Beneficial microbes in agro-ecology. Bacteria and fungi 9780128235584, 0128235586

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BENEFICIAL MICROBES IN AGRO-ECOLOGY Bacteria and Fungi

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BENEFICIAL MICROBES IN AGRO-ECOLOGY BACTERIA AND FUNGI Edited by

N. AMARESAN C.G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Bardoli, Surat, Gujarat, India

M. SENTHIL KUMAR ICAR-Indian Institute of Pulses Research, Kanpur, Uttar Pradesh, India

K. ANNAPURNA Division of Microbiology, ICAR-Indian Agricultural Research Institute, New Delhi, India

KRISHNA KUMAR Pandit Deendayal Upadhyay College of Horticulture & Forestry, Dr. Rajendra Prasad Central Agricultural University,Tirhut College Campus, Dholi, Muzaffarpur, Bihar, India

A. SANKARANARAYANAN C.G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Bardoli, Surat, Gujarat, India

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2020 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-823414-3 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

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Contents Contributors xiii Preface xxi

4. Characterization and identification of the genus 30 5. Plant growth-promoting attributes of Serratia spp. 31 6. Phytoremediation 32 Acknowledgment 33 References 33

I Bacteria

4. Rhizobium

1. Arthrobacter

Renu Verma, Harika Annapragada, Nalini Katiyar, Nalini Shrutika, Krishnasis Das, and Senthilkumar Murugesan

Pratiti Roy and Ashok Kumar

1. Introduction 3 2. Taxonomy 4 3. Isolation and identification of Arthrobacter genus 4 4. Arthrobacter as a plant growth-promoting rhizobacteria 5 5. Siderophore production and metal solubilization by Arthrobacter 5 6. Role of Arthrobacter in bioremediation 6 7. Future prospects 8 8. Conclusion 8 References 9

1. Introduction 37 2. Diversity and taxonomy of rhizobia 3. Physiologic aspects of rhizobia 38 4. Beneficial effect of rhizobia 40 5. Conclusion 48 References 49

5. Streptomyces S. Gopalakrishnan, V. Srinivas, and S.L. Prasanna

1. 2. 3. 4. 5.

Introduction 55 Taxonomy of Streptomyces 55 Isolation of Streptomyces 57 Identification of Streptomyces 58 Beneficial role of Streptomyces in ago-ecology: in vitro PGP and biocontrol traits of the Streptomyces 59 6. In vitro physiologic traits of the Streptomyces 60 7. In planta PGP traits of the Streptomyces 61 8. In planta biocontrol traits of the Streptomyces 61 9. Secondary metabolite production traits of the Streptomyces 62 10. Streptomyces research at ICRISAT 62 11. Conclusion 67 Acknowledgments 67 References 68

2. Alcaligenes Makoto Shoda

1. Taxonomy 13 2. Isolation of A. faecalis No. 4 14 3. Identification of A. faecalis No. 4 14 4. Beneficial properties of A. faecalis No. 4 14 5. Conclusion 25 References 25

3. Serratia Soma Barman, Satya Sundar Bhattacharya, and Narayan Chandra Mandal

1. Introduction 27 2. Taxonomy of the genus Serratia 3. Isolation of the genus 29

38

28

v

vi

CONTENTS

6. Azospirillum Raúl O. Pedraza, María P. Filippone, Cecilia Fontana, Sergio M. Salazar, Alberto Ramírez-Mata, Daniel Sierra-Cacho, and Beatriz E. Baca

1. Taxonomy of Azospirillum 74 2. Isolation of Azospirillum 75 3. Biochemical and genetic methods for the identification of Azospirillum 78 4. Beneficial role of the genus Azospirillum in agroecology 84 5. Concluding remarks 97 References 98

7. Bacillus Rainer Borriss

1. Taxonomy of Bacillus 108 2. Isolation of different Bacillus species 113 3. Morphology and simple biochemical and molecular methods for identification of different Bacillus species 118 4. Beneficial role of bacilli in agroecology 128 References 129

