae M. incognita M. incognita, M. arenaria and Rotylenchulus M. incognita Heterodera cajani M. incognita

Host plant Greengram Greengram Sugar beet

Reference Khan and Kaunsar, [2000] Khan and Kaunsar, [2000]; Khan et al., [2002] Sikora, [1988], Khan and Tarannum, [1999]; El-Sherief and Barakat, [1995]

Tomato Sugar beet

Gautam et al. [1995], Khan and Akram, [2000] Sikora [1988]

Chickpea Pigeonpea Mungbean

Siddiqui and Mahmood, [1995a] Siddiqui and Mahmood, [1995b] Khan and Kounsar, [2000], Khan et al., [2002]

B. firmus B. subtilis Beijerinkia indica Pseudomonas fluorescens P. fluorescens P. fluorescens of Pseudomonas aeruginosa strain IE-6S+ and Pseudomonas fluorescens strain CHA0 P. fluorescens CHA0 or CHA0/pME3424

M. incognita M. incognita M. incognita Heterodera schachtii M. incognita Hirschmanniella gracilis M. javanica

Tomato Ornamental Greengram Sugar beet Mungbean Rice Tomato

Meoloidogyne spp.

P. fluorescens, P. lilacinus and T.T. viride

Globodera rostochiensis and G. pallid M. javanica

Mungbean roots compared with the Potato

Terefe et al., [2009] Khan et al., 2005b Khan and Kaunsar, [2000]; Khan et al., [2002] Oostendrop and Sikora [1989] Khan and Kounsar, [2000], Khan et al., [2002] Seenivasan and Devrajan, [2000] Siddiqui and Shaukat, [2002]; Siddiqui and Shaukat, [2003] Hamid et al., [2003]

B. subtilis. B. subtilis B. subtilis B. subtilis B. subtilis

P. fluorescens [EPS291 and EPS817 P. fluorescens P. fluorescens [PS 07] and T. harzianum [T014] P. fluorescens CHA0 or CHA0/pME3424 P. fluorescens and T. harzianum

Seenivasan et al., [2007]

Banana

Rodríguez-Romero et al., [2007]

Meoloidogyne spp. M. incognita

Brinjal Gladiolus

Anita and Rajendran [2002] Khan and Mustafa, [2005]

Meloidogyne species M. javanica

Mungbean Tomato

Hamid et al., [2003] Siddiqui and Shaukat [2004]

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19.3.1.3. Induced Systemic Resistance Induced resistance may be defined as the process of active resistance dependent on the host plant’s physical or chemical barriers activated by biotic and abiotic agents [Kloepper et al., 1992]. Most of the work has focused on the systemic resistance induced by nonpathogenic rhizosphere colonizing Bacillus and Pseudomonas species in systems where the inducing bacteria and the challenging pathogen remain spatially separate for the duration of the experiment, and no direct interaction occurs between the bacteria and pathogen [Sticher et al., 1997; van Loon, 1997]. Application of IE-6S+ to one half of the split-root system caused a significant [42%] while it was 29% with CHA0 strain. The application of P. fluorescens CHA0 or CHA0/pME3424 applied in conjunction with ammonium molybdate caused greater reduction in nematode penetration on mungbean roots compared with the bacterial application alone [Hamid et al., 2003]. The full range of inducible moieties produced by bacteria is probably not yet known, but lipopolysaccharides [Leeman et al., 1995] and Siderophores [Metraux et al., 1990; Leeman et al., 1996b] are clearly indicated. Van Peer and Schippers [1989] demonstrated that lipopolysaccharides [LPS] extracted from the outer membrane of P. fluorescens WCS417 induced systemic resistance in carnation against Fusarium wilt. Following changes may take place in plant roots exhibiting induced systemic resistance as a result of inoculation with biocontrol bacteria: [i] strengthening of epidermal and cortical cell walls and deposition of newly formed barriers beyond infection sites including cellulose, lignin and phenolics [Benhaumou et al., 1996a, b, c, 2000; Duijff et al., 1997; Jetiyanon et al., 1997; M’ Piga et al., 1997] [ii] increased levels of enzymes such as chitinase, peroxidase, polyphenol oxidase and phenylalaline ammonia lyase [M’Piga et al., 1997; Chen et al., 2000] [iii] enhanced production of phytoalexins [van Peer et al., 1991; Ongena et al., 1999] and [iv] enhanced expression of stress related genes [Timmusk and Wagner, 1999]. However, all these changes generally do not occur in one bacterial-plant combination [Steijl et al., 1999]. Similarly, the ability of the bacteria to colonize the internal tissue of the roots has been considered to be an important feature in many of the bacterial root interactions involving induced systemic resistance, but is not a constant feature [Steijl et al., 1999]. 19.3.1.4. Competition for Iron Although competition between for space or nutrients has been known to exist as a biocontrol mechanism for many years [Whipps, 1997], the greatest interest recently has involved in competition for iron. Under iron limiting conditions, bacteria produce a range of iron chelating compounds or siderophores which have a very high affinity for ferric iron. These bacterial iron chelators are thought to sequester the limited supply of iron available in the rhizosphere making it unavailable to pathogens, thereby restricting their growth [O’ Sullivan and O’Gara, 1992; Loper and Henkens, 1999]. It has been clearly shown that the plant nutrition influences the rhizosphere microbial community [Yang and Crowley, 2000]. As an example, B. subtilis not only produced antibiotics which suppress plant pathogens but also siderophores and the regulation of these products by the gene lpa-14 indicate the possibility of enhanced effectiveness of biocontrol by the manipulation of the gene [Shoda, 2000]. 2, 3- Dihydroxybenzoylglycine [2, 3-DHBG] is known as a siderophore produced by the Gram positive B. subtilis [Leong, 1986; Ito and Neilands, 1958; Shoda, 2000]. The strains of B. subtilis which showed a wide suppressive spectrum on plant pathogens by producing antibiotics, also produce 2, 3-DHBG.

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19.3.1.5. Plant Growth Promotion The plant growth promoting activity of B. subtilis and P. fluorescens results from the contribution of different components acting either directly or indirectly on the plant. Indirect plant growth promotion is due to suppression of soil borne plant parasites and deleterious rhizosphere microorganisms, whereas direct plant stimulation is mainly exerted by release of growth factors. These microorganisms may enhance the plant growth and suppress the nemtodes present in the rhizosphere through the following mechanisms:

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19.3.1.5.1 Siderophore Production Strains of B. subtilis and P. fluorescens can synthesize siderophores that can solubilize and sequester iron in the soil making it available to plant cells [Glick, 1995]. Co-inoculation of chickpea with Pseudomonas strain CRP55b resulted in 68-115% increase in nodule formation at 80 and 100 days after planting. Khan et al. [2002] isolated nine strains of fluorescent Pseudomonas from the rhizospheres of wheat and chickpea and were characterized morphologically as well as biochemically for siderophores activity. All of the strains produced siderophores and were effective in the growth of chickpea. Powell et al. [1980] demonstrated the role of iron in crop development and reported the presence of siderophores, particularly hydroxamate siderophore [10-7 to 10-8 M], in 67 different soils of United States, at concentrations high enough to be useful to plant roots. Akers [1983] also detected siderophore [schizokinen] in paddy crop, suggesting their role in plant growth. Reid et al. [1984] and Barker et al. [1985] provided evidences that plants have an ability to incorporate Fe3+ of siderophores into their biomass. Sharma and Johri [2003] reported that fluorescent Pseudomonas strain GRP3A produced siderophores under iron limited conditions and increased the growth of mungbean. Johri et al. [1997] also reported that fluorescent pseudomonad strain RBT 13 producesdsiderophores which exhibited in vitro antagonism against several bacterial and fungal pathogens and simultaneously increased the growth of four crops. 19.3.1.5.2. Phytohormone Production Several strains of B. subtilis and P. fluorescens synthesize phytohormones such as indole acetic acid, gibberellins, cytokinins and zeatin that promote plant growth [Gracia de Salamone et al., 2001]. Plant growth promotion is due to production of cytokinin [Gracia de Salamone et al., 2001], vitamins [Marek-Kazaczok and Skorupsks, 2001] and IAA [Pal et al., 2001]. Application with the bacterium enhanced growth in tea, pigeonpea, chickpea and maize [Kumar and Bezbaruah, 1997]. Indole acetic acid, gibberellin and zeatin have been detected in the culture medium inoculated with P. fluorescens [Meng et al., 1998]. Application of the culture filtrate promoted wheat growth in a manner similar to application of exogenous growth regulators. A mutant strain of P. fluorescens that overproduced IAA stimulated the root development of blackcurrant softwood cutting [Dubeikovsky et al., 1993]. IAA produced by P. putida strain GR12-2 was found to play a major role in the root growth [Patten and Glick, 2002]. They primary root system of canola seedlings from seeds treated with IAA producing P. putida GR12-2 were on an average 35-50% longer than the roots from the uninoculated seeds. In addition, exposing mungbean cuttings to high levels of IAA by soaking them in a suspension of this bacterial strain stimulated the formation of many, very small adventitious roots. Bacillus subtilis has also been found as a predominant bacteria in the

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rhizosphere of some crops [Kloepper et al., 1992]. Yeun et al. [1985] found that B. subtilis strain improved the growth of many plant species in steamed and natural soils. Seed treatment with B. subtilis increased the yield of carrots by 48%, oats by 33% and peanuts upto 37% [Weller, 1988]. B. subtilis B2g caused significant increases of plant growth of cabbage, cucumber and sunflower [Marten et al., 1999]. Growth promoting effects of B. subtilis and P. fluorescens have also been reported on tomato and chickpea crops [Khan and Akram, 1997; Khan and Khan, 2001; Khan et al., 2004].

19.3.1.5.3. Mineral Solubilization and Synthesis of Other Compounds Phosphorus is one of the major plant nutrients which play a key role in balanced nutrition of plants and thereby in crop production. About 98% of the Indian soils have inadequate supply of available phosphorus [Guar, 1987] and only 0.1% of the total P present in soil is available to plants [Scheffer and Schachtschabel, 1992]. To avoid this deficiency, phosphatic fertilizers are added in soil. But most of phosphorus in the fertilizers becomes insoluble making it unavailable to the crop [Gaur, 1990]. Hence, solubilization of fixed soil P through the use of microorganisms is a viable option to augment the availability of P in easily assimilative form [Parks et al., 190; Dubey and Vaishya, 2000; Vazaquez and Hoguin, 2000]. A number species of Bacillus [Gaur and Gaind, 1987; Kole and Hajra, 1997] and Pseudomonas [Dave and Patel, 1999; Reddy and Swami, 2000] are efficient phosphate solubilizers. Application of Pseudomonas species increased the P uptake in chickpea by 27% [Alagawadi and Gaur, 1988a], sorghum by 18% [Alagawadi and Gaur, 1988b], potato by 26% [Kundu and gaur, 1980] and rice by 15% [Gaur, 1990] leading to significant increase in the crop yield. Khan and Tarannum [1999] have reported that application of B. subtilis enhanced the uptake of nitrogen and phosphorus in tomato plants thus improving the growth and yield of tomato. Yield of wheat was also increased when P. straita was applied along with super phosphate and rock phosphate [Gaur et al., 1980]. Gaur and Ostwal [1972] reported that application of B. polymyxa in the presence of rock phosphate significantly increased the P uptake of wheat and grain and straw yield. In pot experiments where mustard was grown at different rates of Mussoorie rock phosphate with or without P. straita, the microorganism solubilized rock phosphate more efficiently [Dubey and Vaishya, 2000]. Reddy and Swamy [2000] conducted field experiments on blackgram by applying phosphate solubilizing bacteria, farm yard manure and phosphorus and found improved solubilization of inorganic phosphate compounds and increase in seed yields. Growth promoting effects of B. subtilis and P. fluorescens have also been reported on tomato and chickpea crops [Khan and Akram, 1997; Khan and Khan, 2001; Khan et al., 2004]. Phosphate solubilizing microorganisms produce certain organic acids which are considered the most important mechanism of phosphorus solubilization [Illmer and Schinner, 1995; Yadav and Dadarwal, 1997]. Three strains of P. striata have been found to produce seven acids such as malic acid, glyoxalic acid, succinic acid, fumaric acid, citric acid, tartaric acid and ketoglutaric acid [Gaur, 1990]. A commercial formulation, Microphos, has been developed at IARI which is being successfully used by farmers [Gaur and Gaind, 1984]. Both B. subtilis and P. fluorescens have mechanisms for the solubilization of minerals such as phosphorus that become more readily available for plant growth [Pal et al., 2001]. They may also synthesize some low molecular mass compounds, enzymes or vitamins that can modulate plant growth and development [Marek-kazaczuk and Skorupska, 2001].

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19.3.2. Bacillus Subtilis [Cohn] Prazmowski in Nematode Management

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Systematic Position Group Gram positive endospore forming rods and cocci Family Bacillaceae Genus Bacillus Species subtilis The application of B. subtilis in sugar beet considerably reduced the infection by M. incognita, M. arenaria and Rotylenchulus reniformis [Sikora 1988] El-Sherief et. al [1995] have isolated Bacillus, Cornybacterium, Streptomyces and Arthobacterum species from M. incognita egg masses and Heterodera zeae cysts. A liquid culture of these bacteria at 0.1 to 0.6% concentrations was highly toxic to juveniles of M. incognita, R. reniformis and Tylenchulus semipenetrans. Similar effects of B. subtilis were reported by Khan and Tarannum [1999] on root-knot disease of tomato in a field study. Another field trial has shown suppressive effects of B. subtilis on M. incognita infecting tomato [Khan and Akram, 2000]. Effects of seed treatment [Khan and Kounsar, 2000] and soil application [Khan et al., 2002] with B. subtilis were investigated against root-knot nematode on green gram. The bacterial treatment decreased the galling by 33% subsequently increasing the yield of greengram by 22%. Gautam et al. [1995] reported that B. subtilis and P. lilacinus alone or in combination increased plant height and weight and suppressed numbers of root galls, females, eggs and second stage juveniles [J2] on tomato in pots containing sterilized soil. B. subtilis reduced galling and multiplication of M. incognita in chickpea [Siddiqui and Mahmood, 1995] and of Heterodera cajani in pigeonpea [Siddiqui and Mahmood, 1996] and increased the plant growth in both the crops. Gautum et al. [1995] found that the addition of green manure, Eiechornia crassipes to the mixture of P. lilacinum and B. subtilis enhanced the plant growth of nematode inoculated plants. Further, a non-cellular extract of B. subtilis was also reported to have a high degree of larvicidal properties to Heterodera cajani [Gokte and Swarup, 1988]. BioNem a commercial formulation of B. firmus is available in the form of wettable powder [WP] and has shown great potential for the management of root-knot nematode, M. incognita under laboratory, greenhouse and field conditions on tomato plants. BioNem as an aqueous suspension reduced egg hatching from 98% to 100%, 24-days after treatment at 0.5%, 1%, 1.5% and 2% concentration in laboratory. Treatment of second-stage juveniles with 2.5% and 3% concentration of BioNem, caused total inhibition of mobility, 24 hour after treatment. In the green house trials its application @ 8 g/pot reduced gall formation on tomato seedlings by 91%, final nematode populations by 76% and the number of eggs by 45% and subsequently increased the root and shoot weight [Terefe et al., 2009]. It has been found that application of B. subtilis can also control rootknot problem of seasonal ornamental plants [Khan et al., 2005b].

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19.3.3. Pseudomonas Fluorescens [Threvesan] Migula in Nematode Management

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Systematic Position Group Gram negative aerobic rods and cocci Family Pseudomonadaceae Genus Pseudomonas Species fluorescens Oostendrop and Sikora [1989] found that eight isolates of bacteria with three of them being identified as P. fluorescens from rhizosphere of sugar beet suppressed early root infection by Heterodera schachtii when applied to seed in non-sterilized field soil in the green house. In the field, all isolates reduced nematode penetration with some reducing nematode number in roots by as much as 75%. Root-dip treatment of rice seedlings with P. fluorescens effectively reduced the plant damage and population of Hirschmanniella gracilis [Seenivasan and Devrajan, 2000]. Amendment of culture filtrate or methanol extract of the culture filtrate of a T. harzianum strain Th 6 to P. fluorescens growth medium enhanced the production of nematicidal compound [s] by bacterial inoculants in vitro. The rhizobacteria P. aeruginosa [strain IE-6S+] and P. fluorescens [strain CHA0] when used as bare root-dip treatment or as soil drench application substantially reduced M. javanica penetration in tomato roots under glasshouse conditions [Siddiqui and Shaukat, 2002; Siddiqui and Shaukat, 2003]. Application of IE-6S+ to one half of the split-root system caused a significant [42%] while it was 29% with CHA0 strain. The application of P. fluorescens CHA0 or CHA0/pME3424 applied in conjunction with ammonium molybdate caused greater reduction in nematode penetration on mungbean roots compared with the bacterial application alone [Hamid et al., 2003]. The in vitro study showed inhibitory/lethal effect of culture filtrate of P. fluorescens on M. javanica juveniles [Hamid et al., 2003]. The experiment on efficacy of three bio-control agents viz., P. fluorescens, P. lilacinus and T. viride against potato cyst nematodes [PCN], Globodera rostochiensis and G. pallida under field conditions revealed that the application of P. lilacinus [7 × 108 CFU/g] @ 10 kg/ha and P. fluorescens [15 × 108 cfu/g] @ 10 kg/ha reduced the penetration by 68.2 and 63.4%, respectively over control and also resulted in increase the tuber yield by 88.2 and 76.2% [Seenivasan et al., 2007]. P. fluorescens [EPS291 and EPS817] @ 108 CFU/g−1 substrate significantly increased aerial fresh weight, plant length and leaf area in banana. The population of M. javanica showed a marked decline 135 days after the second treatment with the bacteria [Rodríguez-Romero et al., 2007]. Oyekanmi, et al., [2008] observed reduction in nematode density by 79.6% and improvement in plant growth by 46% on application with Trichoderma pseudokoningii, T. viride, P. lilacinus, A. niger, Glomus mosseae and Pseudomonas fluorescens and P. putida. Siddiqui et al. [2003] reported that the culture filtrates of P. fluorescens strain CHAO caused significant mortality [46%] of M. incognita juveniles in vitro compared to control [10%]. Cell concentrations of four strains of P. fluorescens have been found inhibitory to the hatching of eggs and survival of larvae of M. incognita [Channppa et al., 2008]. A cell concentration of 1010 of the bacterium caused significant decrease in the hatching and induced 50% juvenile mortality. Anita and Rajendran [2002] reported a significant reduction in nematode population, number of egg masses and gall indices in tomato and brinjal plants in

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nursery plots where talc based formulation of P. fluorescens with CFU 7 x 108/g was applied @10 g/m2 of plot. Siddiqui and Ehteshamul-Haque [2001] reported that P. aeruginosa [7.4 x 108 CFU/ml] caused the greatest reduction in gall formation due to M. javanica [500 J2/plant]. The cell free culture filtrate of P. fluorescens was found to be toxic to Heterodera avanea as it showed 100% mortality within 24 hours [Kamra et al., 1999]. Sharma and Johri [2003] reported that fluorescent Pseudomonas strain GRP3A produced siderophores under iron limited conditions and increased the growth of mungbean. The probable reason for increase in plant growth may be the production of plant hormones or increased uptake of nutrients by the plant [Chet et al., 1993]; suppression of one or more pathogens [Baker, 1986]; strengthening host defense mechanisms [Zimand et al., 1996], siderophore production, increased synthesis of phytohormones, better mineral solubilization, induce production of enzymes or vitamins that can modulate plant growth and development [Marek-kazaczuk and Skorupska, 2001]. Seed treatment with biopesticides [P. fluorescens, P. chlamydosporia and T. harzianum] @ 2g/kg seeds along with commercial Rhizobium reduced the yield by 31 and 34% [P. fluorescens] and 28 and 25% [P. chlamydosporia] of chickpea cultivar BG 256 in presence of M. incognita. The gall formation and egg mass production was decreased by 23 and 18% [P. chlamydosporia] against 26 and 19% with fenamiphos [Khan et al., 2005a]. Field study on some ornamental crops revealed that P. chlamydosporia, P. fluorescens and B. subtilis suppressed the root-knot nematode population by 37, 27 and 24% respectively, leading to 7-15, 14-36 and 7-33% increase in the flower production of the tested ornamentals [Khan et al., 2005b]. The frequency of colonization by the biocontrol agents was greatest with P. chlamydosporia followed by B. subtilis and P. fluorescens. Khan and Mustafa [2005] evaluated the efficacy of P. fluorescens [PS 07] and T. harzianum [T014] against M. incognita on different cultivars of Gladeoli [Khan and Mustafa, 2005]. Application of P. fluorescens and nemacur decreased the galling and soil population of the nematode.

19.4. MERITS AND DEMERITS The chemical pesticide or biopesticide has both the advantages and disadvantages associated with their application. Environmental contamination and development of resistance are some of the major problems accompanied with the use of chemical pesticides. Similarly, the use of phosphate-solubilizing bacteria or fungi has both merits and demerits. Although, no environmental hazard is linked with the use of biological control agents yet there are certain reasons that restrict their wide scale application in sustainable agriculture. Aspergillus niger, P. digitatum, Trichodertma, Paecilomyces lilacinus etc. can easily and inexpensively be cultured and mass produced, show rhizospheric competence, have wide host range and could be used as seed treatment. The limitation is that they, however, require a soil rich in organic matter contents and high temperature for better performance and has to be applied in large amounts under field conditions [106/g soil]. Furthermore, results vary with the species and density of nematodes. In addition, there are certain strains that have shown pathogencity of Paecilomyces spp. [Kerry, 1987; Stirling, 1991] and Trichodertma spp. [Guarro etal., 1999] to human populations. Phosphate- solubilizing bacteria such as, Pseudomons and Bacillus species are easily mass cultured on synthetic media and could be applied in soil or seeds. Their application may potentially reduce the plant damage by

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suppressing nematode pathogenesis and stimulating the plant growth. However, the inhibitory effects to nematode are of shorter duration; probably for this reason the PSMs are not commercially used in nematode management.