8. Pseudomonas Krishnashis Das, Shrutica Abrol, Renu Verma, Harika Annapragada, Nalini Katiyar and Senthilkumar M

1. Introduction 133 2. Historical perspective of Pseudomonas and their classification 134 3. Role of Pseudomonas as PGPR in agriculture 135 4. Role of Pseudomonas in biodegradation of pesticides 141 5. Conclusion 142 References 142

9. Brevibacillus Sanket Ray, Nafisa Patel, and Dhruti Amin

1. Taxonomy of the genus Brevibacillus 149 2. Isolation of the genus Brevibacillus 153 3. Biochemical methods for identification of the genus Brevibacillus 155 4. Beneficial role of the genus Brevibacillus in agroecology 158 References 162

10. Exiguobacterium Neha Pandey

1. Introduction/taxonomy 169 2. Isolation of the Exiguobacterium genus 171 3. Identification of the Exiguobacterium genus 172 4. Beneficial properties of Exiguobacterium 175 5. Future prospects 179 6. Concluding remarks 180 Acknowledgments 180 References 181

11. Frankia M. Narayanasamy, D. Dhanasekaran, and N. Thajuddin

1. Introduction/taxonomy 185 2. Isolation of the Frankia spp. 187 3. Characterization of Frankia 191 4. Beneficial properties of Frankia spp. 204 5. Conclusions 207 Acknowledgment 207 References 207

12. Kosakonia Janet Jan-Roblero, Juan Antonio Cruz-Maya, and Claudia Guerrero Barajas

1. The order Enterobacteriales 213 2. Genus Kosakonia: biochemical characteristics 217 3. Participation of Kosakonia spp. as plant growth promoter 217 4. Other species of Kosakonia 227 Acknowledgment 228 References 228

13. Klebsiella YingWu Shi, Hongmei Yang, Ming Chu, XinXiang Niu, XiangDong Huo, Yan Gao, Jun Zeng, Tao Zhang, YuGuo Li, KuEr Outi, Kai Lou, XueYan Li, WenFang Dang, and Chun Li

1. Taxonomy of the genus Klebsiella 233 2. Isolation of the genus Klebsiella 237 3. Simple biochemical methods for identification of the genus Klebsiella 239 4. Beneficial role of the genus Klebsiella in agroecology 245 Acknowledgment 251 References 251

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14. Enterobacter

4. Paenibacillus sp. in Exopolysaccharide production 347 5. Genetic engineering-based modification of Paenibacillus 348 6. Paenibacillus in bioremediation of xenobiotics 349 7. Conclusion and future direction 354 References 354

Ashraf Khalifa

1. 2. 3. 4. 5.

Introduction 259 Taxonomy 259 Isolation 262 Identification 262 Characteristics of the Enterobacter as plant growth promoters 262 6. Bioremediation 265 7. Biofortification 266 8. Conclusions 266 References 267

18. Firmicutes Isha Hashmi, Saskia Bindschedler, and Pilar Junier

15. Paraburkholderia Santiago Adolfo Vio, Sabrina Soledad García, Victoria Casajus, Juliana Soler Arango, María Lina Galar, Pamela Romina Bernabeu, and María Flavia Luna

1. Introduction 271 2. Taxonomy and some other features of Burkholderia sensu lato 272 3. The new genus Paraburkholderia 277 4. Paraburkholderia: a genus rich in plant growth promoting species 285 References 301

16. Lysobacter

1. Introduction 363 2. Genus Lysinibacillus 364 3. Genus Aneurinibacillus 377 4. Genus Brevibacillus 378 5. Genus Oceanobacillus 380 6. Genus Planococcus 381 7. Genus Clostridium 383 8. Genus Sporosarcina 384 9. Genus Virgibacillus 385 10. Genus Terribacillus 386 11. Genus Staphylococcus 386 12. Other genera 387 13. Conclusion 390 Acknowledgments 390 References 391

Francesca Brescia, Ilaria Pertot, and Gerardo Puopolo

19. Azotobacter

1. Taxonomy of Lysobacter, a 40-year-old bacterial genus 313 2. Ubiquity of Lysobacter species and their ecology in the agroecosystem 318 3. Correlation of Lysobacter spp. with soil suppressiveness and isolation of bacterial strains belonging to plant beneficial species 319 4. Plant beneficial role of Lysobacter spp. relies on multiple mechanisms of action 323 5. Potential of Lysobacter members for the bioremediation of contaminated soils 331 References 331

1. 2. 3. 4.