19.5. FUTURE THRUST The past three decades have seen a significant increase in the number of scientists working on the biological control of nematodes. Current experience suggests that the integration of phosphate-solubilizing microbes, nematicides and other control measures would be a feasible and viable strategy for nematode management in the developed and developing countries of the world. The urgent need to reduce the dependence on nematicides requires considerable amount of research and development in order to commercialize the successful use of phosphate-dissolving organisms. The future of sustainable agriculture relies heavily on the combination of biotechnology with traditional agricultural practices involving crop rotation, trap crops, intercropping etc. Identification of microorganisms possessing multiple growth promoting and pathogen suppressing potential for nematode management needs to be explored and tested under changing environmental conditions. To acieve these goals, extensive field trials are needed to ascertain their biocontrol potential under natural environment. Biologically-based pest management has the potential to control crop diseases without causing damage to the environment.

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REFERENCES Agrios, G.N. [2005]. Plant Pathology. V Edition, Elsevier Academic Press, California, USA. pp. 922. Akers, H. A. [1983]. Multiple hydroxanic acid microbial iron chelators [siderophores] in soil. Science, 135, 156-159. Alagawadi, A.R. and Gaur, A.C. [1988a]. Associative effect of Rhizobium and phosphate solubilizing bacteria on yield and nutrient uptake of chickpea. Plant Soil, 105, 241-246. Alagawadi, A.R. and Gaur, A.C. [1988b]. Interaction between Azospirillum brasilence and phosphate solubilizing bacteria and their influence on yield and nutrient uptake of sorghum [Sorhum bicolor L.]. Zentralblatt Fur Mikrobiologie, 143, 637-643. Anita, B. and Rajendran, G. [2002]. Nursery application of Pseudomonas fluorescens for the control of Meloidogyne incognita on tomato and brinjal. Nematologia Meditteranea 30, 209-210. Anwer, A. and Khan, M.R. [2005]. Effects of soil isolates of Aspergillus niger for effectiveness against root-knot–root-rot disease complex of egg plant. National Symposium on Recent Advances and Research Priorities in Indian Nematology, 9-10 December 2005, IARI, New Delhi. pp. 38. Ashraf, M.S. and Khan, T. A. [2008]. Biomanagement of reniform nematode, Rotylenchulus reniformis by fruit wastes and Paecilomyces lilacinus on chickpea. World Journal of Agricultural Sciences, 4, 492-494.

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Mujeebur Rahman Khan, Shahana Majid, Fayaz A. Mohidin et al.

Atkinson, G.F. [1892]. Some Diseases of Cotton. Alabama Agricultural Experiment Station Bulletin. 41 pp. 65. Baker, R. [1986]. Biological control: An overview. Canadian Journal of Plant Pathology, 8, 218-221. Baker, R. [1986]. Biological control: An overview. Canadian Journal of Plant Pathology, 8, 218-221. Barker, K.R.; Carter, C.C. and Sasser, J.N. [1985]. Advanced treatise on Meloidogyne species. vol I Methodology. North Carolina State University Graphics Raleigh, USA pp. 223. Benhamou, N.; Belanger, R.R. and Paulitz, T.C. [1996b]. Preinoculation of Ri T-DNA transformed pea roots with Pseudomonas fluorescens inhibits colonization by Pythium ultimum Trow: An ultrastructural and cytochemical study. Planta, 199, 105-117. Benhamou, N.; Belanger, R.R. and Paulitz, T.C. [1996c]. Induction of differential host responses by Pseudomonas fluorescens in Ri T-DNA transformed pea roots after challenge with Fusarium oxysporum f.sp. pisi and Pytium ultimum. Phytopathology, 86, 1174-1185. Benhamou, N.; Kloepper, J.W.; Quadt-Hallman, A. and Tuzum, S. [1996a]. Induction of defense related ultrastructural modifications in pea root tissues inoculated with endophytic bacteria. Plant physiology, 112, 919-929. Benhamou, N.; Gagne, S.; Quere, D.L. and Dehbi, L. [2000]. Bacterial mediated induced resistance in cucumber: beneficial effects of the endophytic bacterium Serratia plymuthica on the protection against infection by Pythium ultimum.Phytopathology, 90, 45-56. Benitez, T.; Delgado-Jarana, J.; Rincon, A.M.; Rey, M. and Limon, M.C. [1998]. Biofungicides: Trichoderma as a biocontrol agent against phytopathogenic fungi. In: Recent Research Developments in Microbiology vol-2, S.G Pandalai [ed.] Research Signpost, Trivendrum pp. 129-150. Benítez, T; Rincón, A.M; Limón, M.C; Codón, A.C. [2004]. Biocontrol mechanisms of Trichoderma strains. International Microbiology, 7, 249-260. Brannen, R. [1995]. Production of antibiotics by Bacillus subtilis and their effect on fungal colonies on various crops. Transactions of British Mycological Society, 65, 203. Campbell, R. [1989]. Biological Control of Microbial Plant Pathogens, Cambridge University Press, Cambridge. pp. 218. Campbell, R. [1989]. Biological Control of Microbial Plant Pathogens, Cambridge University Press, Cambridge. pp. 218. Cannayane I. and Jonathan E.I [2008]. Nematode egg parasitic fungus - II: Pochonia chlamydosporia scope for commercial exploitation – A review. Agricultural Review 29, 93-99. Cannizares Monteros, C. A. [2003]. Estudio sobre pablaciones de hongos endofiticos provenientes de suelos supresivos al nematode barrenadov Radopholus similis [Cobb] Thorne en plantacions commerciales de platoon en la zona de Talamanca, Costa Rica M. Sc Thesis CATTE, Turialba, Costa Rica. Channppa, B.S.; Srivastava, A.N. and Dhavan, S.C. [2008]. Effect of cell concentrations of four straits of rhizobacterium, Pseudomonas fluorescens on hatching and mortality of root-knot nematode, Meloidogyne incognita. Indian Journal of Nematology, 38,189-195.

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Biological Control of Plant Nematodes…

415

Chen, S., and Dickson, D.W. [2004]. Biological Control of Nematodes by Fungal Antagonists. In: Nematology: Advances and Perspectives. vol. 2. Nematode Management and Utilization, Z.X. Chen, S.Y. Chen, and D.W. Dickson [eds.] Cambridge, MA: Tsinghua. University Press and CABI, pp. 656. Chet, I,; Inbar, J. and Hadar, I. [1997]. Fungal antagonists and mycoparasites. In: The Mycota IV: Environmental and microbial relationships, D.T. Wicklow and B. Soderstrom [eds.]. Springervorla, Berlin pp 165-184. Chet, I.; Barak, Z. and Oppenheim, A. [1993]. Genetic engineering of microorganisms for improved biocontrol activity. In: Biotechnology in Plant Disease Control. Chet, I. [ed.]. Willey Liss Inc., U.S.A. pp. 211-235. Chevanet, C. ; Besson, F. and Michel, G. [1985]. Effect of various growth conditions on spore formation and bacillomycin L. Production in B. subtilis. Canadian Journal of Microbiology, 32, 255-258. Dave, A. and Patel, H.H. [1999]. Inorganic phosphate solubilizing soil Pseudomonas. Indian Journal of Microbiology, 39,161-164. Delcambe, L; Peypouz, F.; Besson, F.; Guinand, G. and Michel, G. [1977]. Structure of iturin and iturin like substance. Biochemical Society Transaction, 5, 1122-1124. Dennis, C. and Webster, J. [1971b]. Antagonistic properties of species group of Trichoderma -I. Production of nonvolatile antibiotics. Transactions of British Mycological Society, 157, 41-60. Dubeikovsky, A.N.; Mordukhova, E.A.; Kochetkow, V.V.; Polikarpora, F.Y. and Boronin, A.M. [1993]. Growth promotion of black current softwood cuttings by recombinant strain Pseudomonas fluorescens BSP53a synthesizing an increased amount of indole-3 acetic acid. Soil Biology and Biochemistry, 25,1277-1281. Dubey, A.V.;Vaishya, U.K.; Bapat, P.N. and Tomar, V.S. [2000]. Microbial solubilization of rock phosphate: an alternative phoshorus source for mustard crop. JNKV Research Journal, 31, 54-55. Duijff, B.J.; Gianinazzi-Pearson, V. and Lemanceau, P. [1997]. Involvement of outer membrane lipopolysachrides in the endophytic colonization of tomato roots by biocontrol agent, P. fluorescens strain WCS417r. New Phytologist, 135, 325-334. Duponnois, R; Kisa, M; Plenchette, C. [2006]. Phosphate-solubilizing potential of the nematophagous fungus Arthrobotrys oligospora. Journal of Plant Nutrition and Soil Science, 169, 280-282. Eapen, S.J. and Venugopal, M.N. [1995]. Field evaluation of Trichoderma spp and Paecilomyces lilacinus for control of root-knot nematode and fungal diseases of cardamom nurseries. Indian Journal of Nematology, 25, 15-16. El-Hasan, A; Walker, F; Buchenauer, H. [2007]. Trichoderma harzianum and its metabolite 6pentyl-alpha-pyrone suppress fusaric acid produced by Fusarium moniliforme. Journal of Phytopathology, 156, 79 – 87. El-Sherif, M.A.; Ali, A.H. and Barakat, M.I. [1995]. Suppressive bacteria associated with plant parasitic nematodes in Egyptian agriculture. Japanese Journal of Nematology 24, 558-59. Gaur A.C. 1990. Phosphate Solubilizing Microorganisms as Biofertilizers. Omega Scientific Publishes, New Delhi. pp.176. Gaur, A.C. and Gaind, S. [1987]. Shelf life of phosphate solubilizing microorganisms in charcoal-soil based carrier. Geobios, 11, 227-229.

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416

Mujeebur Rahman Khan, Shahana Majid, Fayaz A. Mohidin et al.

Gaur, A.C. and Ostwal, K.P. [1972]. Influence of phosphate dissolving bacilli on yield and phosphate uptake of wheat crop. Indian Journal of Experimental Biology, 10, 393-394. Gaur, A.C. [1987]. Organic manures and biofertilizers, Division of microbiology, IARI, New Delhi pp. 46. Gaur, A.C.[1990]. Phosphate Solubilizing Microorganisms as Biofertilizers. Omega Scientific Publishers, New Delhi. Gaur, A.C. and Gaind, S. [1984]. Shelf life of phosphate solubilizing microorganisms in charcoal-soil based carrier. Geobios, 11, 227-229. Gaur, A.C.; Mathur, R.S. and Sadosivam, K.V. [1980]. Effect of organic material and phosphate dissolving cultures on the yield of wheat and green gram. Indian Journal of Agronomy, 25, 501-503. Gautam, A.Z.; Siddiqui, Z.A. and Mahmood, I. [1995]. Integrated management of Meloidogyne incognita on tomato. Nematologia Mediterranea 23, 245-247. Glick, B.R [1995]. The enhancement of plant growth by free living bacteria. Canadian Journal of Microbiology, 41, 109-117. Gokte, N. and Swarup, G. [1988]. On the potential of some biocides against root-knot and cyst nematodes. Indian Journal of Nematology, 18, 151-152. Goswami B K, Rao U and Mantoo M A. 1994. Biotechnology in India, 177-198. Gracia de Salamone, I.E.; Hynes, R.K.I. and Nelson, L.M. [2001]. Cytokinin production by plant growth promoting rhizobacteria and selected mutants. Canadian Journal of Microbiology, 47, 404-411. Guarro, J.; Antolin-Ayala, M.I.; Gene, J.; Gutierreg-Calzada, J.; Nieves-Diez, C. and Ortoneda, M. [1999]. Fatal case of Trichoderma harzianum infection in a renal transplant recipient. Journal of Clinical Microbiology, 37, 3751-3755. Hamid, M.; Siddiqui, I.A. and Shaukat, S.S. [2003]. Improvement of Pseudomonas fluorescens CHA0 biocontrol activity against root-knot nematode by the addition of ammonium molybdate. Letters in Applied Microbiology 36, 239 – 244. Hanson, L.E. and Howell, C.R. [2004]. Elicitors of plant defense responses from biocontrol strain of Trichoderma virens. Phytopathology, 94, 171-176. Harman, G.E.; Howell, C.R.; Viterbo, A.; Chet, I. and Lorito, M. [2004]. Trichoderma species opportunistic, avirulent plant symbionts. Nature Reviews, 2, 43-56. Hasan, N. [2004]. Evaluation of native strain of Paecilomyces lilacinus against Meloidogyne incognita in cowpea followed by lucerne. Annals of Plant Protaction Sciences, 12, 121124. Haseeb, A. and Kumar, V. [2005]. Evaluation of bioinoculants, organic amendment materials and pesticides for the management of Meloidogyne incognita – Fusarium solani disease complex of brinjal cv. Pusa Kranti. National symposium on recent advances and research priorities in Indian nematology, 9-10 December 2005, IARI, New Delhi. pp. 6. Hewlett, T. E.; Dickson, D. W.; Mitchell, D. J. and Kannwischer-Mitchell, M. E. [1988]. Evaluation of Paecilomyces lilacinus as a biocontrol agent of Meloidogyne javanica on tobacco. Journal of Nematology, 20, 578–584. Howell, C.R. and Stipanovic, R.D. [1980]. Suppression of Pythium ultimum induced damping off of cotton seedlings by Pseudomonas fluorescens and its antibiotic pyoleutorin. Phytopathology, 70, 712-715. Indian Institute of Spices Research [IISR] [1995]. Annual report 1994-95, Calicut India, pp. 89.

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Ito, T. and Neilands, J.B [1958]. Products of low iron fermentation with Bacillus subtilis: isolation, characterization and synthesis of 2, 3-dihydroxybenzoylglycin Journal of American Chemical Society, 80, 4645-4647. Jatala, P.; Franco J.; Gonzalez, A. and O’Hara, C.M. [1985]. Hatching simulation and inhibition of Globodera pallida eggs by enzymatic and exopathic toxic compounds of some biocontrol fungi. Journal of Nematology, 17, 501. Jatala, P. [1985]. Biological control of nematodes. In: An Advanced Treatise on Meloidogyne.Vol. I. Biology and Control. J. N. Sasser and C. C. Carter [eds.]. North Carolina States University Graphics. Raleigh, N. C., U. S. A. pp. 303-308. Jatala, P. [1986]. Biological control of plant parasitic nematodes. Annual Review of Phytopathology 24, 453-491. Jatala, P.; Kaltenbach, R. and Bocangel M. [1979]. Biological control of Meloidogyne incognita acrita and Globodera pallida on potatoes. Journal of Nematology, 11, 303. Jetiyanon, K.; Tuzun, S. and Kloepper, J.W. [1997]. Lignification, peroxidase and superoxidase dismutases as early plant defense reaction associated with PGPR-mediated induced syatemic resistance. In: Plant Growth Promoting Rhizobacteria: Present Status and Future Prospects. A. Ogoshi, K. Kobayashi, Y. Homma, F. Kodama, N. Kondo and S. Akino [eds.], Japan; Sapporo,pp. 265-268. Johri, B.N.; Rao, C.V.S. and Goel, R. [1997]. Fluorescent Pseudomonas in plant disease management. In: Biotechnological Approaches in Soil Microorganisms for Sustainable Crop Production, ed. K.R. Dadarwal, Scientific Publisher, Jodhpur, India, pp. 193-223. Kang, Y.M.; Carlson, R.; Tharpe, W. and Schell, M.A. [1998]. Characterization of genes involved in biosynthesis of a novel antibiotic from Bukholderia cepacia BC11 and their role in biological control of Rhizoctonia solani. Applied and Environmental Microbiology, 64, 3939-3947. Keel, C. and Defago, G. [1997]. Interactions between the beneficial soil bacteria and root pathogens: mechanisms and ecological impacts. In : Multitrophic interactions in Terrestrial System, A.C. Gange and V.K, Brown [eds.], Oxford, Blackwell Science pp. 27-47. Kerry, B.R. [1982]. The decline of Heterodera avenae populations. EPPO Bulletin, 12, 491496. Kerry, B.R. [1987]. Biological control. In: Principles and practice of nematode control in crops, R.H. Brown and B.R. Kerry [eds.]. Academic Press, Sydney. pp. 233-263. Kerry, B.R.; Crump, D.H. and Mullen, L.A. [1982a]. Studies of the cereal-cyst nematode, Heterodera avenae under continuous cereals, 1974-1978. I. Plant growth and nematode multiplication. Annals of Applied Biology,100, 477-487. Kerry, B.R.; Crump, D.H. and Mullen, L.A. [1982b]. Studies of the cereal-cyst nematode, Heterodera avenae under continuous cereals, 1975-1978. II. Fungal parasitism of nematode eggs and females. Annals of Applied Biology, 100, 489-499. Khan, M.R.; Anwer, M.A; Khan, S.M. and Mohiddin F A. [2006]. A characterization of some isolates of Aspergillus niger and evaluation of their effects against Meloidogyne incognita. Tests of Agrochemicals and Cultivars [U K] 27, 33-34. Khan M R.; Khan, S.M. and Mohiddin, F.A. [2004]. Biological control of fusarial wilts of chickpea through seed treatment with the commercial formulations of Trichoderma harzianum and/or Pseudomonas fluorescens. Phytopathologia Mediterranea [Italy] 43, 20-25.

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

418

Mujeebur Rahman Khan, Shahana Majid, Fayaz A. Mohidin et al.

Khan M R.; Kounsar, K. and Hamid, A. [2002]. Effect of certain rhizobacteria and antagonistic fungi on root nodulation and root-knot nematode disease of green gram. Nematologia Mediterranea, 30, 85-89. Khan M. R., Khan, N. and Khan S M. [2001]. Evaluation of agricultural materials as substrate for mass culture of fungal biocontrol agents of fusarial wilt and root-knot nematode diseases. Annals of Applied Biology [TAC-21 Suppl.] [U.K.] 22, 50-51. Khan, M.R., Khan, S.M. and Mohiddin,F.A. [2007]. Effect of certain fungal and bacterial phosphate-solubilizing microorganisms on the fusarial wilt of tomato. In: Developments in plant and soil sciences, Velazquez-perez, E. [ed.], Kluwer Academic Publishers. pp. 357-361. Khan, M.R. and Akram, M. [2000]. Effect of certain antagonistic fungi and rhizobacteria on wilt disease complex caused by Meloidogyne incognita and Fusarium oxysporium f.sp. lycopersici on tomato. Nematologia Mediterranea, 28, 139-144. Khan M.R. and Anwer M.A. [2007]. Molecular and biochemical characterization of soil isolates of Aspergillus niger and assessment of antagonism against Rhizoctonia solani. Phytopathologia Meditteranea [Italy] 46, 304-315. Khan, A.; Williams, K. and Nevalainen H. [2006]. Testing the nematophagous biological control strain Paecilomyces lilacinus 251 for paecilotoxin production. FEMS Microbiology letters, 227, 107-111. Khan, M.R. [2005]. Biological Control of Fusarial Wilt and Root-Knot of Legumes. Government of India Publication, Department of Biotechnology, Ministry of Science and Technology, New Delhi, India pp. 50. Khan, M R. and Ejaz, M. N. [1997]. Effect of neem leaves and Paecilomyces lilacinus on root-knot nematode disease of okra. Vasundhara 2, 1-5. Khan, M.R. and Tarannum, Z. [1997]. Effect of certain bacteria and fungi on the development of root-knot disease of tomato. Symposium on Herbal and Microbial Pesticides, Banaras Hindu University, Varanasi, pp. 13-14. Khan, M.R. [2008]. Plant Nematodes: Methodology, Morphology, Systematics, Biology and Ecology. Science Publishers, Enfield, USA. pp.1-360. Khan, M.R. and Akram, M. [1997]. Effect of certain bacterial and fungal bioagents on a disease complex caused by Meloidogyne incognita and Fusarium oxysporum f. sp. lycopersici on tomato. Abstracts, International Conference on Integrated Plant Disease Management, New Delhi. pp.252. Khan, M.R. and Kounsar, K. [2000]. Effects of certain bacteria and fungi on the growth of mungbean and reproduction of Meloidogyne incognita. Nematologia Mediterranea, 28, 221-226. Khan, M.R. and Mustafa, U. [2005]. Corm rot and yellows of gladeoli and its biomanagement. Phytopathologica Mediterrranaea, 44, 208-215. Khan, M.R. and Tarannum, Z. [1999]. Effects of field application of various microorganisms on Meloidogyne incognita on tomato. Nematologia Mediterranea 27, 233-238. Khan, M.R.; Khan, S.M. and Mohiddin, F.A. [2005b]. Root-knot problem of some winter ornamental plants and its biomanagement. Journal of Nematology, 37, 198-206. Khan, M.R.; Mohiddin, F.A.; Khan, S.M and. Khan, B. [2005a]. Effect of seed treatment with certain biopesticides on root-knot of chickpea. Nematologia Mediterranea, 33, 107-112. Khan, M.W. and Dasgupta, M.K. [1993]. The concept of interaction. In: Nematode Interactions, M.W. Khan [ed.], Chapman and Hall, New York, pp. 42-54.

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Biological Control of Plant Nematodes…

419

Khan, M.W. and Reddy, P.P. [1993]. Management of disease complexes. In: Nematode Interactions, M.W. Khan [ed.], Chapman and Hall, New York, pp.345-365. Khan, M.W. and Khan, M.R. [1995]. Fungus-nematode interactions. In: Nematode Pest Management An Appraisal of Eco-friendly Approaches. G. Swarup, D.R.Dasgupta, J S Gill [eds ]. Nematological Society of India. pp. 70-78. Kim, B.S.; Moon, S.S. and Hwang, B.K. [1999]. Isolation, identification and antifungal activity of a macrolide antibiotic, oligomycin A, produced by Streptomyces libani. Canadian Journal of Botany,77, 850-858. Kirkpatrick, J.D.; Mai, W.F.; Parker, K.G. and Fisher, E.G. [1964]. Effect of phosphorus and potassium nutrition of sour cherry on the soil population level of five plant parasitic nematodes. Phytopathology, 54, 706-712. Kloepper, J.W.M.; Tsuzum, S. and Kuc, J. [1992]. Proposed definition related to induced disease resistance. Biocontrol Science and Technology, 2, 347-349. Koike, N.; Hyakumachi, M.; Kageyama, K.; Tsuyumu, S. and Doke, N. [2001]. Induction of systemic resistance in cucumber against several diseases by plant growth- promoting fungi: lignification and superoxide generation. European Journal of Plant Pathology, 107, 523-533 Kole, S.C and Hajra, J.N [1997]. Isolation and evaluation of tricalcium phosphate and rock phosphate solubilizing microorganisms from acedic terai and lateritic soil of West Bengal. Journal of International Academicia, 1, 198-205. Kumar, S.S.D.; Balamani, B. and Bezbaruah, B. [1997]. Plant growth promotion and fungal pest control through an antibiotic and siderophore producing fluorescent Pseudomonas strain from tea [Camellia sinesis[L] O Kuntze] plantations. Indian Journal of Experimental Biology, 35, 289-292. Kundu, B.S. and Gaur, A.C. [1981]. Carrier studies on phospho micro-organisms as single and mixed inoculants. Indian Journal of Agricultural Sciences, 51, 252-255. Lal, L. [2002]. Phosphatic Biofertilizers. Agrotech Publishing Academy, Udaipur. pp. 48-50. Leeman, M.; den Ouden, F.M.; Van Pelt, J. A.; Dirkx, FP.N.; Steijl, H.; Bakker, P. A. H. M. and Schippers, B. [1996b]. Iron availability effects induction of systemic resistance to Fusarium wilt of radish by Pseudomons fluorescens. Phytopathology, 86, 149-155. Leeman, M.; Van Pelt, J. A.; Hendrickx, M. J.; Scheffer, R. J.; Bakker, P. A. H. M. and Schippers, B. [1995]. Biocontrol of Fusarial wilt of radish in commercial green house trials by seed treatment with Pseudomonas fluorescens WCS 374. Phytopathology , 85, 1301-1305. Leong, J. [1986]. Siderophores: their biochemistry and possible role in the biocontrol of plant pathogen. Annual Review of Phytopathology, 24, 187-209. Lipping, Y; Jiatao, X; Daohong, J; Yanping, F; Guoqing, L; Fangcan, L. [2008]. Antifungal substances produced by Penicillium oxalicum strain PY-1—potential antibiotics against plant pathogenic fungi. World Journal of Microbiology and Biotechnology, 24, 909–915. Loeffler, W.; Tschen, J.S.M., Vanittanakom, N; Kugler, M.; Knorpp, E; Hsieh, T.F. and Wu, T.G. [1986]. Antifungal effect of bacilysin and fengymycin from Bacillus subtilis F-29-3. A comparison with activities of other Bacillus antibiotics. Journal of Phytopathology, 115, 204-213. Loper, J.E. and Henkens, M.D. [1999]. Utilization of heterologous siderophores enhances levels of iron available to Pseudomonas putida in the rhizosphere. Applied and Environmental Microbiology, 65, 5357-5363.

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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420

Mujeebur Rahman Khan, Shahana Majid, Fayaz A. Mohidin et al.

Luc, M.; Bridge, J. and Sikora, R.A. [2005]. Reflections on plant nematology in tropical and subtropical agriculture. In: Plant Parasitic Nematodes of Tropical and Subtropical Agriculture, M. Luc, R.A.Sikora and J. Bridge [eds.]. CABI, London, U.K. pp. 1-10. Lysek, H. [1966]. Study of biology of geohelminths. II. The importance of some soil microorganisms for the viability of geohelminth eggs in the soil". Acta Universitatis Palackianae Olomucensis, 40, 83–90. M Piga, P.; Belager, R.R.; Paulitz, T.C. and Benhamou, N. [1997]. Increased resistance to Fusarium oxysporum f.sp. radices-lycopersici in tomato plants treated with the endophytic bacterium P. fluoresecns strain 63-28. Physiological and Molecular Plant Pathology, 50, 301-320. Mahadtanapuk, S; Sanguansermsri, M; Cutler, R.W; Sardsud, V; Anuntalabhochai, S. [2007]. Control of anthracnose caused by Colletotrichum musae on Curcuma alismatifolia Gagnep. using antagonistic Bacillus spp. American Journal of Agricultural and Biological Sciences 2, 54-61. Majumdar, S.K. and Bose, S.K. [1970]. γ-Glutamyl and D-and L-peptide linkage in mycobacillin a cyclic peptide antibiotic. Archives of Biochemistry and Biophysiolgy, 90, 154-158. Mankau R. [1969 b]. Nematicidal activity of Aspergillus niger culture filtrates. Phytopathology, 59: 1170. Mankau R. [1969a]. Toxicity of cultures of Aspergillus niger to mycophagous nematode Aphelenchus avenae. Phytopathology, 59, 13. Mannanov, R.N; Sattarova, R.K. [2001]. Antibiotics produced by Bacillus bacteria. Chemistry of Natural Compounds, 37, 17-123. Marek-kozaczuk, M. and Skorupska, A. [2001]. Production of B-group vitamins by plant growth promoting Pseudomonas fluorescens strain 267 and the importance of vitamins in the colonization and nodulation of red clocer. Biology and Fertility of Soils, 33, 146-151. Marten, P.; Bruckner, S. and Luth, P. [1999]. Plant growth promotion of different cultivated plants and biological control of soil borne phytopathogenic fungi by Bacillus subtilis strain 82g. Zeitschrift fur pflanzenkrankheiten and pflanzenschutz, 106, 74-81. Mehmet, O; Cevdet, A; Oral, D; Ali, S.M. [2005]. Single and double inoculation with Azospirillum/Trichoderma : the effects on dry bean and wheat. Biology and Fertility of Soils 41, 262-272. Metraux, J.P.; Signer, H.; Ryals, J.; Ward, E.; Wyss-Benz, M; Gaudin, J.; Raschdorf, K.; Schimid, E.; Blum, W., and Inverardi, B. [1990]. Increase in salicylic acid at the onset of systemic acquired resistance in cucumber. Science, 250, 1004-1006. Meyer, S.L.F.; Massoud, S.I.; Chitwood, D.J. and Roberts, D.P. [2001]. Evaluation of Trichoderma virens and Bukholderia cepacia for antagonistic activity against root-knot nematode, Meloidogyne incognita. Nematology 2, 871-879. Milner, J.L.; Silo-Suh, L.; Lee, J.C.; He, H.; Clady, J. and Handelsman, J. [1996]. Production of kanosamine by Bacillus cereus OW85. Applied and Environmental Microbiology, 62, 3061-3065. Mittal, V; Singh, O; Nayyar, H; Kaur, J; Tewari, R. [2008]. Stimulatory effect of phosphatesolubilizing fungal strains (Aspergillus awamori and Penicillium citrinum) on the yield of chickpea (Cicer arietinum L. cv. GPF2). Soil Biology and Biochemistry, 40, 718-727. Mittal, N.; Saxena,G. and Mukerji, K.G. [1999]. Biological control of root knot nematodedestroying fungi. In: Ethnomycology of Fungal Biotechnology Exploiting from Natural

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

Biological Control of Plant Nematodes…

421

Resources for Novel Products, J. Singh and K.R. Aneja [eds.] Kluwer Academic/ Planum Publishers, New York, USA, pp. 143-172. Morales, A; Alvear, M; Valenzuela, E; Rubio, R; Borie, F. [2007]. Effect of inoculation with Penicillium albidum, a phosphate-solubilizing fungus, on the growth of Trifolium pratense cropped in a volcanic soil. Journal of Basic Microbiology 47, 275-280. Nakayamna, T.; Homma, Y.; Hashidoko, Y.; Mizutani, J. and Tahara, S. [1999]. Possible role of xanthobaccins produced by Stenotrophomonas sp. Strain SD-K88 in suppression of sugarbeet damping off disease. Applied and Environmental Microbiology 65, 4334-4339. Nielson, M.N. ; Sorensen, J. ; Fels, J. ; Pedersen, H.C. [1998]. Secondary metabolite an endochitinase vdependent anatgonsm towards plant pathogenic amacro fungi of pseudomaos flurescens isolated from sugarbeet rhizosphere. Applied and environmental amicrobio 64 :3563-3569. O’ Sullivan, D.J. and O’Gara, F. [1992]. Traits of fluorescent Pseudomonas spp. Involved in suppression of plant root pathogens. Microbiological Reviews, 56, 662-676. O’Hara, C.M. [1985]. Efectos de compuestos enzimaticas y exopaticos difusibles de hongos en el control biologico de neatodos. Ms thesis. Univ. Peru, Cayetano Heredia, Fac. Cienc. Filosof, Lima, Peru. pp. 63. Okholm, L J. [1960]. Kem Maanesdbl, 41: 17-21[Chemicals Abstr. 61, 1226-1964]. Ongena, M.; Daayf, F.; Jacques, P.; Thonart, P.; Benhamou, N.; Paulitz., T.C.; Cornelis, P.; Koedam, N. and Belanger, R.R. [1999]. Protection of cucumber against Pythium root rot by fluorescent pseudomonads: predominant role of induced resistance over siderophores and antibiosis. Plant Pathology 48, 66-76. Oostendrop, M. and Sikora, R. A. [1989]. Seed treatment with antagonistic rhizobacteria for the suppression of Heterodera schachtii early root infection of sugarbeet. Review de Nematologia, 12, 77-83. Oyekanmi, Z.D.L.; Coyne and Fawole L.B. [2008]. Utilization of the potentials of selected microorganisms as biocontrol and biofertilizer for enhanced crop improvement. Journal of Biological Sciences, 8, 746-752. Pal, K.K. and Gardener, B.M. [2006]. Biological Control of Plant Pathogens The Plant Health Instructor, 1117-02. Pal, K.K.; Tilak, K.V.B.R.; Saxena, A.K.; Dey, R. and Singh, C.S. [2001]. Suppression of maize root diseases caused by Macrophomina phaseolina, Fusarium moniliforme and Fusarium graminearum by plant growth promoting rhizobacteria. Microbiology Research, 156, 209-223. Pandey, A; Namrata, D; Bhavesh, K; Rinu, K; Pankaj, T. [2008]. Phosphate solubilization by Penicillium spp. isolated from soil samples of Indian Himalayan region. World Journal of Microbiology and Biotechnology 24, 97-102. Pant, H. and Pandey, G. [2001]. Efficacy of biocontrol agents for the management of rootknot nematode on chickpea. Annals of Plant Protection Sciences, 9, 117-170. Pant, H. and Pandey, G. [2002]. Use of Trichoderma harzianum and neem cake alone and in combination on Meloidogyne incognita galls in chickpea. Annals of Plant Protection Sciences, 10, 134-178. Peypoux, F.; Besson, F.; Michel, G.; Delcambe, L. and Das, B.C. [1978]. Structure of iturin A, a peptidolipid antibiotic from B. subtilis. Biochemistry, 17, 3992-3996. Peypoux, F.; Besson, F. and Michel, G. [1980]. Characterization of a new antibiotic of iturin group by bacillocycin D. Journal of Antibiotics 33, 1146-1149.

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

422

Mujeebur Rahman Khan, Shahana Majid, Fayaz A. Mohidin et al.

Peypoux, F.; Pommmier, M.T.; Marion, D.; Ptak, M. and Michel, G. [1986]. Revised structure of mycosubtilin a lipidolipid from B. subtilis. Journal of Antibiotics 39, 636641. Phister, T.G., Sullivan, DJO; McKay, L.L. [2004]. Identification of bacilysin, chlorotetaine, and iturin A produced by Bacillus sp. strain CS93 isolated from Pozol, a Mexican fermented maize dough. Applied and Environmental Microbiology, 70, 631–634. Pocasangre Enamorado, L.E.; zum Felde, A.; Cañizares, C.; Muñoz, J.; Suarez, P.; Jimenez, C.; Riveros, A.S.; Rosales, F.E.; Sikora, R.A. [2007]. Field evaluation of the antagonistic activity of endophytic fungi towards the burrowing nematode, Radopholus similis, in plantain. In: Latin America Pro Musa Symposium, Greenway Woods Resort, White River [ZAF], 2007/09/10-14. Powell, P.E.; Cline, G.R.; Reid, C.P.P. and Szaniszlo, P.J. [1980]. Occrrence of hydroxamate siderophore iron chelators in soil. Nature, 287, 833-834. Rudresh, D.L; Shivaprakash, M.K; Prasad, R.D. [2005]. Tricalcium phosphate solubilizing abilities of Trichoderma spp. in relation to P uptake and growth and yield parameters of chickpea (Cicer arietinum L.). Canadian Journal of Microbiology 51, 217–222. Rao, A.B. [1990]. Role of microorganisms in plant nutrition under acid conditions. In: Biofertilizers, Vyas, L.L. [ed.], Scientific Publishers Jodhpur. pp. 67-84. Reddy, B.P. and Swamy, S.N. [2000]. Effect of farmyard manure, phosphate solubilizing bacteria and phosphorus on yield and economics of black gram. Indian Journal of Agricultural Sciences, 70, 694-696. Reid, R.K.; Reid, C.P.P.; Powell, P.E. and Szaniszlo, P.J. [1984]. Comparison of siderophore concentration on aqueous extracts of rhizosphere and adjacent bulk soils. Pedobiologia, 26, 263-266. Rekha, A. and Saxena, S K. [1999]. Influence of certain rhizosphere fungi together with Rhizoctonia solani and Meloidogyne incognita on germination of ‘Pusa Ruby’ tomato seeds. Indian Phytopathology, 52, 121-126. Robinson, P.M. and Park, D. [1966]. Volatile inhibitors of spore germination produced by fungi. The Transactions, 49, 639-649. Rodriguez-Kabana, R.; Morgan-Jones, G.; Godoy, G. and Gintis, B. O. [1984]. Effectiveness of species of Gliocladium, Paecilomyces and Verticillium for control of Meloidogyne arenaria in field soil. Nematropica, 14,155-170. Rodríguez-Romero, A.S.; Badosa, E.; Montesinos E. and Jaizme-Vega M.C. [2007]. Growth promotion and biological control of root-knot nematodes in micropropagated banana during the nursery stage by treatment with specific bacterial strains Annals of Applied Biology, 152, 41 – 48. Roger, H.J.; Lomalina, N. and Abraham, E.P. [1965]. Observation on the structure of bacilysin. Biochemistry Journal, 97, 579-586. Sahebani, N. and Hadavi N. [2008].Biological control of the root-knot nematode Meloidogyne javanica by Trichoderma harzianum. Soil Biology and Biochemistry, 40, 2016-2020. Saifullah [1996a]. Fungal parasitism of young females of Globodera rostochiensis and G. pallida. Afro Asian Journal of Nematology, 6, 17-22. Saifullah [1996b]. Killing potato cyst nematode males, a possible control strategy. Afro Asian Journal of Nematology, 6, 23-28.

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Biological Control of Plant Nematodes…

423

Sasser, J. N. [1989]. The Farmers Hidden Enemy- Plant Parasitic Nematodes. North Carolina State University Graphics. Raleigh, U. S. A., pp. 115. Schenek, C. [2004]. Control of nematodes in tomato with Paecilomyces lilacinus. Hawaii Agriculture Research Center Vegetable Report 5. Seenivan, N. and Devarajan, K. [2000]. Biocontrol potential of P. fluorescens against rice root nematode, Hierschmanniella gracilis on rice. Current Nematology 13, 35-38. Seenivasan, N., Devrajan,K. and Selvaraj, N. [2007]. Management of potato cyst nematodes, Globodera spp. through biological control. Indian Journal of Nematology 37[1]. Sharma, A. and Johri, B.N [2003]. Combat of iron deploydation through a plant growth promoting fluorescent Pseudomonas strain GRP3A in mungbean [Vigna radiata L. Wilzeck]. Microbiological Research, 158, 77-81. Sharma, H.K.; Prasad, D. and Sharma, P. [2005]. Compatibility of fungal bioagents as seed dressers with carbofuran in okra against Meloidogyne incognita. National Symposium on Recent Advances and Research Priorities in Indian Nematology, 9-11, December, 2005. IARI New Delhi.pp. 72. Sharon, E.; Chet, I. and Spiegel, Y.[2009]. Improved attachment and parasitism of Trichoderma on Meloidogyne javanica in vitro. European Journal of Plant Pathology, 123, 291-299. Shin, W; Ryu, J; Kim, Y; Yang, J; Madhaiyan, M; Sa, T. [2006]. Phosphate solubilization and growth promotion of maize (Zea mays L.) by the rhizosphere soil fungus Penicillium oxalicum.18th World Congress of Soil Science. July 9-15, Philadelphia, Pennsylvania, USA. Shishido, M.; Breuil, C. and Chanway, C.P. [1999]. Endophytic colonization of spruce by plant growth promoting rhizobacteria. FEMS Microbiology Ecology, 29, 191-196. Shoda, M. [2000]. Bacterial control of plant diseases [review]. Journal of Bioscience and Bioengineering, 89, 515-521. Siddiqi, IA. Shaukat, S.S. and Hamid, M. [2003]. Suppression of Meloidogyne incognita by Pseudomonas fluorescens strain CHAO and its genetically modified derivatives: I. The influence of oxygen. Nematologia Mediterranea 31, 105-109. Siddiqi, I.A. and Shaukat, S.S. [2003]. Impact of biocontrol P. fluorescens CHAO and its genitically modified [GM] derivatives on penetration of Meloidogyne javanica in mungbean roots. Nematologia Mediterranea 31, 43-45. Siddiqi, I.A. and Ehteshamul-Haque, S. [2001]. Suppression of root rot-root-knot disease complex by Pseudomonas aeruginosa in tomato: the influence of inoculum density, nematode population, moisture and other plant associated bacteria. Plant and Soil, 237, 81-89. Siddiqi, I.A. and Shaukat, S.S. [2004]. Trichoderma harzianum enhances the production of nematicidal compounds in vitro and improve biocontrol of M. javanica by Pseudomonas fluorescens in tomato. Letters in Applied Mycology 38, 169-175. Siddiqi, Z.A. and Mahmmod I. [1996]. Biological control of Heterodera cajani and Fusarium udum on pigeonpe by Glomus mossae, Trichoderma harzianum and Verticillium chlamydosporium . Israel Journal of Plant Sciences 44, 49-56. Siddiqi, Z.A. and Mahmood I. [1995]. Management of Meloidogyne incognita race 3 and Macrophomina phaseolina by fungus culture filtrates and Bacillus subtilis on chickpea. Fundamentals of Applied Nematology, 18, 71-76.

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424

Mujeebur Rahman Khan, Shahana Majid, Fayaz A. Mohidin et al.

Siddiqui I. A. and Shaukat S.S. [2002]. Rhizobacteria-mediated Induction of Systemic Resistance [ISR] in Tomato against Meloidogyne javanica Journal of Phytopathology, 150, 469 – 473. Sikora, R.A. [1988]. Interrelationship between plant growth promoting rhizobacteria, plant parasitic nematodes and soil microorganisms. Medidlingen Faculteit and bouwwetenschappen Rijksuniversiteit Gent 53, 867-878. Sikora, R.A. and Reimann, S. [2004]. Suppressive soils, the edge of chaos and multitrophic strategies for biocontrol of pests and diseases in soil ecosystems. Bulletin OILB/SROP 27, 251-258. Singh, S.M.; Azam, M.F.; Khan, A.M. and Saxena, S K. [1991]. Effect of Aspergillus niger and Rhizoctonia solani on development of Meloidogyne incognita on tomato. Current Nematology, 2, 163-166. Sitaramaiah, K. and Pathak, K.N. [1993]. Nematode bacterial disease interaction. In: Nematode Interactions, M.W. Khan[ed.]. Chapman and Hall, New York. pp. 232-250. Steijl, H.; Niemann, G.J. and Boon, J.J. [1999]. Changes in chemical composition related to fungal infection and induced resistance in carnation and radish investigated by pyrolysis mass spectrometry. Physiological and Molecular Plant Pathology, 55, 297-311. Sticher, L.; Mauch,-Mani, B. and Metraux, J.P. [1997]. Systemic acquired resistance. Annual Review of Phytopathology, 35, 235-270. Stirling, G.R. [1991]. Biological Control of Plant Parasitic Nematodes. UK: CABI Publishing. pp. 282. Suarez, B.; Rey, M.; Castillo, P.; Monte, E. and Llobell, A. [2004]. Isolation and characterization of PRA1, a trypsin like protease from the biocontrol agent Trichoderma harzianum CECT 2413 displaying nematicidal activity. Applied Microbiology and Biotechnology, 65, 46-55. Taylor, C.E. and Brown, D.J.F. [1997]. Nematode Vectors of Plant Viruses. CABI, London, U.K. pp. 286. Thomas, G.V; Shantaram, M.V; Saraswathy, N. [2005]. Occurrence and activity of phosphate solubilizing fungi from coconut plantation soils. Journal of Plant and Science, 87, 357364. Thrane, C.; Olsson, S.; Nielsen, T.H. and Sorensen, J. [1999]. Vital fluorescent strains for detection of stress in Pythium ultimum and Rhizoctonia solani challenged with viscosinamide from Pseudomonas fluorescens DR54. FEMS Microbiology Ecology, 30, 11-23. Timmusk, S. and Wagner, E.G.H. [1999]. The plant growth promoting rhizobacterium Paenibacillus polymyxa induces changes in Arabidopsis thaliana gene expression: apossible connection between biotic and abiotic stress responses. Molecular-PlantMicrobe Interactions 12, 951-959. Toohey, J.I.; Netson, C.D. and Krotkov, G. [1965]. Isolation and identification of two phenazines from a strain of Pseudomonas aureofasciens. Canadian Journal of Botany, 43, 1055-1062. Troxler, J.; Berling, CH.; Moenne-Loccox, Y.; Keel, C. and Defago, G. [1997]. Interaction between the biocontrol agent Pseudomonas fluorescens CHAO and Thielaviopsis basicola in tobacco roots observed by immunofluorescence microscopy. Plant Pathology, 46, 62-71.