Introduction 397 Isolation of the genus 403 Identification of the genus 405 Beneficial role of the Azotobacter in agroecology 414 5. Outlook 421 Acknowledgment 421 References 421

20. Stenotrophomonas Ranjan Ghosh, Sohini Chatterjee, and Narayan C. Mandal

17. Paenibacillus Rupshikha Patowary and Hemen Deka

1. Introduction 339 2. Paenibacillus taxonomy 340 3. Agroecological applications of Paenibacillus

Satish V. Patil, Bhavana V. Mohite, Chandrashekhar D. Patil, Sunil H. Koli, Hemant P. Borase, and Vikas S. Patil

342

1. Introduction 427 2. Taxonomy of the genus Stenotrophmonas 428 3. Isolation and maintenance of Stenotrophomonas spp. 428

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4. Morphological and biochemical properties of Stenotrophomonas spp. 430 5. Plant growthepromoting attributes of Stenotrophomonas spp. 432 6. Bioremediation by Stenotrophomonas spp. 437 7. Biofortification 438 References 439

21. Actinobacteria Shabiha Nudrat Hazarika and Debajit Thakur

1. Introduction 443 2. Habitat of Actinobacteria 444 3. Morphological characteristics of Actinobacteria 446 4. Taxonomy and molecular morphology of Actinobacteria 451 5. Isolation of Actinobacteria 454 6. Biochemical methods for identification of Actinobacteria 457 7. Beneficial role of Actinobacteria in agriculture 464 8. Conclusion 470 Acknowledgment 470 References 470

22. Clostridium Guilherme Grodzki Oliveira Figueiredo, Valéria Rosa Lopes, Tales Romano, and Marcela Candido Camara

1. 2. 3. 4. 5.

Introduction 477 Taxonomy of the Clostridium genus 478 Isolation and identification methods 479 Plant growthepromoting Clostridium 480 Agroecological use in industry of Clostridium sp. 483 6. Conclusions 486 Acknowledgments 486 References 487

23. Herbaspirillum Filipe Pereira Matteoli, Fabio Lopes Olivares, Thiago Motta Venancio, Letícia Oliveira da Rocha, Luiz Eduardo Souza da Silva Irineu, and Luciano Pasqualoto Canellas

1. Taxonomy 493 2. Ecology 495 3. Isolation procedures

498

4. Simple biochemical and molecular methods for identification 499 5. Beneficial roles in agroecology 502 References 505

24. Methylobacterium Hardik Naik Jinal, N. Amaresan, and A. Sankaranarayanan

1. 2. 3. 4. 5.

Taxonomy of Methylobacterium 509 Methylobacterium interaction with plants 510 Isolation of Methylobacterium 511 Selective media 511 General method for identification of Methylobacterium 512 6. Identification of Methylobacterium at species level 513 7. Identification of Methylobacterium at molecular level 513 8. Beneficial role of Methylobacterium in agroecology 513 9. Conclusion 516 Acknowledgment 516 References 516

25. Gluconobacter Mitesh Dwivedi

1. Introduction 521 2. Natural habitats of Gluconobacter 522 3. Isolation and growth of Gluconobacter 522 4. Taxonomy of Gluconobacter 524 5. Gluconacetobacter diazotrophicus 533 6. Role of Gluconobacter in the agroecosystem 534 7. Conclusion 539 Acknowledgment 539 References 539

26. Thiobacillus Murugan Kumar, Mohammad Tarique Zeyad, Prassan Choudhary, Surinder Paul, Hillol Chakdar, and Mahendra Vikram Singh Rajawat

1. Introduction 545 2. Taxonomy and reorganization of the genus Thiobacillus 545 3. Thiobacillus and its interactions with the physical environment 546 4. Agricultural importance 549

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5. Industrial importance 551 6. Conclusion and future perspectives References 554

6. Evolution of entomopathogenesis 601 7. Mechanism of pathogenicity in insect 601 8. Enzymes associated with pathogenesis or virulence 602 9. Characterization of cyclodepsipeptides 602 10. Applications of bioformulations of Metarhizium and their metabolic products 603 11. Future prospects 604 12. Conclusion 605 References 606

553

II Fungi 27. Glomus Kim Maria Rodrigues and Bernard Felinov Rodrigues

1. 2. 3. 4. 5.