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Biological Control of Plant Nematodes…

425

Turan, M. [2006]. Evaluation of the capacity of phosphate solubilizing bacteria and fungi on different forms of phosphorus in liquid lulture. Journal of Sustainable Agriculture 28, 99108. Varenyam, A; Savant, V.V; Sudhakara Reddy, M. [2007]. Phosphate solubilization by a wild type strain and UV-induced mutants of Aspergillus tubingensis. Soil Biology and Biochemistry.39, 695-699. Van Loon, L.C. [1997]. Induced resistance in the plant and the role of pathogenesis related proteins. European Journal of Plant Pathology, 103, 753-765. Van Peer, R.; Niemann, G.J. and Schippers, B. [1991]. Induced resistance and phytoalexin accumulation in biological control of Fusarium wilt of carnation by Pseudomonas sp. WCS417r. Phytopathology 81, 728-734. Vassilev, N; Vassileva, M; Nikolaeva, I. [2006]. Simultaneous P solubilizing and bio-control activity of microorganisms: potentials and future trends. Applied Microbiology and Biotechnology 71, 137-144. Vesper, S.J. [1987]. Production of pili fimbriae by Pseudomonas fluorescens and a correlation with attachment to corn roots. Applied and Environmental Microbiology 53:1397-1405. Vey, A.; Hoagland, R.E. and Butt, Y.M. [ 2001]. Toxic metabolites of fungal agents. In: Fungi as Biocontrol Agents: Progress, Problems and Potential. T.M. Butt, C. Jackson and N. Magan [eds.] CAB International pp. 311-346. Viaene, N.M. and Abanoi, G. S. [2000]. Hirsutella rhossiliensis and Verticillium chlamydosporium as biocontrol agents of the root-knot nematode Meloidogyne hapla on lettuce. Journal of Nematology, 32, 85-100. Wakelin, S.A; Gupta, V.V.S.R; Harvey, P.R; Ryder, M.H. [2007]. The effect of Penicillium fungi on plant growth and phosphorus mobilization in neutral to alkaline soils from southern Australia. Canadian Journal of Microbiology 53, 106-115. Walia, R.K. [1994]. Biological control of phytonematodes-principles and prospects. In: Nematode Pest Management in Crops. D.S. Bhatti and R.K. Walia [eds]. CBS Publishers and Distributors, Delhi. pp. 228-238. Webster, J.M. [ed.]. [1972]. Economic Nematology. Academic Press, London and New York. pp. 563. Wei, G.; Kloepper, J.W. and Tuzum, S. [1996]. Induced systemic resistance to cucumber disease and increased plant growth by plant growth promoting rhizobacteria under field conditions. Phytopathology, 86, 221-224. Weller, D. M. and Thomashow, L.S. [1994]. Current challenges in introducing beneficial microorganisms into the rhizosphere. In: Molecular ecology of rhizosphere Microorganisms, F. O. Gara, D.N. Dowling and B. Boesten [eds.] V.C.H., Weinheim pp. 1-18. Weller, M. [1988]. Biological control of soil borne plant pathogens in the rhizosphere with bacteria. Annual Review of Phytopahtology, 26, 379-407. Whipps, J.M. [1997]. Developments in the biological control of soil borne plant pathogens. Advances in Botanical Research, 26, 1-134. Windham, G.I.; Windham, M.T. and Williams, W.P. [1989]. Effects of trichoderma spp on maize growth and Meloidogyne arenaria reproduction. Plant diseases, 73, 493-496. Yadav, K.S. and Dadarwal, K.R. [1997]. Phosphate solubilization and mobilization through soil microorgansims. In: Biotechnological Approaches in Soil Microorganisms for

Phosphate Solubilizing Microbes for Crop Improvement, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

426

Mujeebur Rahman Khan, Shahana Majid, Fayaz A. Mohidin et al.

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Sustainable Crop Production. K.R. Dadrwal [ed.] Scientific publishers, Jodhpur pp 293308. Yang, C.H. and Crowely, D.E. [2000]. Rhizosphere microbial community structure in relation to root location and plant iron nutritional status. Applied and Environmental Microbiology, 66, 345-351. Zareen, A.; Khan, N. J. and Zaki, M.J. [2001]. Biological Control of Meloidogyne javanica [Treub] Chitwood, root knot nematodes of Okra {Abelmoschus esculentus [L] Moench. Pakistan Journal of Biological Sciences , 4, 990-994. Zimand, G.; Elad, Y. and Chet, I. [1996]. Effect of Trichoderma harzianum on Botrytis cinerea pathogenicity. Phytopathology 86, 1255-1260. Zum Felde A.; Pocasangre, L.E.; Carñizares Monteros, C.A.; Sikora, R.A.; Rosales, F.E. and Riveros, A.S. [2006]. Effect of combined inoculations of endophytic fungi on the biocontrol of Radopholus similis Info Musa 15 [1-2], 12-17. Zum Felde, A. [2002]. Screening of Endophytic Fungi from Banana [Musa] for Antagonistic Effects towards the Burrowing Nematode, Radopholus similis [Cobb] Thorne. M.Sc. Thesis, University of Bonn, Germany.

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INDEX

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A AAC, 312 ABC, 383 abiotic, 63, 79, 81, 83, 107, 161, 186, 216, 386, 391, 400, 407, 424 Abiotic, 392 absorption, 45, 47, 70, 71, 72, 162, 174, 179, 219, 291, 338 Abundance, 159 ACC, 8, 67, 68, 72, 95, 97, 98, 100, 108, 115, 125, 157, 178, 215, 250, 342 accessibility, 45 accounting, 173, 208 accuracy, 28 acetate, 7, 117, 206, 238, 271 acetic acid, 8, 48, 69, 71, 95, 115, 122, 151, 178, 214, 215, 250, 337, 354, 361, 408, 415 acetylene, 314, 347 ACF, 10, 277 acidic, 5, 38, 49, 55, 70, 107, 113, 146, 147, 148, 161, 162, 187, 201, 205, 206, 221, 242, 256, 262, 282, 285, 310, 329, 330, 372 acidification, 2, 48, 122, 133, 141, 157, 206, 218, 236, 237, 344, 362 acidity, 162, 176, 186, 188, 206, 207, 223, 253, 254, 255, 360, 372 Acinetobacter, 16, 18, 19, 20, 23, 28, 32, 33, 36, 38, 39, 40, 112, 119, 123, 147, 157, 211, 219, 228, 250, 267, 343 ACM, 10, 277 ACS, 241 actinobacteria, 93 actinomycetes, vii, 1, 2, 10, 38, 65, 66, 69, 72, 75, 78, 88, 91, 94, 96, 97, 102, 163, 202, 203, 208, 233, 247, 252, 265, 268, 271, 277, 278, 281, 282, 287, 292, 298, 303, 326 activation, 2, 14, 72, 79, 177, 179, 224

activators, 344 active site, 8 Adams, 31 adaptability, 149, 157, 311 adaptation, 89, 103, 139, 146, 147, 217, 358 adenine, 287 adenosine, 3, 146, 208, 389 adenosine triphosphate, 3, 146, 389 adhesion, 81 adipate, 168 adjustment, 47, 176 ADP, 146 ADS, 38 adsorption, 8, 29, 44, 45, 141, 146, 283, 360 adults, 398, 404 aerobic, 71, 164, 255, 271, 411 Africa, 97, 195, 244, 283, 324, 359 Ag, 158 agar, 49, 76, 114, 115, 116, 118, 132, 136, 182, 183, 202, 203, 218, 312, 313, 333, 343, 370, 381, 388 age, 251 agent, 32, 48, 77, 78, 91, 93, 97, 122, 206, 216, 265, 268, 269, 270, 271, 273, 275, 276, 277, 278, 294, 296, 342, 343, 402, 404, 414, 415, 416, 424 agents, 38, 66, 77, 79, 86, 87, 91, 98, 102, 103, 105, 126, 168, 250, 266, 267, 277, 297, 304, 329, 359, 398, 407, 411, 412, 418, 421, 425 aggregates, 176, 401 aggregation, 90, 193, 194 agrarian, viii, 140, 325, 360 agricultural, 1, 2, 36, 51, 55, 59, 65, 66, 88, 91, 111, 112, 113, 120, 136, 138, 139, 142, 147, 151, 157, 162, 173, 184, 199, 200, 203, 210, 215, 231, 248, 252, 256, 277, 281, 282, 283, 287, 299, 305, 310, 326, 330, 334, 335, 338, 343, 348, 349, 357, 358, 368, 370, 383, 389, 395, 396, 397, 398, 399, 402, 406, 413, 418 agricultural crop, 2, 55, 65, 283, 395, 396, 399, 406 agricultural residue, 112

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agriculture, viii, 5, 10, 15, 16, 33, 51, 55, 56, 59, 64, 65, 69, 82, 87, 88, 89, 92, 96, 98, 109, 121, 124, 129, 130, 140, 141, 142, 143, 148, 151, 159, 162, 195, 200, 207, 210, 216, 217, 222, 242, 243, 249, 250, 254, 259, 282, 294, 299, 302, 303, 308, 310, 311, 320, 323, 324, 350, 351, 352, 353, 370, 372, 373, 386, 397, 412, 413, 415, 420 agrochemicals, 147, 240, 248, 255, 268, 281, 299, 338 agroforestry, 89, 188, 261 agroindustrial, 108 aid, 120, 332, 341, 378, 396 aiding, 326 air, 93, 174, 181, 249, 258 air-dried, 93, 181 Alabama, 414 alanine, 400 Alberta, 5, 11, 12, 13, 304 Albino, 101 alcohols, 77 aldehydes, 77 alfalfa, viii, 4, 31, 109, 132, 194, 196, 263, 276, 305, 306, 309, 311, 313, 315, 316, 317, 318, 320, 339, 387, 392 algorithm, 312 alkali, 207, 306, 314 alkaline, 6, 8, 10, 13, 49, 70, 102, 112, 125, 131, 132, 133, 146, 148, 159, 201, 208, 221, 240, 242, 244, 259, 260, 262, 358, 360, 386, 394, 425 alkaline phosphatase, 8, 49, 70, 132, 208, 240 Allah, 277 Allium cepa, 182 alluvial, 13, 57, 149, 187, 216, 218, 223, 262, 327, 372 alpha, 61, 72, 415 alternative, vii, 2, 48, 69, 87, 88, 118, 130, 139, 145, 164, 167, 197, 211, 230, 231, 248, 266, 282, 297, 299, 339, 344, 388, 415 alternatives, 147 aluminium, 44, 146, 148, 201, 203, 206, 218, 220, 222, 255, 283, 285, 325 aluminum, 5, 44, 70, 112, 131, 188, 252, 255, 265, 267, 290, 310, 325, 337, 358, 383 amendments, 54, 57, 79, 87, 185, 379 AMF, 174, 184, 287, 290, 291, 293, 295 amino, 46, 47, 77, 80, 169, 178, 180, 211, 251, 256, 285, 291, 361, 381, 386 amino acid, 46, 47, 77, 80, 169, 180, 211, 251, 256, 291, 361, 381, 386 amino acids, 46, 47, 77, 80, 169, 180, 251, 256, 291, 361, 381, 386 ammonia, 72, 78, 164, 178, 250, 314, 337, 342, 343, 359, 400, 401, 403, 405, 407

ammonium, 112, 119, 170, 194, 206, 214, 254, 314, 351, 359, 407, 411, 416 ammonium borate, 314 ammonium salts, 207, 254 ammonium sulphate, 207, 254 amorphous, 206, 219 Amsterdam, 189, 333, 334 amylase, 250, 252, 287 anaerobes, 164 anaerobic, 31, 166, 207 anaerobic sludge, 31 analysis of variance, 314 anger, 207 Angiosperms, 171 animal waste, 252, 287 animals, 131, 209, 360, 385, 394 annealing, 19 ANOVA, 314 antagonism, 77, 78, 90, 104, 115, 271, 279, 304, 342, 398, 401, 408, 418 antagonist, 98, 270 antagonistic, 36, 72, 77, 81, 88, 102, 105, 109, 121, 122, 123, 163, 220, 250, 269, 270, 271, 273, 275, 277, 279, 397, 399, 402, 403, 418, 420, 421, 422 antagonists, 98, 116, 269, 415 Antarctic, 158 anthocyanin, 358 anthropogenic, 189 Anthyllis cytisoides, 305 antibiotic, 76, 83, 84, 94, 97, 103, 106, 115, 124, 126, 247, 275, 345, 416, 417, 419, 420, 421 antibiotic resistance, 83, 84 antibiotics, 63, 66, 72, 76, 77, 94, 107, 117, 318, 342, 345, 348, 398, 407, 414, 415, 419 antibody, 403 antigen, 79, 81, 93 antimicrobial protein, 79 antioxidant, 176 antisense, 22, 105 antisense RNA, 105 apatite, 61, 142, 193, 202, 227, 245, 253, 267, 358, 359 apatites, 6, 112, 201, 267, 360 API, 119 apples, 98 aqueous solutions, 218 aqueous suspension, 410 Arabidopsis thaliana, 72, 103, 380, 383, 384, 385, 394, 424 ARB, 37 arbuscular mycorrhizal fungi, viii, 57, 90, 95, 106, 107, 112, 114, 186, 187, 189, 190, 192, 193, 194,

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Index 195, 196, 197, 220, 223, 244, 261, 278, 279, 290, 293, 302, 304, 305, 307, 308, 332 arbuscular mycorrhizal fungus, 14, 92, 106, 127, 190, 191, 192, 245, 263, 307, 308, 349, 352, 355 Arctic, 159 Argentina, 127, 309, 311, 313, 314, 315, 319, 320, 321 arginine, 117 arid, 14, 147, 148, 200, 228, 239, 245, 253, 254, 308 aromatic compounds, 151 arsenic, 385, 391, 393 arsenite, 385 arthropods, 112, 191, 200 ash, 123, 189, 194, 252, 259 Asia, 324 Asian, 422 asparagin, 134 aspergillosis, 275 Aspergillus niger, 103, 127, 204, 210, 213, 215, 221, 238, 240, 243, 245, 252, 256, 257, 278, 285, 286, 289, 297, 300, 306, 307, 308, 395, 398, 399, 400, 412, 413, 417, 418, 420, 424 Aspergillus terreus, 364 assessment, 18, 21, 28, 30, 64, 92, 129, 136, 229, 254, 339, 418 assignment, 26 assimilation, 46, 71, 119, 168, 172, 185, 206, 228, 257, 259, 267, 353, 363, 384, 388 atmosphere, 16, 165, 314 ATP, 2, 3, 146, 166, 384, 393 ATPase, 48 attachment, 81, 403, 423, 425 Australia, 13, 104, 195, 225, 324, 331, 334, 353, 359, 369, 425 automation, 22 autotrophic, 202 awareness, 130, 369 Azospirillum lipoferum, 95, 213, 215, 219, 347, 371, 406

B B. licheniformis, 203, 210, 238, 362 B. subtilis, 67, 68, 73, 74, 77, 79, 80, 85, 86, 90, 93, 94, 146, 171, 210, 270, 286, 292, 298, 318, 344, 362, 406, 407, 408, 409, 410, 412, 415, 421, 422 bacilli, 16, 18, 25, 29, 318, 416 bacillus, 261 Bacillus subtilis, 31, 33, 72, 91, 94, 97, 98, 100, 104, 107, 109, 171, 180, 192, 203, 211, 213, 215, 216, 221, 222, 228, 250, 252, 261, 270, 271, 278, 292, 298, 318, 342, 344, 355, 395, 398, 403, 406, 408, 414, 417, 419, 420, 423

429

Bacillus thuringiensis, 38, 343 bacterial cells, 116, 179, 181, 182 bacterial strains, viii, 9, 11, 23, 25, 38, 120, 133, 134, 151, 157, 199, 200, 202, 203, 209, 211, 216, 217, 220, 232, 237, 253, 284, 287, 295, 313, 314, 315, 317, 319, 345, 368, 370, 422 bacteriocin, 363 bacterium, 8, 13, 33, 34, 37, 39, 59, 77, 82, 92, 94, 95, 100, 104, 106, 115, 125, 126, 147, 151, 160, 189, 196, 206, 209, 224, 226, 227, 258, 259, 269, 270, 290, 291, 295, 298, 302, 306, 320, 323, 326, 330, 333, 339, 341, 342, 343, 354, 374, 408, 411, 414, 420 balance sheet, 226 bananas, 97, 193 Bangladesh, 374 barley, 49, 62, 79, 84, 92, 105, 125, 157, 170, 171, 187, 194, 195, 215, 228, 231, 243, 295, 306, 311, 349, 380, 387, 392, 394, 396 barrier, 79 barriers, 407 base pair, 24, 26 basidiomycetes, 8, 11, 171, 241 beef, 31 behavior, 11, 20, 23, 45, 248, 251, 383 bell, 403 beneficial effect, 39, 176, 183, 184, 216, 289, 292, 295, 309, 310, 318, 323, 367, 414 benefits, 30, 43, 87, 151, 174, 182, 186, 230, 248, 276, 300, 330, 342, 390 beta lactam, 83 bias, 31 bicarbonate, 50, 352 binding, 82, 122, 166, 225, 253, 256, 360, 379, 385, 403 bioassay, 269, 270, 277 bioassays, 403 bioavailability, 147, 175, 219, 228, 229, 307, 333, 357, 388 biochemistry, 102, 220, 419 biocontrol, 29, 31, 32, 39, 64, 66, 72, 77, 78, 79, 80, 86, 87, 91, 93, 94, 96, 97, 98, 100, 101, 102, 103, 105, 107, 108, 109, 113, 114, 121, 122, 125, 126, 157, 174, 188, 237, 241, 250, 262, 265, 267, 268, 271, 276, 277, 278, 279, 285, 287, 294, 295, 304, 307, 337, 342, 343, 344, 345, 349, 367, 373, 398, 400, 403, 404, 407, 412, 413, 414, 415, 416, 417, 418, 419, 421, 423, 424, 425, 426 biodegradation, 82, 111, 113, 151, 378 biodiversity, 2, 15, 16, 18, 28, 104, 120, 216, 228, 249, 282 biofilm formation, 64, 80, 81, 91, 100, 101, 106 biofilms, 81, 93

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Index

biogas, 363 biogeography, 21, 28 bioinformatics, 390 biological activity, 102, 107, 360 biological control, 32, 65, 76, 78, 80, 91, 94, 95, 102, 103, 104, 105, 106, 107, 109, 111, 123, 159, 215, 258, 268, 277, 278, 343, 345, 350, 412, 413, 417, 418, 420, 422, 423, 425 biological control agents, 91, 412 biological nitrogen fixation, 64, 132, 141, 165, 285, 309, 311, 335, 338, 369 biomass, 8, 9, 27, 47, 51, 59, 60, 64, 82, 85, 87, 88, 125, 132, 136, 148, 149, 151, 152, 172, 173, 174, 193, 212, 215, 216, 225, 231, 236, 241, 243, 269, 270, 275, 282, 285, 287, 289, 290, 292, 293, 295, 299, 304, 305, 310, 313, 317, 318, 324, 332, 344, 346, 347, 378, 379, 381, 383, 384, 385, 387, 389, 392, 393, 408 biometric, 318 biomineralization, 209 bioreactor, 241, 243 bioremediation, xi, 15, 16, 82, 209 biosurfactant, 77, 102, 106 biosynthesis, 46, 60, 117, 123, 124, 125, 178, 192, 197, 224, 269, 340, 352, 380, 381, 384, 386, 387, 392, 400, 417 biosynthetic pathways, 383 biota, 16, 64 biotechnological, 209, 217, 242, 311, 344, 361 biotechnology, viii, 16, 61, 126, 189, 195, 226, 374, 389, 391, 413 biotic, 79, 81, 83, 88, 161, 186, 216, 386, 407, 424 biotic factor, 81 biotransformation, 389 blood, 193 bonding, 45 bonds, 8, 49, 113, 208, 239 bootstrap, 120 borate, 314 boric acid, 314 Bose, 202, 222, 405, 420 bradyrhizobial, 193 Bradyrhizobium, 9, 37, 39, 59, 65, 68, 122, 124, 129, 130, 135, 138, 139, 140, 141, 166, 178, 185, 186, 187, 196, 203, 215, 219, 221, 262, 293, 301, 307, 308, 318, 321, 347, 351, 352 branching, 71 Brassica rapa, 8 Brazil, 29, 51, 57, 61, 106, 142, 188, 306, 324, 359, 369 Brazilian, 12, 38, 324 breakdown, 59, 124, 200, 208 breeding, 89, 195, 379, 386

brevis, 170, 362 broad spectrum, 63, 72, 269, 403 Buenos Aires, 311 buffer, 44, 253, 312 building blocks, 3 Bulgaria, 377 Burkholderia, 9, 12, 16, 17, 18, 19, 20, 23, 29, 30, 31, 33, 34, 36, 40, 65, 67, 72, 74, 76, 112, 113, 123, 135, 192, 203, 209, 212, 213, 220, 226, 238, 244, 250, 254, 259, 284, 310, 346, 368, 402 butyric, 204, 338 bypass, 211 by-products, 324

C Ca2+, 2, 5, 48, 113, 133, 167, 168, 253 cabbage, 8, 12, 36, 261, 305, 409 cadmium, 9, 11, 72, 105, 106, 107, 258, 350, 380, 381, 382, 383, 386, 387, 391, 392, 393, 394 calcium, 2, 5, 6, 29, 44, 55, 70, 114, 118, 131, 133, 134, 136, 145, 146, 148, 149, 152, 158, 168, 185, 190, 193, 200, 201, 202, 206, 208, 209, 216, 218, 219, 220, 221, 222, 232, 237, 241, 242, 244, 252, 254, 258, 265, 267, 283, 301, 310, 325, 333, 339, 358, 359, 364, 380 calcium carbonate, 5 calcium oxalate, 222 calibration, 27, 234 Cameroon, 206, 220, 221, 242 Canada, 4, 13, 169, 359, 369 candida, 398 Candida, 203 cane sugar, 324, 328, 329, 330 capillary, 25 carbohydrate, 3, 112, 204, 311 carbohydrates, 3, 39, 46, 80, 112, 142, 256, 331, 381, 393 carbon, 13, 47, 49, 59, 64, 69, 70, 81, 94, 96, 99, 101, 106, 113, 117, 119, 121, 169, 170, 181, 185, 186, 194, 202, 222, 252, 253, 258, 297, 306, 339, 353, 357, 373 Carbon, 105, 190, 193, 196, 197, 253 carboxyl, 207, 237 carboxyl groups, 207, 237 carboxylates, 205, 256 carboxylic, 7, 76, 108, 110, 116, 121, 168, 178, 204, 205, 206, 269, 363, 405 carboxylic acids, 204, 205, 206 carboxylic groups, 7 carcinogenic, 9 cardamom, 403, 415 Carica papaya, 404

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Index carrier, 85, 179, 180, 181, 182, 191, 195, 231, 234, 235, 236, 361, 415, 416 CAS, 114, 343 case study, 169 catalase, 250 catalytic activity, 49 catechol, 343 category a, 201 cation, 8, 48, 118, 236, 253, 388 cattle, 190 CBS, 425 CCC, 312 cDNA, 21, 381 cell, 3, 20, 22, 47, 48, 64, 72, 78, 79, 81, 84, 92, 93, 94, 95, 101, 116, 121, 146, 166, 167, 169, 171, 173, 177, 178, 180, 181, 197, 200, 208, 209, 211, 212, 220, 239, 265, 268, 269, 270, 275, 325, 358, 368, 390, 407, 411, 414 cell adhesion, 81 cell division, 72, 166, 178, 200, 325 cell growth, 181 cell signaling, 64 cell surface, 47, 212 cellulose, 112, 116, 169, 271, 407 Cellulose, 112 ceramics, 359 cereals, 51, 87, 136, 163, 185, 203, 396, 397, 417 CES, 38 CGT, 312 changing environment, 413 channels, 284, 360 chaos, 424 charcoal, 95, 106, 180, 361, 415, 416 chelates, 255 chelators, 379, 387, 407, 413, 422 chemical composition, 424 chemical industry, 89 chemical properties, 9, 10, 251, 255, 289, 291, 316, 348, 377 chemical reactions, 3, 148, 199 chemicals, 23, 50, 64, 78, 147, 236, 240, 248, 249, 255, 277, 299, 324, 363, 377, 399 chemisorption, 218 Chemotherapy, 32 chicken, 185 Chile, 188, 241, 311, 315 China, 11, 100, 106, 172, 204, 254, 324, 329, 359 chitin, 86, 100, 116, 269 Chitin, 78, 100 chloride, 115, 255, 260, 371 chlorine, 107 chlorophyll, 3, 152, 153, 178, 231, 318, 358 chloroplast, 388, 390, 391, 394

431

chloroplasts, 381, 391, 393 cholera, 391 chromatin, 72 chromatography, 270 chromium, 9, 262, 342, 354, 393 Chromium, 13, 262, 354 chromosome, 368 chromosomes, 3, 22 CHS, 400 cis, 225, 299 citrus, 90, 176, 197, 353, 396 classes, 212, 383 classical, 386, 398 classification, 17, 24, 31, 102, 193 clay, 162, 172, 176, 182, 223, 291, 295, 358 cleavage, 113, 208 climatic factors, 107 clone, 32, 217 cloning, 60, 87, 98, 123, 211, 221, 222, 228, 230, 350, 372 closure, 325 clustering, 38 clusters, 32, 117 CMV, 73 Co, 62, 71, 82, 83, 109, 196, 245, 262, 295, 308, 310, 321, 335, 347, 348, 354, 365, 373, 374, 375, 408 CO2, 3, 207, 208, 363 cocoa, 93 coconut, 232, 269, 373, 424 codes, 209, 211, 368 coding, 35, 40, 118, 210, 384, 388 codon, 388 coenzyme, 60, 123, 124, 192, 352, 368 coffee, 177, 188, 196, 284, 304, 307, 308 coil, 212 collateral, 122, 218, 277, 349 colloids, 51 Colombia, 194, 319 colon, 226 colonization, viii, 29, 33, 63, 64, 81, 82, 83, 84, 91, 92, 93, 95, 96, 100, 101, 103, 105, 107, 122, 134, 140, 175, 177, 183, 184, 185, 193, 194, 216, 217, 225, 236, 251, 268, 276, 285, 286, 290, 291, 292, 293, 295, 298, 305, 306, 319, 332, 340, 347, 390, 398, 400, 405, 412, 414, 415, 420, 423 colonizers, 79, 81, 145 Columbia, 324 combined effect, 184, 205, 206 commerce, 324 commercialization, 64, 85, 89, 157, 169, 240 commodity, 324 communication, 81, 105, 166, 167

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432

Index

communities, viii, 15, 16, 17, 20, 21, 22, 23, 24, 25, 26, 28, 30, 31, 32, 34, 37, 81, 87, 92, 97, 105, 109, 129, 151, 216, 229, 230, 240, 248, 251, 255, 256, 258, 260, 265, 270, 286, 305, 317, 325, 338, 342, 348, 360, 390 community, 17, 21, 22, 24, 25, 26, 27, 31, 32, 33, 34, 35, 38, 39, 47, 64, 80, 84, 92, 94, 108, 109, 140, 151, 185, 209, 249, 251, 255, 256, 257, 260, 286, 308, 407, 426 compaction, 92, 349 compatibility, 234, 240, 290 competence, 47, 69, 83, 282, 298, 303, 340, 405, 412 competition, 8, 63, 66, 74, 81, 94, 177, 182, 268, 271, 296, 345, 385, 407 competitiveness, 20, 47, 88, 161, 174 compilation, ix, 201 complement, 161 complex interactions, 217, 286, 333 complexity, 16, 19, 26, 121, 211 components, 3, 7, 45, 46, 84, 99, 132, 180, 201, 211, 231, 243, 270, 293, 295, 325, 328, 359, 363, 408 composition, 10, 22, 25, 46, 229, 249, 251, 256, 260, 342, 379, 424 compost, 7, 12, 79, 85, 88, 90, 140, 159, 180, 181, 185, 192, 193, 202, 203, 223, 224, 252, 285, 305, 324, 363, 372, 373 composting, 142, 217, 252 compounds, 3, 4, 5, 6, 8, 14, 44, 46, 48, 51, 61, 71, 73, 77, 78, 83, 89, 94, 100, 103, 125, 130, 132, 133, 138, 147, 149, 151, 162, 166, 167, 169, 179, 190, 200, 201, 203, 206, 207, 208, 220, 229, 236, 239, 245, 247, 250, 251, 254, 255, 256, 265, 267, 268, 269, 270, 271, 274, 275, 278, 283, 310, 337, 338, 348, 353, 359, 360, 361, 370, 384, 388, 394, 398, 400, 402, 407, 409, 417, 423 computer software, 314 concentrates, 325 concentration, 1, 2, 3, 5, 6, 7, 9, 10, 27, 44, 70, 71, 72, 81, 113, 115, 131, 132, 134, 136, 137, 138, 141, 148, 154, 157, 170, 175, 176, 201, 216, 231, 237, 239, 244, 253, 255, 258, 283, 287, 290, 293, 295, 310, 311, 313, 318, 330, 344, 346, 358, 360, 384, 388, 403, 410, 411, 422 conceptual model, 46 conductivity, 249 confidence, 120 confusion, 146 Congress, 244, 307, 423 conifer, 185 conjugation, 212, 297, 368, 383 consensus, 19, 29, 30, 120 conservation, 33, 101, 309 constraints, 1, 147, 283

constructed wetlands, 392 construction, 17, 87 consumption, 163, 164 contaminant, 378 contaminants, 181, 369, 378, 390 contaminated soils, 9, 98, 259, 381 contamination, 31, 181, 183, 216, 268, 390, 412 conversion, 5, 7, 113, 146, 178, 204, 208, 282, 310, 340, 360, 362, 393 copper, 104, 174, 175 corn, 3, 88, 95, 180, 209, 210, 223, 301, 403, 425 correlation, 48, 133, 134, 168, 175, 201, 204, 208, 237, 425 corrosion, 359 Corynebacterium, 147, 202, 362 cosmetics, 359 Costa Rica, 414 cost-effective, 182, 240, 324 costs, 21, 147, 163, 299, 353 cotton, 76, 80, 94, 97, 109, 172, 187, 195, 215, 222, 223, 235, 270, 271, 273, 276, 278, 371, 396, 400, 416 covering, 201 CPS, 363 CRC, 101, 104, 105, 107, 108, 194, 390, 391 critical analysis, 307 criticism, ix CRM, 145, 149, 151, 152, 153, 154 Crop loss, 397 crop production, vii, 2, 5, 44, 51, 56, 63, 64, 66, 87, 104, 111, 147, 156, 161, 162, 170, 184, 185, 195, 196, 200, 228, 281, 282, 297, 325, 370, 386, 409 crop residues, 87, 96, 112, 185, 307, 374 cross-talk, 81 crust, 142, 194, 241, 373 crystal structure, 147, 360 crystallization, 58, 147 crystals, 147, 391 Cuba, 324 cultivation, 29, 38, 51, 59, 61, 181, 248, 332, 333, 334, 339, 354, 364, 365 cultural practices, 217 culture, 14, 15, 17, 21, 22, 35, 57, 63, 87, 90, 97, 106, 109, 116, 117, 119, 133, 134, 136, 138, 140, 143, 151, 152, 169, 179, 181, 182, 183, 187, 188, 189, 197, 204, 206, 210, 217, 222, 224, 226, 232, 233, 234, 236, 237, 241, 245, 253, 257, 263, 270, 277, 286, 288, 299, 301, 313, 332, 343, 344, 349, 351, 364, 369, 372, 402, 403, 408, 410, 411, 418, 420, 423 culture conditions, 288 culture media, 204, 313 cutters, 26

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Index cyanide, 94, 122, 247, 250, 261, 342 cyanobacteria, 16, 98, 163, 165, 166, 226, 285 Cyanobacteria, 68, 362 cycles, 16, 21, 27, 47, 66, 134, 216, 294, 312, 313, 344 cycling, 8, 13, 15, 60, 93, 101, 132, 158, 162, 197, 216, 228, 231, 248, 271, 283, 303, 304, 305, 307, 312 cyclohexane, 299 cyclohexanol, 271 cyclohexanone, 272 cyst, 396, 397, 404, 411, 416, 417, 422, 423 cysteine, 380, 381, 389, 391 Cysteine, 393 cysts, 397, 402, 410 cytochemistry, 244 cytochrome, 77, 117 cytochrome oxidase, 117 cytometry, 34 cytoplasm, 388 cytoplasmic membrane, 147, 205 cytosol, 331, 380, 381 cytosolic, 391 cytotoxic, 344 Czech Republic, 61, 129, 136, 138, 140

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D dairy, 35, 36 dairy products, 36 damping, 32, 76, 90, 94, 97, 102, 105, 258, 270, 271, 278, 416, 421 data analysis, 31 data set, 120 database, 26, 120, 312, 314, 368 death, 80 decay, 163, 207, 396 decomposition, 2, 14, 47, 66, 106, 112, 175, 190, 207, 242, 271, 314 defense, 78, 79, 82, 99, 121, 127, 177, 178, 268, 269, 386, 400, 412, 414, 416, 417 defense mechanisms, 79, 269, 412 defenses, 64 deficiency, vii, 2, 3, 4, 44, 46, 51, 71, 111, 147, 175, 200, 229, 230, 248, 256, 266, 267, 282, 283, 299, 320, 325, 387, 396, 409 deficit, 31, 176, 286, 295, 302 definition, 38, 88, 146, 163, 419 deforestation, 29 deformation, 402 degradation, 7, 39, 60, 112, 121, 215, 240, 268, 269, 271, 363

433

degrading, 24, 34, 36, 78, 88, 94, 95, 116, 122, 151, 218, 265, 268, 270, 275, 277, 349 dehydrogenase, 9, 11, 58, 70, 95, 117, 123, 211, 219, 220, 223, 228, 302, 340, 341, 352 dehydrogenases, 205 delivery, 64, 89, 130, 209, 217, 300, 331, 369 denaturing gradient gel electrophoresis, 35, 38 denitrifying, 258 density, 72, 80, 81, 92, 105, 107, 135, 168, 176, 181, 184, 188, 231, 249, 251, 291, 297, 334, 378, 411, 412, 423 deoxyribonucleic acid, 146 dephosphorylating, 49 dephosphorylation, 113, 208 deposition, 407 deposits, 172, 299, 359 deprivation, 13, 160 derivatives, 90, 108, 224, 423 desert, 103, 189, 225, 251 desiccation, 81, 85, 251, 254 desorption, 5, 12, 44, 55 destruction, 248, 255 detection, 21, 22, 23, 24, 25, 26, 27, 29, 34, 35, 36, 37, 38, 39, 40, 84, 118, 126, 243, 262, 424 detection techniques, 262 detoxification, 379, 380, 381, 384, 385, 387, 389 Detoxification, 388 detoxifying, 383 developed countries, 369, 397 developing countries, 230, 283, 397, 413 dextrose, 116, 117, 118, 170 diazotrophs, 69, 93, 98, 163, 166, 183, 202, 214, 283, 299, 372 dietary, 209, 311 diets, 209, 223 differentiation, 17, 19, 24, 29, 146, 321 diffusion, 131, 174, 176 digestion, 26, 34, 120 dimer, 24 discriminant analysis, 39 discrimination, 25, 27 diseases, viii, 76, 98, 102, 103, 106, 112, 167, 214, 265, 266, 267, 273, 276, 353, 396, 413, 415, 419, 421, 423, 424 displacement, 8 distillation, 314 distilled water, 119, 134, 234, 312, 313 distribution, 13, 35, 39, 47, 60, 65, 84, 131, 168, 181, 182, 225, 231, 240, 255, 390, 393 disulfide, 299 divergence, 148 diversity, 10, 12, 15, 16, 17, 18, 20, 21, 22, 23, 26, 27, 28, 29, 30, 31, 33, 34, 35, 36, 38, 39, 40, 64,

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434

Index

65, 84, 87, 98, 101, 104, 111, 114, 125, 126, 151, 188, 222, 223, 224, 228, 247, 248, 249, 260, 261, 277, 278, 300, 348, 373, 377 division, 72, 122, 178, 200, 308, 325, 400, 401, 402 DNA, 3, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 31, 34, 35, 37, 38, 39, 40, 84, 87, 89, 119, 120, 127, 146, 183, 187, 208, 210, 211, 216, 223, 224, 228, 262, 312, 317, 352, 388, 414 DNA polymerase, 312 dominance, 37, 81, 360 donor, 115, 212 dosage, 323, 328 drought, 176, 187, 188, 195, 216, 393 drugs, 16 dry matter, 8, 9, 82, 106, 138, 139, 150, 213, 214, 215, 288, 289, 290, 291, 293, 295, 298, 307, 315, 318, 324, 363, 366, 373 DSM, 37 dung, 185, 234 duration, 324, 329, 360, 385, 407, 413 dust, 173, 180, 181, 241 dyes, 20, 27, 202, 220

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E E. coli, 118, 158, 210, 211, 212, 224, 340, 341, 368 E.coli, 83, 170, 271 earth, 16, 200, 267, 324, 360 earthworms, 132 ecological, 1, 8, 10, 16, 28, 65, 83, 89, 101, 106, 121, 140, 248, 249, 250, 251, 255, 256, 282, 300, 361, 368, 417 ecological systems, 255 ecologists, 229 ecology, 15, 22, 25, 35, 83, 89, 103, 190, 390, 425 economic efficiency, 150, 293 economic problem, 140, 338 economic sustainability, 307 economically disadvantaged, 282 economics, 85, 360, 369, 422 ecosystem, viii, 104, 161, 162, 184, 249, 305 ecosystems, vii, 9, 10, 28, 29, 43, 58, 82, 88, 156, 176, 185, 216, 230, 231, 240, 248, 282, 283, 287, 303, 317, 338, 346, 370, 424 ectoparasites, 396 Education, 57 effluents, 324 egg, 270, 401, 402, 403, 410, 411, 412, 413, 414 Egypt, 186, 219, 265, 278, 359, 370 electrical conductivity, 249 electron, 228 electrophoresis, 18, 19, 22, 23, 24, 25, 26, 30, 33, 35, 36, 38, 39, 84, 120, 123, 127, 286

elongation, 68, 100, 147, 151, 178, 325, 363 e-mail, 309 embryo, 387 embryonic development, 402 emission, 27 encapsulated, 241, 278, 344 encapsulation, 354 encoding, 34, 58, 83, 118, 126, 127, 208, 220, 226, 227, 230, 337, 340, 380, 381, 383, 384, 385, 389 encouragement, ix energy, 1, 3, 43, 132, 148, 165, 179, 180, 200, 250, 257, 265, 324, 378 energy transfer, 1, 3, 43, 148, 200 England, 195 enlargement, 178 environment, vii, viii, 2, 15, 16, 17, 21, 23, 30, 38, 40, 69, 81, 86, 87, 89, 121, 131, 133, 138, 145, 148, 151, 156, 157, 161, 162, 166, 167, 182, 189, 190, 195, 216, 217, 229, 231, 235, 236, 247, 251, 252, 255, 258, 266, 268, 271, 282, 290, 299, 300, 325, 339, 342, 344, 360, 365, 369, 373, 377, 388, 389, 390, 391, 393, 394, 398, 413 environmental advantage, 277 environmental change, 217, 251 environmental conditions, 81, 86, 139, 155, 213, 257, 276, 295, 296, 396 environmental control, 81 environmental factors, 71, 146, 231, 249, 342 Environmental Protection Agency, 360, 391 enzymatic, 2, 87, 167, 218, 237, 384, 417 enzymatic activity, 384 enzymes, vii, 3, 8, 26, 46, 49, 63, 66, 71, 72, 78, 84, 94, 95, 99, 113, 121, 132, 146, 176, 199, 205, 206, 208, 209, 210, 218, 230, 239, 240, 242, 253, 256, 265, 268, 269, 270, 271, 285, 287, 310, 357, 368, 379, 383, 384, 387, 389, 393, 399, 405, 407, 409, 412 EPA, 360 epidemic, 32 epidermal cells, 396 equilibrium, 1, 4, 7, 45, 132, 208, 236 ERIC, 19, 32, 120 Escherichia coli, 32, 40, 58, 60, 118, 123, 124, 192, 221, 222, 223, 226, 228, 312, 340, 351, 352, 368, 380 ester, 4, 8, 92, 113, 208, 239, 270, 278 ester bonds, 8 esters, 4, 239 estimating, 124, 197, 319 estuarine, 38 ethane, 123 ethanol, 122, 313, 324 ethanolamine, 201

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Index Ethiopia, 97, 281, 304 ethyl acetate, 117 ethylene, 46, 71, 72, 79, 83, 95, 178, 282, 314, 317, 342 eucalyptus, 96 eukaryotes, 19 Europe, 397 eutrophication, 282, 360 evolution, 61, 87, 103, 126, 158, 226, 314, 374, 394 excision, 24 exclusion, 72, 80 excretion, 2, 112, 206, 209, 236, 291, 371 exploitation, 64, 69, 82, 87, 88, 108, 145, 161, 186, 318, 395, 414 exposure, 180, 387 Exposure, 402 extraction, 7, 25, 49, 61, 380 extrusion, 194, 363 exudate, 80

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F F. solani, 401 family, ix, 32, 39, 61, 127, 227, 338, 383, 402 family members, ix FAO, 61, 308, 360, 369, 371 farmers, vii, 44, 64, 80, 130, 132, 157, 158, 190, 195, 231, 282, 293, 300, 324, 326, 338, 365, 369, 396, 397, 409 farming, 98, 129, 158, 184, 201, 212, 213, 240, 243, 282, 390 farms, 241 fatty acid, 77, 83, 257, 278 fauna, 64, 87, 104, 162, 176 fear, 88 feeding, 396, 400 feedstock, 324 feet, 106 females, 397, 402, 410, 417, 422 fencing, 389 fermentation, 85, 89, 117, 181, 297, 344, 354, 417 fermentation technology, 85 fertiliser, 60 fertility, viii, 10, 13, 64, 88, 107, 111, 112, 113, 120, 130, 133, 138, 157, 162, 163, 188, 191, 216, 221, 225, 244, 248, 255, 256, 278, 281, 288, 309, 311, 317, 349, 377 fertilization, 2, 134, 175, 191, 192, 307, 334, 344, 371, 374, 379 fiber, 324, 392 field trials, 9, 129, 136, 138, 154, 169, 177, 214, 215, 217, 283, 343, 346, 361, 366, 390, 401, 404, 413 Fiji, 324

435

film, 180, 183 filtration, 134 finance, 378 financial support, 122, 300 fingerprinting, 15, 19, 21, 24, 25, 26, 27, 30, 35, 39, 40, 109, 117, 123 fingerprints, 19, 32, 37, 84, 124 FISH, 15, 22, 40, 84 fitness, 108, 301 fixation, vii, 6, 7, 16, 44, 46, 48, 67, 68, 82, 97, 99, 131, 138, 148, 162, 167, 175, 185, 186, 188, 189, 200, 201, 250, 257, 271, 281, 282, 283, 292, 310, 311, 318, 320, 323, 325, 327, 329, 338, 342, 348, 352, 353, 357, 358, 367 flavonoid, 103, 353 flavonoids, 83, 166, 347 flooding, 32, 50, 207 flora, 64, 71, 87, 104, 162, 285, 286, 292, 342 flora and fauna, 64, 87, 162 flow, 34, 185, 190, 233, 354, 360 fluorescence, 20, 22, 25, 27, 34, 40, 85, 146, 147, 153, 157, 354 fluorescence in situ hybridization, 34, 40 fluorine, 267 fluorophores, 20 folding, 26 food, 30, 94, 162, 186, 190, 230, 248, 253, 260, 266, 281, 283, 359, 371, 378, 385, 390 food production, 281, 283 forage crops, 260 forestry, 282, 290 Forestry, 92, 196, 277, 321 forests, 174, 284 formamide, 23 Fox, 49, 58, 350 fractionation, 50, 54, 58 fragmentation, 84, 121 France, 58, 99, 119, 320, 369 freezing, 147, 159, 160 freshwater, 31 fructose, 117, 253 fruits, 51, 358 fuel, 324 fumarate, 168 fumaric, 46, 133, 204, 205, 206, 238, 267, 338, 409 fungal, 12, 37, 70, 76, 78, 79, 80, 81, 95, 102, 116, 121, 123, 134, 142, 174, 176, 178, 182, 183, 185, 188, 193, 202, 207, 214, 216, 220, 233, 237, 240, 252, 258, 268, 269, 276, 278, 285, 286, 288, 289, 291, 292, 304, 331, 341, 343, 344, 345, 403, 404, 408, 414, 415, 418, 419, 420, 423, 424, 425 fungal infection, 344, 345, 424 fungal spores, 81, 182, 183, 188, 331

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436

Index

fungicides, 266, 299, 302, 359 fungus, 12, 14, 48, 72, 76, 79, 83, 92, 94, 106, 127, 143, 169, 170, 172, 173, 175, 177, 183, 184, 185, 186, 190, 191, 192, 197, 222, 225, 237, 240, 241, 243, 244, 245, 254, 257, 259, 261, 263, 270, 275, 276, 277, 288, 290, 291, 293, 295, 296, 297, 298, 300, 302, 303, 307, 308, 318, 332, 333, 341, 344, 349, 352, 355, 363, 367, 398, 400, 401, 403, 404, 414, 415, 421, 423 Fur, 413 Fusarium, 66, 73, 74, 75, 76, 77, 90, 91, 93, 95, 104, 106, 177, 197, 203, 215, 238, 254, 259, 269, 271, 273, 276, 278, 285, 343, 344, 351, 353, 362, 407, 414, 415, 416, 418, 419, 420, 421, 423, 425 Fusarium oxysporum, 73, 74, 77, 91, 106, 203, 238, 269, 271, 273, 276, 414, 418, 420 fusion, 20 fusion proteins, 20