Introduction 561 Phylogenetic relationships 561 Isolation of AM fungal spores 562 Identification of AM fungal spores 562 General morphological characters used for identification of AM fungal spores 563 6. Beneficial role 565 7. Conclusion 566 References 566

30. Aspergillus Raphael Sanzio Pimenta, Drielly Dayanne Monteiro dos Santos Baliza, and Juliana Fonseca Moreira da Silva

1. Introduction 611 2. The genus Aspergillus 612 3. Aflatoxins 613 4. The biocontrol 615 5. Conclusion 619 References 619

28. Trichoderma

31. Ganoderma

Pralay Shankar Gorai, Soma Barman, Surendra K. Gond, and Narayan C. Mandal

Ritu Mawar, Ladhu Ram, Deepesh, and Tanu Mathur

1. Introduction 625 2. Taxonomy 627 3. Diversity and distribution of Ganoderma 632 4. Host range 634 5. Isolation 639 6. Biochemical method for identification 640 7. Benefits 641 8. Culture collections of G. lucidum 645 9. Future prospects 645 References 646

1. 2. 3. 4.

Introduction 571 Characteristic features of Trichoderma 573 Isolation of Trichoderma spp. 574 Beneficial role of Trichoderma in agroecology 576 5. Trichoderma used as biocontrol agent in agriculture 579 6. Roles in biofortification 582 7. Roles in bioremediation 583 8. Roles in phytoremediation 584 Acknowledgment 584 References 584

32. Penicillium R. Srinivasan, G. Prabhu, M. Prasad, M. Mishra, M. Chaudhary, and R. Srivastava

29. Metarhizium Tarun Kumar Patel

1. Introduction 593 2. Classification and taxonomic status of entomopathogenic fungi 594 3. Strategies for the isolation of Metarhizium 4. Identification and characterization of entomopathogenic fungi 597 5. Secondary metabolites 598

594

1. 2. 3. 4. 5. 6. 7. 8.

Introduction 651 Identification of Penicillium 652 PlantePenicillium interactions 654 Plant endophytic Penicillium 654 Phosphate solubilization by Penicillium 655 Plant disease suppression 656 Penicillium in soil suppressiveness 657 Penicillium in alleviation of abiotic stresses 658

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3. Response to drought 715 4. Response to soil contamination 717 Acknowledgments 721 References 722

9. Penicillium for a green environment 659 10. Bad members of Penicillium for human beings 660 11. Conclusion 662 References 662

33. Pochonia

37. Beauveria

Thalita Suelen Avelar Monteiro, Paulo Victor Magalhães Pacheco, Angélica Souza Gouveia, Huarlen Marcio Balbino, and Leandro Grassi de Freitas

Lizzy A. Mwamburi

1. Taxonomy 659 2. Isolation and mass production 672 3. Pochonia chlamydosporia for the control of nematodes 674 4. The plant-growth promotion by Pochonia chlamydosporia 676 5. Final considerations 678 References 679

34. Duddingtonia Thalita Suelen Avelar Monteiro, Angélica Souza Gouveia, Huarlen Marcio Balbino, Túlio Morgan, and Leandro Grassi de Freitas

1. Taxonomy 683 2. Isolation and mass production 684 3. The action of Duddingtonia flagrans on gastrointestinal nematodes 685 4. Duddintonia flagrans in the biological control of plant-parasitic nematode 689 5. Duddingtonia flagrans promoting plant growth 690 6. Final considerations 691 References 692

35. Paxillus Aqib Sayyed and Anwar Hussain

1. Introduction 695 2. Distribution 696 3. Taxonomy of the genus 697 4. Isolation 697 5. Molecular identification 697 6. Beneficial role of the genus in agroecology References 704