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G G8, 171 gas, 69 gases, 70, 314 gastrointestinal, 209 gastrointestinal tract, 209 gaur, 409 GCC, 312 GC-content, 24 gel, 22, 23, 24, 25, 26, 35, 36, 38, 84, 180, 286, 354 gelatin, 117 gels, 19, 24, 25, 27, 39, 120, 312, 344 GenBank, 120, 314 gene, 17, 18, 20, 21, 22, 23, 26, 27, 30, 35, 36, 37, 38, 39, 40, 58, 60, 71, 72, 81, 82, 83, 87, 89, 92, 96, 98, 99, 102, 105, 108, 114, 116, 117, 119, 120, 122, 123, 124, 126, 127, 192, 203, 209, 210, 211, 212, 217, 220, 221, 222, 223, 224, 226, 240, 258, 312, 321, 340, 341, 347, 349, 350, 352, 354, 368, 372, 379, 380, 381, 382, 383, 384, 385, 387, 388, 391, 393, 394, 402, 407, 424 gene amplification, 35 gene arrays, 21, 40 gene expression, 20, 22, 72, 81, 82, 84, 92, 108, 210, 379, 387, 424 gene silencing, 388 gene transfer, 89, 118 generation, 1, 3, 324, 378, 419 genes, 3, 17, 18, 20, 21, 22, 23, 25, 26, 27, 28, 30, 33, 34, 35, 38, 40, 72, 79, 82, 83, 84, 87, 90, 102, 110, 116, 117, 119, 120, 123, 125, 126, 127, 145, 166, 199, 204, 208, 209, 210, 211, 212, 217, 222, 225, 227, 230, 312, 319, 337, 340, 341, 352, 367,

368, 370, 379, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 400, 407, 417 genetic diversity, 10, 16, 18, 20, 28, 30, 114, 261, 277, 300 genetic information, 3 genetic marker, 40 genetic traits, 148, 230, 338 genetics, viii, 89, 120, 217, 220, 301, 385 genome, 18, 19, 20, 25, 28, 32, 39, 89, 120, 379, 388, 390 genome sequencing, 18, 89 genomes, 28, 30, 32, 40, 388 genomic, 18, 27, 28, 31, 38, 84, 87, 100, 117, 119, 120, 124, 211, 216, 340, 390 genomic DNA library, 340 genomics, 39, 108 genotype, 225, 256 genotypes, 9, 35, 37, 60, 105, 119, 168, 230, 251, 260, 276, 349 genus Streptomyces, 271 geochemical, 58 Georgia, 319 Germany, 159, 258, 334, 426 germination, 71, 72, 121, 151, 152, 154, 177, 178, 182, 239, 244, 252, 268, 271, 295, 327, 330, 367, 372, 400, 401, 403, 422 GFP, 15, 20, 84 gibberellin, 400, 408 gibberellins, 95, 178, 367, 384, 408 girth, 330 glass, 21, 85, 297, 331 global resources, 358 Glomus fasciculatum, 83, 109, 142, 216, 291, 308, 318, 321, 332, 349 Glomus intraradices, 69, 94, 175, 179, 184, 192, 292, 293, 295 glucose, 11, 47, 58, 60, 70, 95, 112, 117, 123, 124, 134, 205, 206, 211, 220, 223, 228, 232, 239, 241, 253, 258, 302, 312, 340, 341, 344, 352, 353 glucose oxidase, 344, 353 glutathione, 121, 127, 268, 350, 379, 380, 383, 389, 394 glycerol, 115, 117, 181, 253 glycine, 77 glycopeptides, 158 glycoprotein, 173 goals, 299, 413 gold, 28 Gordonia, 16, 59, 238 Gore, 91 government, 360 GPS, 85 gracilis, 406, 411, 423

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Index graduate students, xi grain, 3, 9, 82, 86, 93, 126, 139, 149, 199, 213, 214, 215, 289, 290, 292, 293, 294, 295, 299, 302, 318, 338, 345, 346, 348, 353, 363, 365, 366, 372, 387, 409 grains, 90, 292, 346, 372, 396 Gram-negative, 118, 205, 208, 211, 218, 220, 224, 250, 284, 302 gram-negative bacteria, 11, 58, 123 Gram-positive, 77, 284 granules, 31 grapes, 4 grass, 97, 106, 135, 182, 184, 187 grasses, 5, 59, 93, 106, 124, 125, 163, 261 grassland, 61 Great Britain, 352 green revolution, 255, 282 greenhouse, 9, 11, 50, 76, 95, 103, 109, 136, 140, 143, 196, 197, 214, 217, 220, 251, 269, 270, 271, 281, 283, 287, 288, 289, 290, 291, 297, 319, 348, 403, 410 Greenhouse, vi, 213, 281, 287, 345 ground water, 66, 130, 282, 39 grouping, 19, 40 groups, 7, 16, 18, 19, 21, 22, 25, 26, 27, 28, 65, 77, 88, 101, 113, 120, 148, 165, 166, 202, 207, 208, 234, 237, 245, 248, 277, 326, 363, 368, 389 growth factor, 122, 349, 400, 408 growth hormone, 121, 178 growth inhibition, 343 growth rate, 46, 47, 93, 181, 340, 379 growth temperature, 59, 146 Guinea, 195, 196 gums, 86

H H2, 46, 51, 52, 53, 54, 55, 56, 133, 134 habitat, 266 halophyte, 218 halophytes, 373 halos, 114, 116, 118, 203 handling, 24, 86 harm, 81 harvest, 54, 98, 132, 154, 155, 295 harvesting, 2, 364 Hawaii, 35, 333, 353, 404, 423 hazards, 129, 130, 156, 163, 240, 282, 299, 338 health, 63, 82, 88, 89, 104, 107, 111, 112, 148, 182, 200, 216, 229, 240, 258, 262, 278, 282, 299, 311, 343, 345, 385 heat, 146, 160 heating, 312

437

heavy metal, xi, 9, 33, 71, 92, 209, 250, 251, 257, 259, 261, 378, 379, 380, 381, 383, 389, 391, 392, 393 heavy metals, xi, 71, 251, 261, 378, 379, 380, 381, 383, 389, 391, 392, 393 height, 8, 273, 275, 288, 289, 291, 293, 295, 313, 330, 332, 345, 346, 364, 367, 410 hemicellulose, 112, 170 herbivores, 177 heterocyst, 166 heterogeneity, 25, 26, 228 heterogeneous, 230 heterotrophic, 189, 202 heterotrophic microorganisms, 189 high resolution, 22, 25, 27 high temperature, 180, 195, 251, 253, 412 high-tech, 369 hip, 48 histidine, 211 holistic, 89 holoenzyme, 118 homeostasis, 197, 237, 387, 388 homogenized, 311 homology, 40, 118, 211 horizon, 50, 55, 56, 176, 240 horizontal gene transfer, 89 hormone, 72, 178, 383, 392 hormones, vii, 71, 121, 178, 195, 271, 291, 400, 412 Horticulture, 220 host, vii, 30, 63, 64, 79, 80, 85, 89, 102, 125, 126, 148, 163, 166, 167, 171, 173, 174, 176, 182, 183, 185, 190, 209, 212, 268, 270, 276, 311, 331, 339, 345, 368, 387, 396, 398, 400, 407, 412, 414 host tissue, 396 House, 335, 350 HPLC, 117, 206, 225, 243 human, 23, 87, 182, 277, 281, 324, 385, 412 humic acid, 207, 217 humus, 4, 201, 208 husbandry, 88 hybrid, 387, 404 hybridization, 17, 21, 22, 28, 34, 40, 84, 387, 391, 392 hybrids, 379, 387 hydrogen, 77, 79, 92, 116, 123, 131, 147, 151, 176, 218, 250, 269, 285, 337, 344, 401 hydrogen bonds, 147 hydrogen cyanide, 77, 92, 116, 151, 250, 269, 285, 337, 401 hydrogen peroxide, 79, 176, 344 hydrolysis, 8, 12, 49, 87, 117, 208 hydrolyzed, 48, 210, 239, 331 hydroponics, 388

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438

Index

hydrothermal, 35 hydroxide, 50, 219 hydroxides, 71, 112, 162, 201, 206, 385 hydroxyapatite, 6, 58, 113, 133, 141, 167, 170, 203, 211, 212, 222, 252, 259, 283, 351, 360 hydroxyapatites, 112 hydroxyl, 113, 175, 176, 201, 205, 207, 267 hypersensitive, 399 hypersensitivity, 393 hypothesis, 8 hysteresis, 158, 160

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I ice, 88, 147, 158, 159, 160 id, 78, 79, 326 identification, 12, 17, 18, 19, 21, 22, 23, 24, 26, 29, 30, 34, 35, 36, 84, 91, 93, 119, 120, 125, 138, 206, 261, 277, 279, 312, 419, 424 identity, 17, 27 illumination, 120 imaging, 20 imaging techniques, 20 immobilization, 5, 13, 47, 48, 121, 149, 208, 283, 326 immunofluorescence, 424 impurities, 183 in situ, 22, 29, 34, 35, 84, 88, 122, 150, 151, 229, 251, 289, 332, 334, 377 in situ hybridization, 22, 34, 40, 84 in vitro, 31, 35, 47, 76, 77, 88, 101, 115, 124, 125, 129, 136, 151, 182, 183, 210, 214, 216, 225, 229, 231, 234, 236, 237, 239, 243, 251, 260, 268, 270, 271, 287, 304, 309, 313, 317, 318, 332, 339, 342, 344, 345, 352, 395, 401, 403, 404, 405, 408, 411, 423 in vivo, 15, 82, 126, 129, 183, 242, 270, 271, 372, 401, 405 inactivation, 268 inactive, 84, 391 incentive, 88 incidence, 86, 90, 177, 276, 343 inclusion, 24, 26, 132 income, 248 incompatibility, 86 incubation, 85, 114, 115, 116, 134, 136, 149, 150, 179, 232, 239, 314, 358, 402 incubation period, 115 incubation time, 85, 239 incurable, 396 India, xi, 1, 38, 57, 63, 90, 93, 108, 111, 119, 122, 145, 147, 158, 169, 172, 178, 196, 199, 221, 228, 229, 239, 242, 244, 245, 247, 254, 258, 296, 302,

306, 323, 324, 325, 326, 327, 330, 332, 334, 335, 337, 350, 353, 359, 361, 371, 373, 374, 375, 395, 402, 416, 417, 418, 419 Indian, 9, 12, 36, 57, 67, 68, 89, 91, 96, 102, 124, 125, 140, 142, 143, 147, 148, 151, 157, 158, 160, 190, 201, 217, 218, 219, 220, 221, 223, 224, 225, 226, 227, 228, 244, 254, 257, 258, 260, 261, 279, 294, 295, 301, 302, 305, 306, 307, 308, 334, 335, 337, 351, 353, 361, 371, 373, 374, 375, 380, 385, 389, 390, 393, 394, 409, 413, 414, 415, 416, 419, 421, 422, 423 indication, 332, 345 indicators, 43 indices, 88, 107, 141, 276, 411 indigenous, 47, 60, 188, 193, 252, 291, 292, 293, 298, 300, 304, 342, 369 indirect effect, 185, 271 indole, 8, 71, 95, 115, 122, 151, 178, 214, 215, 287, 337, 343, 354, 361, 408, 415 Indonesia, 30, 324 inducer, 343 induction, 79, 83, 92, 103, 178, 210, 267, 268, 271, 353, 419 industrial, 16, 100, 127, 209, 300, 324, 335, 359, 362, 383 industrial application, 100 industrial production, 362 industrial wastes, 127, 300 industry, 87, 89, 296, 324 inert, 44, 182, 325 infection, 64, 66, 79, 83, 84, 91, 141, 167, 177, 186, 191, 193, 216, 285, 291, 298, 333, 339, 344, 345, 396, 397, 401, 403, 407, 410, 411, 414, 416, 421, 424 infections, 79 infertile, 151, 195 infrastructure, 369 ingestion, 389 inheritance, 388 inhibition, 64, 72, 116, 134, 141, 177, 193, 210, 215, 268, 271, 274, 343, 410, 417 inhibitors, 422 inhibitory, 9, 124, 270, 403, 411, 413 inhibitory effect, 9, 270, 413 initiation, 175, 178, 300, 311 injury, 159 Innovation, 195 inoculum, 105, 176, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 195, 282, 296, 298, 313, 396, 423 inorganic salts, 131 inositol, 60, 113, 117, 201, 209, 210, 226, 240, 244, 339

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Index insecticide, 215, 255 insects, 121, 267, 404 insertion, 35, 76, 209, 224, 230, 368 insight, 64, 300, 385 instability, 118, 134, 233, 297 integration, 64, 180, 388, 404, 413 interaction, xi, 12, 63, 64, 79, 80, 88, 90, 95, 141, 158, 172, 175, 177, 182, 184, 185, 190, 195, 216, 220, 255, 259, 261, 268, 270, 271, 279, 296, 307, 319, 331, 354, 357, 361, 390, 403, 407, 418, 424 interactions, viii, 22, 57, 60, 64, 65, 77, 81, 90, 98, 109, 114, 147, 161, 177, 186, 193, 195, 212, 215, 216, 217, 245, 251, 260, 261, 262, 277, 286, 291, 294, 300, 301, 333, 342, 351, 354, 388, 407, 417, 419 intercalation, 218 interface, 58, 159, 331 interrelationships, 5 intrinsic, 16 inulin, 117 inversion, 84 invertebrates, 132 investment, 244 ion transport, 383 ionic, 45, 91, 133 ions, 2, 5, 6, 20, 44, 70, 76, 107, 132, 133, 136, 179, 194, 201, 205, 208, 236, 239, 243, 247, 255, 256, 340, 360, 383, 394 Iran, 352 iron, 5, 44, 66, 70, 71, 76, 78, 94, 100, 105, 109, 112, 121, 125, 131, 135, 148, 175, 190, 201, 207, 220, 225, 242, 243, 245, 250, 252, 255, 259, 265, 267, 268, 283, 310, 325, 337, 349, 350, 358, 359, 371, 386, 392, 394, 407, 408, 412, 413, 417, 419, 422, 423, 426 iron deficiency, 71 iron transport, 125 irradiation, 181 irrigation, 225, 254, 360 Islam, 181, 190 isolation, vii, 15, 19, 24, 35, 36, 87, 89, 123, 139, 146, 200, 220, 232, 233, 292, 341, 350, 417 Israel, 252, 359, 423 Italy, 417, 418

J Japan, 188, 417 Japanese, 190, 415 jellyfish, 84 Jordan, 99, 166, 191, 204, 222, 259, 303, 351, 352, 359 juveniles, 402, 403, 410, 411

439

K K-12, 123 kaolinite, 85, 241 Kashmir, 395 Kenya, 191 kernel, 231 ketones, 77 kidney, 72, 273 kinase, 80 kinetics, 112, 172, 175, 181 King, 25, 33, 59, 76, 119, 124, 234, 242 KOH, 312 Korea, 92, 179

L labeling, 20, 40, 179 lactic acid, 178 lactones, 81 lactose, 83, 117, 253 lagoon, 160, 245, 308 lakes, 360 land, 88, 207, 243, 254, 291, 300, 305, 308, 331, 333, 352, 369, 386 land-use, 243, 305 large-scale, 32, 136, 148, 229, 370 larvae, 395, 398, 411 laser, 15, 20, 25 Latin America, 324, 422 leachate, 38, 188 leaching, 66, 71, 132, 241, 290, 360 lectin, 403 legume, xi, 18, 33, 69, 82, 91, 95, 104, 148, 163, 165, 166, 167, 175, 180, 182, 185, 195, 214, 220, 294, 296, 305, 306, 309, 311, 320, 337, 338, 339, 352, 371, 375 legumes, 20, 31, 40, 69, 82, 87, 122, 135, 157, 162, 163, 165, 178, 185, 186, 193, 203, 227, 285, 289, 291, 301, 335, 339, 347, 348, 351, 352, 358 Legumes, 90, 148, 185, 188, 195, 311, 418 Lepidoptera, 259 lesions, 77, 212, 396 lettuce, 30, 69, 82, 92, 157, 301, 339, 350, 425 liberation, 178, 207 life cycle, 7, 163 ligand, 46, 205 ligands, 250, 268, 384 lignin, 170, 269, 271, 387, 394, 407 lignocellulose, 271 likelihood, 105 limitation, 21, 22, 28, 175, 208, 239, 412

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440

Index

limitations, 11, 21, 26, 84, 236, 378, 379 linear, 27, 48, 78, 133, 168 linkage, 420 links, 185 lipase, 250, 252, 269, 287 lipid, 147,151, 244 lipopolysaccharides, 79, 93, 101, 407 liquid chromatography, 225, 237, 243 livestock, 311 LMW, 19, 125, 127 localization, 20, 84, 104 locus, 30, 38, 211 logging, 333 London, 12, 158, 195, 259, 307, 333, 335, 351, 352, 373, 420, 424, 425 long distance, 276 long period, 255, 326, 360 longevity, 141 losses, 255, 257, 266, 360, 396 Louisiana, 4 love, 106 Lovelock, 173 low molecular weight, 7, 19, 30, 32, 39, 76, 78, 120, 123, 245, 251, 268 low temperatures, 149, 150, 387 LPS, 79, 81, 407 LSD, 367 LSU, 4 luciferase, 83 Lycopersicon esculentum, 50, 62, 68, 69, 73, 74, 75, 94, 385 lysimeter, 350 lysimeter experiments, 350 lysis, 121, 269

M M.O., 320 machinery, 167 macromolecules, 46, 357 macronutrients, 299 Madison, 13, 140, 244, 303 magnesium, 189, 200, 208, 209, 265, 325 maintenance, 200, 248, 278 Maintenance, 176, 360 maize, 9, 11, 13, 30, 31, 32, 36, 49, 51, 69, 71, 91, 92, 93, 96, 102, 103, 104, 105, 133, 140, 142, 143, 157, 172, 175, 185, 189, 191, 192, 193, 194, 197, 203, 210, 214, 235, 242, 244, 255, 269, 285, 286, 287, 288, 292, 294, 295, 298, 301, 302, 303, 304, 306, 308, 339, 345, 346, 350, 351, 353, 355, 366, 372, 396, 408, 421, 422, 423, 425 Malaysia, 161, 168, 190, 194, 195, 197

males, 422 malic, 132, 133, 204, 205, 206, 237, 238, 338, 361, 371, 409 maltose, 117, 253 management, viii, 7, 11, 12, 13, 35, 45, 49, 72, 87, 96, 100, 104, 130, 142, 147, 162, 184, 192, 195, 201, 215, 230, 248, 261, 266, 267, 269, 270, 277, 299, 302, 324, 326, 334, 343, 360, 371, 374, 379, 395, 397, 398, 402, 404, 405, 410, 413, 416, 417, 421 management practices, 35, 45, 324, 326, 379, 397 manganese, 112, 191, 242, 243, 320 Manganese, 193 mangroves, 160, 245, 308 manipulation, 89, 139, 200, 216, 217, 230, 367, 368, 383, 389, 392, 407 mannitol, 117, 170, 313 manpower, 369 mantle, 171, 174 manufacturing, 240, 324 manure, 2, 3, 4, 7, 14, 44, 88, 163, 185, 191, 207, 234, 236, 297, 324, 327, 334, 335, 339, 360, 363, 374, 409, 410, 422 mapping, 278 marine environment, 232 marker genes, 26 market, 80, 179, 184, 240, 377, 378 marketing, 395 MAS, 11, 241 mass spectrometry, 424 matrix, 8, 46, 72, 120, 248, 403 maturation, 130 Mauritius, 324 MCA, 236 measurement, 11, 221, 234, 320 measures, 43, 92, 413 meat, 31 media, viii, xi, 46, 47, 108, 113, 117, 127, 134, 179, 180, 182, 183, 202, 204, 253, 255, 270, 271, 283, 285, 296, 313, 314, 343, 361, 363, 388, 412 Mediterranean, 188, 254, 305 melting, 23, 159 membranes, 147, 244 mercury, 350, 380, 383, 384, 389, 393, 394 Mercury, 384 meristem, 396 metabolic, 2, 81, 82, 83, 95, 130, 146, 200, 201, 212, 247, 251, 252, 290, 298, 383, 386, 391 metabolic pathways, 2, 82, 146, 200 metabolism, 1, 29, 46, 66, 71, 81, 82, 103, 197, 250, 299, 319, 325, 349, 350, 357, 361, 379, 381, 384, 387, 389, 394 metabolite, 77, 127, 250, 269, 415, 421

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Index metabolites, 15, 66, 72, 79, 83, 87, 93, 97, 100, 103, 110, 116, 121, 245, 251, 254, 267, 268, 269, 271, 279, 285, 291, 343, 344, 386, 398, 400, 405, 425 metagenomics, 15, 64, 87, 89, 100 metal chelators, 379, 388 metal ions, 70, 107, 394 metallothioneins, 382, 392 metallurgy, 359 metals, 218, 224, 258, 261, 377, 378, 379, 380, 381, 383, 384, 385, 387, 390, 391, 392, 393, 394 methanol, 411 methylcellulose, 86 Mexican, 58, 422 Mexico, 188, 324 Mg2+, 113 MgSO4, 117, 118, 134, 232 mica, 26, 172, 187, 193 microarray, 20, 29, 39, 82 Microarrays, 20, 34, 37 Microbes, i, iii, v, vi, vii, viii, 1, 57, 150, 158, 161, 172, 214, 231, 237, 239, 247, 265, 267, 268, 281, 289, 298, 326, 361, 365 Microbial, vi, 16, 17, 21, 33, 35, 36, 46, 47, 61, 70, 92, 93, 98, 103, 105, 107, 109, 112, 113, 114, 140, 142, 178, 187, 189, 190, 192, 196, 197, 221, 224, 225, 231, 243, 248, 257, 260, 278, 279, 281, 293, 303, 304, 306, 307, 308, 323, 324, 334, 349, 351, 371, 375, 414, 415, 418 microbial cells, 2, 29, 78 microbial communities, 15, 16, 21, 22, 23, 25, 26, 28, 34, 87, 92, 97, 216, 229, 230, 240, 248, 251, 255, 256, 265, 348 microbial community, 17, 22, 24, 25, 27, 31, 34, 38, 47, 80, 92, 94, 109, 185, 249, 256, 260, 407, 426 microbiota, 22 microcosms, 31, 252 microenvironment, 80, 88, 296 microflora, 36, 47, 90, 92, 107, 129, 132, 134, 142, 201, 216, 220, 292, 303, 343, 349, 369 microhabitats, 249 micronutrients, 13, 106, 159, 174, 223, 226, 241, 242, 277, 304, 349 microorganism, 43, 47, 50, 82, 86, 113, 121, 133, 187, 250, 296, 300, 313, 332, 400, 409 micro-organisms, 102, 148, 149, 150, 151, 156, 157, 159, 187, 191, 244, 248, 260, 282, 337, 338, 341, 353, 3363, 371, 419 microscopy, 15, 20, 22, 424 migration, 24, 30 milk, 65, 114 millet, 68, 96, 103, 185, 187, 258, 318 mine soil, 175, 381, 393 mine tailings, 390

441

mineralization, 2, 4, 5, 13, 47, 49, 112, 113, 121, 132, 133, 149, 151, 199, 208, 230, 236, 239, 241, 243, 254, 256, 259, 283, 372 mineralized, 5, 55, 162, 175, 208, 282, 344 mineralogy, 8, 172 minerals, 5, 6, 60, 71, 94, 99, 107, 113, 148, 162, 172, 189, 201, 214, 225, 237, 243, 304, 310, 311, 344, 358, 405, 409 mines, 11, 96, 140, 141, 143, 242, 249, 258, 278, 303 mining, 162, 175, 360, 381 Minnesota, 10, 277 misconceptions, 11 Missouri, 92 mixing, 204, 231, 235, 236 mobility, 24, 131, 141, 283, 326, 338, 410 models, 17 moieties, 407 moisture, 5, 81, 147, 172, 179, 180, 181, 236, 249, 251, 338, 396, 423 moisture content, 179, 251 molasses, 324 mold, 97, 273 molecular biology, viii, 11, 16, 63, 241 molecular markers, 17 molecular mass, 227, 409 molecular mechanisms, 386 molecular weight, 7, 19, 30, 32, 39, 76, 78, 120, 123, 209, 245, 251, 268, 398 molecules, 2, 7, 12, 19, 20, 22, 39, 46, 49, 63, 64, 79, 81, 83, 120, 132, 166, 178, 205, 271, 350, 400 molybdenum, 227 momentum, 162, 323 monoclonal, 403 monomeric, 20 montmorillonite, 85, 241 Moon, 419 morning, 188 Morocco, 204, 359 morphogenesis, 350 morphological, 78, 140 morphology, 22, 66, 71, 174, 256, 349 mortality, 395, 401, 403, 411, 414 mosaic, 73, 109 Moscow, 35, 101 mouse, 381, 393 movement, 1, 58, 81, 131, 132, 176, 360, 378, 383, 402, 403 MPS, 70, 189, 205, 206, 210, 211, 212 multidisciplinary, 300, 389 multiples, 300 multiplication, 63, 85, 182, 395, 404, 405, 410, 417 mushrooms, 174 Muslim, xi, 1, 229, 245, 247, 395

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442

Index

mutagenesis, 157, 211, 212, 224, 350, 373 mutagenic, 9, 145 mutant, 71, 76, 77, 98, 100, 105, 145, 147, 149, 150, 152, 153, 154, 155, 156, 159, 210, 254, 259, 385, 408 mutants, 59, 76, 79, 83, 99, 109, 145, 149, 151, 157, 158, 159, 179, 189, 194, 206, 212, 223, 225, 243, 244, 251, 259, 345, 353, 354, 383, 416, 425 mutation, 24 mycelium, 86, 121, 127, 174, 268, 271, 291, 404 Mycobacterium, 67, 164, 267, 310 mycology, viii mycorrhiza, 91, 174, 175, 176, 184, 185, 187, 188, 190, 191, 195, 216, 228, 231, 243, 285, 301, 302, 303, 305, 307, 320, 323, 332, 333, 335, 339, 349, 351, 353 myo-inositol, 244

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N N-acety, 78 NaCl, 117, 232 NADH, 93 NAS, 380, 381 native species, 291 natural, vii, 7, 28, 58, 63, 82, 86, 87, 108, 126, 131, 138, 149, 151, 161, 163, 200, 204, 227, 230, 231, 236, 240, 244, 247, 251, 255, 266, 282, 284, 290, 300, 303, 338, 345, 357, 367, 372, 378, 379, 381, 387, 397, 409, 413 natural environment, 7, 86, 87, 126, 251, 255, 413 natural resources, 87, 164, 240 necrosis, 276, 397, 404 neem, 193, 293, 304, 402, 403, 404, 418, 421 negative relation, 48 nematicides, 397, 403, 404, 413 nematode, 177, 190, 261, 270, 298, 303, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 425 nematodes, 81, 177, 196, 267, 395, 396, 397, 398, 400, 402, 403, 404, 405, 411, 412, 413, 415, 416, 417, 419, 422, 423, 424, 426 Nepal, 354 nested PCR, 23, 37 Netherlands, 11, 33, 35, 36, 94, 186, 188, 189, 190, 192, 194, 196, 303, 306, 307, 308, 334, 335, 392 network, 185 New Jersey, 196 New South Wales, 195 New York, 11, 13, 57, 92, 102, 106, 157, 158, 159, 187, 188, 195, 257, 258, 262, 354, 371, 390, 391, 394, 418, 419, 421, 424, 425

NFT, 183 Ni, 71, 260, 380, 381, 387 nickel, 109, 209, 218, 255, 257, 355, 387 258, 380, 384, 388, 392 Nielsen, 77, 99, 102, 146, 159, 424 Niger, 400 Nigeria, 185, 196 nitrate, 100, 164, 170, 254, 351, 358 nitric acid, 206 nitrogen compounds, 202 nitrogen fixation, 64, 69, 71, 91, 93, 103, 109, 114, 130, 132, 141, 165, 166, 179, 191, 193, 217, 262, 271, 285, 289, 309, 311, 319, 333, 335, 338, 339, 350, 352, 353, 364, 369 nitrogen fixing, 14, 62, 65, 69, 127, 129, 135, 161, 193, 219, 223, 245, 262, 310, 311, 316, 317, 327, 339, 347, 352, 355 nitrogen gas, 69 nitrogen-fixing bacteria, 188, 214, 293, 304, 318 nodulation, 9, 20, 33, 35, 82, 83, 95, 101, 109, 149, 166, 178, 185, 196, 213, 215, 216, 217, 219, 220, 221, 226, 261, 292, 293, 295, 300, 302, 308, 309, 311, 318, 319, 320, 347, 348, 349, 350, 354, 364, 418, 420 nodules, 65, 82, 165, 166, 202, 243, 293, 295, 298, 311, 313, 314, 317, 347, 371, 374 non-destructive, 84 non-enzymatic, 176 non-enzymatic antioxidants, 176 non-invasive, 20 non-renewable, 201, 358, 374 normal, 3, 56, 66, 153, 155, 164, 266, 311, 313, 329, 384, 396 normalization, 24 North America, 38 North Carolina, 254, 414, 417, 423 nuclear, 38, 388 nuclease, 8 nucleation, 160 nucleic acid, 2, 3, 17, 20, 49, 66, 84, 207, 208, 267, 357 nucleosides, 46 nucleotide sequence, 24, 25, 123, 124, 127, 192, 211, 312, 352 nucleotide sequencing, 312, 317 nucleotides, 208, 312 nutrient cycling, 8, 93, 132, 162, 228, 271, 305, 307 nutrient flow, 189 nutrients, vii, 16, 46, 47, 59, 62, 63, 64, 66, 74, 80, 81, 87, 88, 99, 121, 124, 132, 136, 147, 148, 151, 159, 163, 172, 174, 175, 178, 179, 181, 182, 185, 197, 199, 202, 208, 215, 216, 230, 239, 248, 256, 276, 282, 283, 291, 299, 304, 309, 311, 324, 326,

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Index 337, 341, 342, 345, 348, 354, 357, 378, 398, 400, 407, 409, 412 nutrition, 2, 9, 10, 43, 44, 48, 57, 58, 60, 69, 70, 71, 91, 95, 109, 131, 140, 141, 162, 175, 200, 204, 208, 210, 216, 219, 221, 222, 239, 240, 256, 258, 268, 276, 291, 292, 303, 307, 319, 320, 323, 326, 327, 331, 332, 333, 334, 335, 339, 345, 350, 351, 354, 371, 402, 407, 409, 419, 422

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O oat, 47, 203, 212, 213, 255, 396 obligate, 66, 182, 402 observations, 66, 210 OECD, 188 oil, viii, 61, 62, 112, 131, 168, 172, 178, 180, 186, 187, 190, 195, 218, 219, 289, 295, 369 Oil palm, 181 oils, 151 oilseed, 132, 394 oligomers, 269, 391 oligonucleotides, 21 oligosaccharides, 79, 127, 245 olive, 245, 297, 308 onion, 216, 276 online, 26, 27, 394 oospore, 271 opacity, 312 operon, 25, 82, 83, 384, 387, 388, 391 optical, 119 optical density, 119 optimization, 108, 181, 388 OR, 244 Oregon, 93 ores, 221, 359 organ, 183, 188, 291 organelle, 383, 388 organelles, 20 organic C, 201, 289 organic compounds, 2, 3, 5, 72, 113, 130, 131, 163, 199, 201, 208, 209, 239, 256, 265, 266, 368 organic matter, 5, 13, 45, 47, 51, 55, 56, 61, 64, 65, 66, 112, 113, 132, 147, 148, 162, 175, 176, 201, 207, 208, 224, 239, 271, 281, 291, 326, 339, 412 organism, 20, 25, 51, 53, 83, 85, 89, 101, 234, 238, 269, 345, 348, 372, 397, 398 orthophosphates, 202 osmotic, 176, 254, 369 overproduction, 221, 382, 388 oxalate, 7, 70, 206, 222, 326 oxalic, 46, 48, 113, 133, 206, 237, 238, 241, 253, 258, 301, 326, 338, 361, 398 oxalic acid, 133, 241, 253, 258, 301, 399

443

oxidation, 60, 70, 117, 169, 205, 206, 211 oxidative, 78, 383, 392 oxidative stress, 383, 392 oxide, 241, 314, 358 oxides, 6, 46, 55, 56, 112, 385 oxygen, 4, 92, 166, 176, 197, 423

P packaging, 179, 180, 240 pairing, 292, 332, 367 Pakistan, 225, 302, 304, 324, 351, 357, 370, 373, 375, 426 Panama, 402 parameter, 120, 181 parasite, 190, 268, 402, 404 parasites, 163, 398, 404, 408 Parkinson, 388, 391 particles, 5, 6, 44, 132, 133, 159, 176, 256, 358, 360 partnerships, 161 pasture, 59, 60, 106, 124, 195, 244, 255, 304 pathogenesis, 405, 413, 425 pathogenic, vii, 23, 66, 78, 79, 91, 96, 103, 121, 177, 259, 266, 268, 270, 275, 276, 277, 285, 320, 321, 343, 400, 403, 404, 407, 419, 421 pathogens, 27, 63, 64, 65, 72, 76, 77, 78, 79, 80, 85, 89, 95, 98, 102, 103, 109, 113, 115, 123, 125, 157, 177, 216, 220, 237, 250, 265, 266, 267, 268, 269, 271, 276, 277, 304, 343, 344, 345, 348, 353, 363, 396, 397, 399, 400, 401, 407, 408, 412, 417, 421, 425 pathology, 102, 275 pathways, 2, 69, 79, 82, 106, 146, 200, 383, 386 Pb, 381, 387 PBL, 335 PCA, 76, 116, 121 PCM, 76 PCR, 17, 18, 19, 22, 23, 24, 25, 26, 27, 30, 31, 32, 34, 35, 37, 38, 39, 40, 84, 100, 116, 117, 120, 124, 243, 286, 312, 368 PCs, 379 peanuts, 409 peat, 32, 85, 86, 162, 180, 182, 197, 234, 313, 334 pectin, 116 Pennsylvania, 244, 307, 423 peptide, 77, 385, 420 peptides, 79, 101, 158 periodic, 50 permeability, 402 permit, 19 peroxide, 97 Peru, 38, 404, 421 pest control, 419

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444

Index

pest management, 413 pesticide, xi, 121, 147, 412 pesticides, 85, 112, 120, 182, 251, 363, 397, 412, 416 pests, 63, 66, 343, 395, 397, 424 Petri dish, 115, 269, 270 petroleum, 31 PGPR, 10, 15, 64, 65, 69, 72, 76, 77, 82, 83, 84, 85, 90, 92, 94, 95, 98, 101, 104, 138, 139, 149, 159, 163, 167, 170, 179, 183, 186, 187, 189, 204, 209, 210, 212, 215, 217, 234, 282, 291, 305, 310, 341, 342, 343, 345, 346, 348, 349, 350, 375, 417 pH, 2, 5, 6, 8, 10, 12, 44, 48, 49, 55, 59, 61, 71, 76, 81, 113, 117, 118, 119, 124, 131, 133, 136, 137, 139, 148, 162, 168, 181, 185, 190, 201, 202, 203, 204, 205, 206, 208, 209, 211, 221, 227, 230, 232, 234, 236, 237, 242, 249, 251, 252, 253, 255, 259, 260, 267, 279, 288, 289, 291, 295, 296, 310, 312, 313, 330, 338, 339, 340, 344, 352, 358, 361, 363, 364, 370, 386 pH values, 49, 230, 237 phenazine, 76, 82, 92, 110, 116, 121, 125, 269, 405 phenol, 29, 270 phenolic, 76, 100, 106, 151, 166, 207, 363 phenotype, 205, 211, 212, 340, 341 phenotypes, 33, 87, 223 phenotypic, viii, 29, 30, 114, 117, 203, 212, 228 phenylalanine, 78, 403 Philadelphia, 244, 307, 423 Philippines, 191, 324 phosphatases, 8, 12, 49, 57, 60, 61, 113, 118, 126, 199, 203, 208, 209, 217, 226, 227, 230, 239, 256, 363, 368, 374 Phosphatases, 11, 208, 239, 370 phospholipase C, 126 phospholipids, 2, 3, 49, 201, 207, 208, 267, 357 phosphonates, 49 phosphor, 208 photon, 354 photosynthesis, 1, 3, 130, 148, 200, 230, 325, 338, 358 photosynthesize, 174 photosynthetic, 176, 324 phototrophic, 321 phycocyanin, 345 phyllosphere, 86, 90, 232 phylogenetic, 17, 21, 22, 24, 25, 27, 28, 29, 32, 33, 114, 120, 126, 127, 203 phylogenetic tree, 120, 126 phylogeny, 18, 87 phylum, 22 physical factors, 369 physical properties, 174

physicochemical, 378 physico-chemical properties, 9, 251, 255, 289, 377 physiological, 43, 71, 78, 81, 82, 91, 148, 155, 177, 187, 210, 230, 291, 310, 318, 319, 325, 327, 337, 338, 345, 385 physiological regulation, 210 physiology, viii, 30, 61, 64, 80, 89, 126, 226, 333, 345, 351, 374, 375, 386, 402, 414 phytopathogens, 77, 78, 108, 112, 114, 121, 163, 267, 268, 269, 273, 345 phytoremediation, 98, 377, 378, 379, 380, 381, 383, 384, 385, 388, 389, 390, 391, 392, 393, 394 Phytotoxicity, 378 pig, 226 pigments, 107 pigs, 223 Pisum sativum, 5, 38, 67, 68, 299, 302 plagioclase, 197 plant growth promoting rhizobacteria, viii, 10, 64, 90, 92, 103, 123, 125, 163, 189, 192, 204, 209, 210, 215, 218, 256, 259, 267, 277, 301, 339, 346, 350, 351, 352, 353, 355, 357, 368, 416, 421, 423, 424, 425 plasma, 237 plasma membrane, 237 plasmid, 166, 192, 209, 212, 368, 387 plasmids, 212, 251 plastid, 388 play, 10, 15, 16, 47, 49, 64, 66, 72, 78, 82, 85, 111, 112, 113, 118, 119, 121, 130, 134, 146, 147, 172, 200, 216, 229, 230, 231, 237, 248, 266, 268, 271, 282, 283, 300, 337, 344, 361, 380, 405, 408, 409 ploughing, 236 policy makers, viii pollen, 209, 388 pollutants, 111, 112, 113, 151, 251, 377, 378, 379, 384, 393 pollution, 66, 112, 200, 209, 210, 240, 393, 394 polyacrylamide, 180 polyamine, 102 polycrystalline, 218 polyethylene, 180, 181, 313 polymer, 78, 112 polymerase, 30, 35, 37, 38, 40, 124, 243, 286, 321 polymerase chain reaction, 30, 35, 37, 38, 40, 124, 243, 286, 321 polymorphism, 21, 22, 24, 25, 30, 31, 33, 34, 35, 37, 38, 84, 117 polymorphisms, 18, 34, 40 polypeptides, 227 polyphosphates, 49 polysaccharide, 237, 344 polysaccharides, 197, 344

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Index polythene, 234 pomegranate, 404 pools, 11, 43, 45, 46, 50, 51, 55, 56, 58, 194, 218, 236 poor, vii, 2, 47, 69, 82, 84, 112, 133, 172, 175, 182, 253, 255, 296, 369 population, 16, 18, 19, 21, 26, 29, 38, 64, 80, 86, 88, 92, 149, 151, 179, 180, 181, 185, 204, 214, 216, 228, 230, 252, 255, 284, 285, 290, 293, 296, 297, 298, 299, 327, 328, 329, 332, 341, 344, 395, 397, 401, 402, 403, 411, 412, 419, 423 population density, 86, 181, 185, 255, 290 population size, 297 pore, 176 pores, 175, 404 porosity, 70 Portugal, 37, 188 positive correlation, 70 positive interactions, 300 potassium, 66, 71, 96, 106, 119, 134, 161, 163, 172, 186, 187, 189, 193, 195, 196, 215, 254, 293, 314, 335, 419 potato, 4, 37, 84, 86, 105, 116, 127, 157, 170, 186, 189, 196, 223, 268, 409, 411, 422, 423 potatoes, 212, 277, 417 poultry, 209, 297 powder, 86, 91, 101, 234, 354, 410 powders, 93 power, 22, 24, 25, 26, 27, 193, 253, 324 precipitation, vii, 6, 29, 44, 70, 133, 141, 146, 205, 208, 239, 261, 283, 310, 338 predators, 398 prediction, 26 pre-existing, 79 preservatives, 359 pressure, 249, 251, 314 preventive, 125 priming, 103, 235 printing, ix, 21 probe, 22, 35 producers, 72, 121, 147, 168, 325 production costs, 299 productivity, viii, 9, 55, 56, 63, 80, 82, 88, 89, 108, 123, 130, 145, 147, 148, 157, 181, 184, 186, 189, 199, 200, 217, 227, 229, 230, 240, 248, 249, 254, 257, 277, 282, 299, 300, 301, 309, 324, 325, 357, 360, 370, 371, 372, 379, 400 profit, 155 program, 34, 120, 182, 270 prokaryotes, 16, 17, 19, 146, 387 prokaryotic, 16, 17, 34, 39 proliferation, 64, 76, 78, 89, 249, 255, 345 promoter, 83, 224, 379, 391

445

property, iv, 46, 326, 327, 379, 386 prosperity, 324 proteases, 78, 92, 121, 270 protection, viii, 65, 78, 81, 85, 98, 112, 138, 177, 224, 257, 276, 277, 360, 363, 395, 404, 414 protective role, 177 protein, 20, 31, 40, 58, 72, 79, 84, 92, 118, 147, 158, 160, 166, 209, 211, 219, 223, 226, 269, 289, 295, 343, 347, 366, 368, 372, 381, 383, 387 protein synthesis, 72, 387 proteins, 15, 20, 46, 47, 66, 78, 79, 80, 82, 145, 146, 147, 158, 160, 211, 251, 256, 325, 379, 381, 383, 388, 400, 402, 425 proteobacteria, 312 proteome, 40 proteomics, 15, 17, 278, 390 protocol, 385 protocols, 85 protons, 34, 168, 192, 205, 244, 256 protoplasm, 201, 208 pruning, 396 PSA, 327 PSB, 15, 16, 18, 19, 25, 27, 28, 50, 55, 56, 71, 85, 96, 150, 162, 167, 168, 170, 171, 183, 202, 214, 215, 216, 217, 219, 221, 232, 253, 255, 290, 293, 294, 295, 297, 299, 310, 323, 327, 328, 329, 330, 332, 335, 364, 365, 366, 367, 368, 370, 373, 374 pseudo, 31 Pseudomonas aeruginosa, 9, 29, 33, 39, 76, 90, 91, 92, 107, 122, 125, 127, 203, 205, 212, 228, 250, 269, 270, 278, 406, 423 Pseudomonas fragi, 146, 151, 160, 171 Pseudomonas spp, 21, 24, 31, 60, 125, 126, 203, 212, 220, 293, 309, 316, 317, 319, 320, 338, 373, 421 publishers, xi, 426 Puerto Rico, 40 pulses, 235, 396, 401 pumps, 383, 392 purification, 16, 130 purines, 46 P-value, 162 pyrimidine, 208 pyrolysis, 424 pyrrole, 76 pyruvic, 178, 287

Q quality assurance, 182 quality control, 240, 300, 369 quinine, 220, 224, 228 quinone, 33, 60, 117, 123, 124, 192, 205, 211, 340, 368

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446

Index

quorum, 64, 81, 105, 125

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R race, 423 radiation, 324 rain, 226, 343 rainfall, 162, 360 Ralstonia solanacearum, 79 Raman, 12, 35, 126, 224, 278 random, 18, 36, 37 random amplified polymorphic DNA, 37 range, vii, 6, 27, 48, 76, 106, 113, 118, 138, 147, 176, 201, 202, 203, 209, 212, 249, 281, 283, 287, 323, 326, 357, 360, 368, 373, 380, 387, 407, 412 RAPD, 18, 19, 34, 35, 120 rape, 59, 132, 141, 172, 195, 224 raw material, 180, 324, 325, 329, 359 raw materials, 325 reactive oxygen, 176 reactive oxygen species, 176 reactivity, 70, 162, 255, 283, 310 reading, 100, 211 reagent, 115 real time, 22 reclamation, 162, 174 recognition, 78, 350 recombinant DNA, 20, 209 recombination, 388 reconciliation, 40 recovery, 44, 218, 261, 299, 326 recycling, 16, 87, 96 redox, 207, 249 regeneration, 385 regular, 56, 70, 266, 310 regulation, 2, 58, 82, 118, 123, 146, 176, 179, 197, 200, 210, 212, 221, 242, 350, 353, 370, 372, 386, 392, 407 regulators, 103, 161, 163, 177, 179, 186, 187, 218, 243, 255, 287, 342, 367, 408 Reimann, 424 relationships, 12, 28, 32, 91, 187, 188, 276, 292, 319, 367, 371, 415 relevance, 298, 305 reliability, 26, 86 remediation, 170, 259, 377, 378, 393 renal, 416 repression, 210, 288 reproduction, 172, 177, 270, 395, 401, 418, 425 reproductive organs, 358 research and development, 413 reserves, vii, 44, 45, 51, 56, 244, 310, 325, 337, 358, 359

reservoir, 43, 148, 199, 201 residues, 45, 47, 60, 85, 87, 96, 112, 132, 161, 185, 186, 200, 294, 297, 307, 374 resilience, 249 resin, 45, 47, 50, 51, 55 resistance, 3, 46, 63, 64, 70, 71, 72, 78, 79, 83, 84, 85, 90, 91, 92, 99, 100, 103, 107, 108, 109, 174, 176, 178, 267, 268, 269, 271, 278, 343, 345, 381, 384, 386, 387, 394, 396, 398, 400, 407, 412, 414, 417, 419, 420, 421, 424, 425 resolution, 19, 21, 22, 23, 25, 26, 30, 32, 123 resorcinol, 76 resources, 17, 87, 164, 201, 229, 240, 311, 358 respiration, 13, 87, 160, 168, 311, 358, 363 respiratory, 126, 225 restriction enzyme, 26, 84 restriction fragment length polymorphis, 18, 22, 25, 31, 34, 35, 37, 84 returns, 131, 299 reverse transcriptase, 84 RFLP, 18, 22, 25, 26, 27, 35, 84 rhizobia, 16, 18, 19, 20, 25, 26, 27, 30, 31, 33, 34, 35, 37, 38, 40, 71, 82, 101, 104, 114, 134, 135, 138, 148, 163, 165, 166, 167, 180, 181, 185, 188, 296, 300, 301, 306, 311, 319, 320, 339, 348, 353 Rhizoctonia solani, 75, 76, 100, 159, 177, 191, 270, 271, 276, 278, 279, 417, 418, 422, 424 Rhodococcus, 16, 238 ribonucleic acid, 146 ribose, 117 ribosomal, 22, 23, 25, 26, 30, 31, 34, 37, 39, 77, 84, 101, 127, 312, 317 ribosomal RNA, 30, 34 ribosome, 122 rice, 30, 34, 39, 59, 81, 88, 90, 99, 103, 107, 108, 109, 122, 126, 157, 186, 191, 194, 196, 203, 207, 213, 214, 224, 226, 242, 286, 289, 292, 293, 294, 297, 302, 307, 339, 345, 346, 350, 352, 353, 354, 364, 374, 387, 394, 396, 409, 411, 423 risk, 183, 282, 360, 389, 390 RNA, 3, 15, 19, 22, 30, 32, 35, 39, 72, 82, 84, 120, 123, 125, 127, 146, 208 rods, 410, 411 Rome, 61, 308 room temperature, 115, 182, 313 root elongation, 100, 178, 363 root hair, 3, 71, 166, 175, 178, 190, 257, 301, 347, 396 ROS, 176 rotations, 60, 304 runoff, 71, 360 rural, 248, 324 rural areas, 248

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Index Russia, 326, 359 rust, 101 rye, 124

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S Saccharomyces cerevisiae, 146, 204, 210, 211, 221, 225, 285, 362 safety, 89, 368 saline, 201, 254, 306, 311, 389 salinity, 97, 121, 254, 257, 261, 383 Salmonella, 32, 228 salt, 34, 115, 208, 209, 242, 255, 259, 361, 373 salts, 115, 131, 135, 200, 201, 205, 258, 265 sample, 21, 22, 24, 26, 27, 311, 314 sand, 152, 162, 182, 194, 252, 305, 313, 358 SAR, 79, 400 saturation, 7, 237 savings, 9 sawdust, 37, 85 scaling, 28 scarcity, 248 Schmid, 102, 104 scientific knowledge, 88 Sclerotinia sclerotiorum, 74, 75, 77, 89, 94, 257, 301, 343 sea ice, 159 search, vii, 2, 26, 88, 120, 147, 194, 240, 281, 282 searching, 2, 248 seasonal variations, 151 secrete, 70, 71, 147, 384 secretion, 72, 73, 80, 104, 163, 226, 243, 244, 261, 265, 266, 267, 340, 347 security, 230, 248, 371 sediment, 389, 390 sediments, 33, 242 seed, 3, 9, 10, 30, 64, 71, 72, 85, 86, 91, 101, 107, 130, 138, 150, 154, 159, 163, 178, 191, 196, 212, 214, 215, 217, 218, 224, 226, 230, 235, 236, 240, 250, 252, 262, 268, 278, 288, 293, 295, 297, 313, 319, 320, 335, 346, 347, 348, 363, 364, 367, 372, 395, 396, 397, 400, 401, 403, 409, 410, 411, 412, 417, 418, 419, 423 seeding, 138, 288, 403 seedlings, 8, 59, 76, 97, 122, 151, 174, 176, 183, 185, 196, 197, 210, 215, 218, 224, 236, 293, 298, 319, 393, 408, 410, 411, 416 seeds, 76, 86, 130, 138, 152, 177, 183, 209, 215, 231, 235, 236, 241, 252, 268, 270, 288, 289, 296, 299, 302, 310, 313, 318, 334, 346, 358, 400, 408, 412, 422 selecting, 63, 89, 270 selectivity, 127, 245

447

selenium, 378, 389, 390, 392 semiarid, 58, 94, 200, 222, 245, 253, 254, 291, 309 semi-arid, 14, 51, 55, 56, 57, 61, 148, 239, 245, 305, 306 Senegal, 188, 204, 254, 359 senescence, 3, 79, 178 sensing, 64, 81, 92, 100, 105, 125 sensitivity, 25, 26, 27, 142 separation, 19, 24, 26, 84, 120, 215 sequencing, 17, 18, 19, 21, 23, 24, 28, 30, 39, 40, 87, 89, 92, 98, 126, 222, 226, 312, 321 serine, 380, 402 severity, 77, 78, 268, 270, 275, 276, 344 sewage, 377 shade, 235 Shahid, 90 shape, 312, 396 shock, 146, 160 shoot, 3, 9, 50, 72, 149, 151, 152, 154, 175, 176, 178, 213, 215, 252, 273, 285, 288, 289, 290, 294, 297, 299, 315, 318, 327, 343, 346, 347, 363, 365, 380, 381, 385, 396, 403, 410 short period, 180, 266, 402 short-term, 253 shrubs, 174, 175 SI, 234 signal transduction, 82, 110 signaling, 64, 69, 80, 88, 95, 106, 108, 178, 189 signaling pathway, 72 signals, 20, 21, 71, 106, 194, 196 silicate, 6, 71, 100, 106, 193 similarity, 17, 117, 211, 269 simulation, 417 Singapore, 57 single-nucleotide polymorphism, 20 sites, 5, 8, 26, 66, 79, 81, 83, 86, 122, 174, 177, 225, 268, 291, 360, 377, 383, 385, 390, 407 sludge, 34, 35, 310 SMR, 302 SMS, 57 sodium, 115, 254, 255, 260, 371 software, 37, 120, 124 soil erosion, 71, 176 soil particles, 44, 133, 176, 256, 358, 360 soil pollution, 112, 200 solar, 324, 378 solid phase, 6, 44, 131 solid-state, 344 solubility, 6, 112, 120, 133, 134, 151, 159, 175, 200, 214, 223, 242, 297, 304, 335, 358, 359, 360 solubilization ability, 217, 368 somatic cell, 387 sorbitol, 117, 253

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448

Index

Sorghum, 74, 97, 171, 172, 182, 187, 190, 192, 222, 255, 311 sorption, 5, 7, 12, 131, 132, 205, 360 South Africa, 97, 324, 359 Soviet Union, 169, 296, 333, 361 soy, 182 soybean, 9, 11, 33, 57, 95, 109, 123, 138, 139, 143, 148, 150, 158, 159, 176, 185, 186, 188, 189, 191, 193, 209, 210, 212, 214, 216, 218, 219, 220, 223, 227, 263, 274, 285, 286, 289, 293, 297, 300, 301, 302, 306, 307, 308, 319, 343, 347, 350, 351, 364, 365, 366, 367, 370, 371, 372, 373, 374, 375 soybean seed, 319 soybeans, 33, 297, 364 spacers, 22, 25 Spain, 15, 33, 41, 140, 308 spatial, 23, 24, 26, 28, 89, 249 specificity, 21, 22, 26, 49, 81, 89, 151, 174, 185, 209, 385 spectrum, 63, 72, 76, 77, 98, 109, 127, 269, 300, 403, 407 speed, 131 spore, 107, 116, 121, 182, 184, 191, 268, 271, 291, 334, 415, 422 stability, 12, 89, 109, 176, 183, 197, 206, 249, 368 stabilization, 176, 385 stabilize, 381 stages, 86, 121, 214, 231, 327, 328, 332, 358, 398, 402, 403 standard deviation, 152, 154, 315 standard error, 153 standardization, 25 standards, 80 Staphylococcus, 271 starch, 148, 230, 253, 257, 338 starches, 1 starvation, 78, 80, 92, 95, 211 statistics, 190 steel, 31 sterile, 38, 47, 105, 179, 181, 313, 354, 369 stimulus, 82 stock, 131 stoichiometry, 241 storage, 3, 86, 90, 97, 172, 179, 180, 181, 197, 250, 369, 384, 385, 395 strain improvement, 64, 89 strategies, vii, 2, 10, 82, 89, 105, 175, 186, 216, 217, 233, 378, 384, 385, 388, 424 streams, 360, 378 strength, 50, 130 streptococci, 35

Streptomyces, 16, 66, 70, 72, 75, 78, 94, 95, 102, 103, 109, 203, 210, 250, 252, 258, 263, 271, 273, 274, 277, 279, 410, 419 stress, 31, 35, 82, 95, 97, 121, 145, 146, 172, 174, 176, 182, 186, 187, 188, 197, 206, 210, 249, 251, 254, 261, 262, 286, 295, 302, 306, 381, 383, 387, 391, 392, 394, 407, 424 stress factors, 387 structural gene, 60 students, vii, viii, xi sub-Saharan Africa, 283 subsistence, 51 substances, 48, 71, 78, 101, 132, 133, 162, 177, 201, 202, 207, 237, 248, 249, 262, 266, 287, 306, 330, 333, 338, 342, 343, 345, 348, 361, 369, 398, 419 substitution, 2, 48, 167, 168 substrates, 46, 48, 86, 96, 113, 147, 170, 178, 186, 208, 251, 284, 296, 297, 368, 388 Succinic, 238 sucrose, 117, 170, 218, 253, 328, 329, 333, 370 sugar, 3, 49, 58, 61, 76, 86, 91, 94, 99, 102, 105, 107, 148, 192, 200, 208, 230, 253, 258, 295, 297, 306, 307, 323, 324, 325, 327, 328, 329, 330, 332, 334, 335, 338, 354, 410, 411 sugar beet, 76, 91, 94, 99, 102, 105, 107, 258, 295, 297, 306, 324, 354, 410, 411 sugar cane, 192, 334 sugar industry, 324 sugarcane, viii, 4, 40, 57, 61, 91, 100, 102, 105, 108, 148, 180, 203, 224, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335 sugars, 1, 3, 46, 47, 98, 148, 157, 180, 207, 243, 251, 338 sulfate, 254, 314, 384 sulfur, 13, 106, 201, 206, 364, 380 sulfuric acid, 206, 314 sulphate, 115, 162, 172, 207, 254, 387, 394 sulphur, 38, 59, 207, 226, 374, 393 summer, 13, 160, 261 Sun, 100, 147, 149, 160, 197, 221 sunflower, 69, 90, 214, 225, 303, 385, 409 sunlight, 378 supernatant, 115, 234 superoxide, 176, 419 supplements, 359 supply, 2, 3, 7, 43, 48, 51, 56, 58, 70, 71, 80, 132, 138, 139, 146, 150, 163, 175, 179, 186, 195, 216, 219, 249, 268, 282, 287, 311, 324, 329, 331, 353, 358, 372, 374, 378, 407, 409 suppression, 27, 32, 63, 95, 103, 105, 108, 111, 114, 121, 125, 126, 177, 184, 261, 267, 269, 271, 300, 345, 353, 397, 398, 403, 408, 412, 421

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Index surface area, vii, 3, 172, 174, 178, 181, 310, 313, 315, 316, 317, 318, 319, 339, 383 surface layer, 51, 56, 131 surface properties, 212 surplus, 7, 46, 361 surveillance, 20 survivability, 236, 247, 251, 296 survival, 47, 63, 82, 85, 88, 139, 145, 146, 160, 162, 179, 180, 181, 191, 192, 195, 213, 257, 276, 292, 298, 299, 302, 332, 341, 369, 395, 411 survival rate, 85, 181, 332 surviving, 65 survivors, 145 susceptibility, 105 suspensions, 86, 101, 114, 241 sustainability, viii, 87, 88, 96, 98, 162, 240, 248, 282, 303, 307 Sweden, 304, 308, 314 symbiont, 166, 182, 339 symbioses, vii, 97, 172, 304, 331 symbiosis, 31, 104, 109, 112, 127, 132, 138, 157, 165, 173, 175, 185, 187, 189, 191, 196, 218, 220, 221, 262, 291, 296, 300, 302, 306, 307, 308, 320, 321, 338, 339, 345, 348, 349, 351, 352, 367 symbiotic, 63, 64, 65, 69, 70, 79, 82, 98, 99, 102, 135, 156, 161, 163, 166, 186, 193, 262, 289, 291, 310, 313, 317, 320, 338, 342, 345, 350, 352 symptoms, 4, 276, 396 synergistic, 81, 82, 83, 114, 161, 183, 214, 237, 256, 290, 292, 295, 296, 297, 332 synergistic effect, 83, 214, 237, 290, 295, 296, 332 Synergy, 185 synthesis, 2, 35, 72, 76, 79, 117, 126, 169, 178, 179, 206, 209, 211, 212, 242, 268, 270, 272, 282, 340, 345, 352, 368, 372, 379, 383, 387, 400, 412, 417 Syria, 359 systematics, 40

T talc, 85, 86, 412 targets, 24, 138 taxa, 17, 21, 28, 173 taxonomic, 17, 22, 24, 37, 120, 188 taxonomy, 28 TCC, 312 TCE, 391 TCP, 6, 134, 136, 152, 168, 202, 203, 206, 214, 216, 237, 252, 288, 289, 330, 361 tea, 262, 408, 419 teachers, vii, viii teaching, xi teeth, 360

449

Tehran, 231 temperature, 5, 22, 23, 36, 81, 84, 98, 145, 146, 147, 149, 151, 155, 157, 158, 159, 160, 172, 179, 180, 181, 188, 197, 220, 231, 242, 249, 251, 253, 259, 296, 351, 369, 392, 412 temperature gradient, 22, 23, 36, 84 temporal, 23, 26, 36, 89 Thailand, 98, 324 thawing, 159 thermodynamic, 222 Thomson, 134, 142 threat, 240, 254, 377, 389 threats, 181 threshold, 27, 81, 175 time use, 146 timing, 177 tissue, 97, 172, 178, 183, 318, 328, 379, 384, 385, 393, 396, 407 tobacco, 76, 77, 212, 224, 380, 381, 382, 385, 387, 388, 392, 393, 397, 405, 416, 424 Togo, 359 Tokyo, 304 tolerance, 13, 35, 121, 151, 160, 174, 176, 177, 180, 182, 254, 257, 258, 261, 262, 285, 286, 330, 350, 379, 380, 381, 382, 383, 384, 386, 387, 388, 389, 391, 392, 393, 394 tomato, 3, 5, 31, 50, 62, 70, 77, 80, 90, 92, 93, 95, 98, 99, 104, 106, 107, 109, 184, 187, 191, 212, 216, 222, 250, 258, 261, 270, 271, 273, 275, 276, 278, 290, 303, 344, 351, 385, 386, 391, 393, 394, 401, 403, 404, 405, 409, 410, 411, 413, 415, 416, 418, 420, 422, 423, 424 topsoil, 6 total organic carbon, 252 toxic, 9, 181, 266, 377, 378, 383, 384, 385, 388, 390, 391, 392, 394, 398, 410, 412, 417 toxic effect, 384 toxic metals, 377, 383, 384, 390, 391, 392, 394 toxic products, 377 toxic substances, 266, 398 toxicities, 176 toxicity, vii, 9, 71, 72, 92, 121, 176, 193, 254, 259, 285, 389, 390, 392 toxin, 30, 99, 352, 391 toxins, 268, 270, 275 trace elements, 285, 338, 348, 369, 388, 392 tracking, 83, 84 trade, 169, 324 tradition, 138 traits, 28, 47, 87, 88, 89, 112, 114, 117, 121, 126, 127, 179, 217, 229, 250, 281, 285, 288, 300, 305, 310, 342, 365, 370, 379, 384 trans, 206, 370

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450

Index

transcriptase, 84 transcription, 30 transcriptional, 58, 82, 89, 211, 224, 387 transcriptomics, 15 transcripts, 15, 39 transduction, 69, 97, 228 transfer, 1, 3, 40, 43, 46, 118, 148, 181, 185, 190, 200, 204, 209, 210, 212, 217, 228, 230, 237, 265, 283, 300, 332, 368, 379, 383, 384, 387, 390 transformation, 1, 13, 37, 61, 133, 148, 151, 164, 230, 283, 338, 379, 384, 385, 387, 388, 389, 394 transformations, 15, 57, 243, 305 transgene, 388, 390 transgenic, 29, 224, 377, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394 Transgenic, 380, 381, 384, 387, 389, 390, 391, 392, 394 transgenic plants, 377, 379, 381, 382, 383, 384, 385, 388, 389, 392, 393, 394 translocation, 48, 291, 379, 384 transmission, 231, 388 transparent, 159, 269 transplant, 416 transplantation, 236 transport, 3, 69, 86, 91, 125, 132, 147, 149, 190, 191, 200, 209, 218, 237, 331, 341, 368, 369, 379, 383, 384, 385, 388, 389 transportation, 10, 148, 182 transposon, 224, 350 trees, 17, 20, 32, 39, 84, 101, 120, 163, 170, 174, 175, 384, 385, 392 TRF, 26 trial, 54, 143, 197, 214, 287, 290, 293, 295, 306, 365, 366, 390, 401, 410 tricarboxylic acid, 237 Trichoderma viride, 94, 203, 275, 277, 288, 363 trifolii, 37, 77, 134, 135, 166, 347 Trp, 386 trypsin, 402, 424 tryptophan, 98, 115, 117, 178, 179, 343, 386, 394 Tryptophan, 386 TSA, 311, 313, 314 TTM, 307 tubers, 86 tumorigenic, 37 Tunisia, 359 Turkey, 43, 52, 54, 60, 61 turnover, 48, 149

U uniform, 181, 182

United Nations, 61, 308 United States, 2, 140, 296, 319, 324, 326, 358, 360, 408 universality, 25 uranium, 209, 224 urea, 23, 254 Uruguay, 311, 315 USDA, 13 USSR, 212, 326 UV, 81, 120, 194, 225, 243, 244, 353, 425 Uzbekistan, 94

V vacuole, 331, 388 values, 27, 49, 120, 139, 181, 230, 237, 292, 315, 317, 365, 366, 367 variability, 37, 89, 117, 119, 148, 159, 172, 217, 296 variables, 251, 401 variance, 314 variation, 25, 26, 28, 35, 80, 120, 176, 202, 230, 252, 370 vasculature, 331 vector, 209, 212, 368, 396, 397 vegetables, 98, 130, 135, 235, 401 vegetation, 51, 52, 60, 162, 201, 207 velvet, 94 Venezuela, 192, 327, 334, 373 Venus, 302, 372 vermiculite, 85, 180, 182, 194, 305 Verticillium dahliae, 276 Vicia faba, 38, 243, 257 Victoria, 225 Vietnam, 346 virulence, 38, 99, 160 virus, 73, 397 viruses, 98, 396, 397 visible, 114, 203, 332 vitamin B1, 47 vitamin E, 181 vitamins, 46, 80, 83, 101, 121, 247, 285, 291, 361, 408, 409, 412, 420 volatilization, 388, 393

W Wales, 195 warfare, 38 warrants, 28 waste water, 209, 224, 380 wastes, 31, 139, 297, 300, 344, 354, 404, 413 wastewater, 16, 29, 31, 35, 243, 310, 319

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Index

X xenobiotics, 151 xylem, 392

Y yeast, 102, 116, 118, 134, 147, 181, 207, 228, 232, 268, 307, 312, 313, 375, 383, 393 yield loss, 396, 397

Z Zea mays, 9, 31, 49, 69, 102, 104, 105, 133, 172, 182, 189, 192, 214, 244, 255, 286, 287, 294, 302, 307, 339, 423 zinc, 174, 241, 258, 338, 387, 391 Zinc, 175 zinc oxide, 241

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wastewater treatment, 29, 35, 243, 310 water, 5, 6, 7, 13, 15, 16, 23, 31, 34, 46, 51, 70, 72, 94, 119, 130, 131, 133, 134, 138, 142, 147, 158, 159, 172, 174, 176, 180, 181, 182, 187, 197, 207, 209, 218, 224, 234, 236, 254, 286, 295, 302, 308, 311, 312, 313, 358, 359, 360, 372, 378, 380, 383, 390, 391, 392, 393 water absorption, 72, 174 water quality, 13, 142 water supplies, 254 water-soluble, 5, 46, 94 Weathered, 176 weathering, 55, 56, 103, 162, 225 web, 34 web-based, 34 West Africa, 187, 195 wild type, 29, 93, 95, 145, 150, 151, 152, 155, 212, 341, 380, 381, 384, 388, 389, 425 wind, 70 winter, 8, 36, 147, 364, 374, 418 Wisconsin, 13, 140, 303 wood, 361, 378 wool, 98 workers, 162, 176, 251, 267, 291, 364

451

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