38. Stropharia 699

36. Pisolithus Mónica Sebastiana, Ana Corrêa, Paula Castro, and Miguel Ramos

1. Introduction 707 2. Effect on plant growth, nutrition, and physiology 709

1. Introduction 727 2. Beauveria bassiana (Balsamo) Vuillemin 727 3. Mechanism of action of Beauveria bassiana 728 4. Beauveria bassiana in agriculture 728 5. Economic importance of pests in livestock and poultry production systems 729 6. Potential for biological control of livestock and poultry pests 730 7. Beauveria bassiana as a biological control agent of pests of livestock and poultry 731 8. Combinatorial studies of Beauveria bassiana with other entomopathogens for pest control 731 9. Economic importance of pests and diseases in crop production systems 734 10. Beauveria bassiana as a biological control agent of pests of crops 734 11. Beauveria bassiana as an endophyte of plants 736 12. Endophytic B. bassiana as a biological control agent of plant pests 736 13. B. bassiana as an endophytic biological control agent of pests with other entomopathogens 737 14. Endophytic B. bassiana as a biological control of plant pathogens 737 15. Conclusions 738 References 739

Judá Ben-Hur de Oliveira, Paula Roberta Costalonga Pereira, Vanessa Silva dos Santos, Jean Moisés Ferreira, and Jean Carlos Vencioneck Dutra

1. Stropharia 749 2. Stropharia taxonomy 750 3. Isolation of Stropharia fungi 751 4. Beneficial uses of Stropharia 752 Acknowledgments 753 References 753

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CONTENTS

39. Entomophthoran

42. Dactylella

Surendra K. Dara and Richard A. Humber

T.R. Kavitha, R.U. Priya, and T.R. Sunitha

1. Introduction 757 2. Taxonomy of the most agriculturally important entomophthoralean genera 758 3. Fungal isolation and multiplication 760 4. Microbial control potential of entomophthoroid fungi 760 5. Influence of environmental factors on fungal epizootics 767 6. Artificial introduction of entomophthoroid fungi 768 7. Conclusion 769 References 769

40. Pythium A. Sankaranarayanan and N. Amaresan

1. Introduction 777 2. Characteristic features, taxonomy, and identification of Pythium 778 3. Beneficial activities of Pythium spp. 779 4. Other promising Pythium candidates 786 5. Futuristic approach on Pythium spp. 786 6. Conclusion 787 Acknowledgments 787 References 787

41. Paecilomyces M. Senthilkumar, R. Anandham, and R. Krishnamoorthy

1. Introduction 793 2. General description 795 3. Taxonomy and phylogeny 795 4. Classification 798 5. Description of genus Purpureocillium 798 6. Identification 799 7. Colony morphological features 799 8. Key features 800 9. Life cycles 801 10. Biocontrol agent 801 11. Conclusion 804 References 805

1. Introduction 809 2. Taxonomy 810 3. Isolation of Dactylella 811 4. Beneficial role of the genus Dactylella References 815

814

43. Hirsutella Narasa Reddy, G. Mahesh, M. Priya, R.U. Shailendra Singh, and L. Manjunatha

1. Introduction 817 2. Phylum ascomycota and dueteromycota 817 3. Nutritional studies on the genus Hirsutella 822 4. Compatibility 827 5. Conclusion 829 References 830

44. Ampelomyces L. Manjunatha, R.U. Shailendra Singh, B.M. Ravikumara, G. Narasa Reddy, and M. Senthilkumar

1. Introduction 833 2. Historical developments in ampelomyces research 834 3. Taxonomy of ampelomyces 835 4. Identification, isolation, and cultivation of mycoparasite 837 5. Biology and life cycle of mycoparasite 840 6. Diversity of ampelomyces species and its host range 842 7. Applications of ampelomyces in agriculture 844 8. Mode of action of Ampelomyces quisqualis on mycohost 847 9. Ampelomyces quisqualis production and formulation development 852 10. Compatibility with fungicides 853 11. Future prospects 853 12. Conclusion 854 Acknowledgments 854 References 854 Index

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Contributors Shrutica Abrol Division of Plant Biotechnology, ICAR-Indian Institute of Pulses Research (ICAR-IIPR), Kanpur, Uttar Pradesh, India N. Amaresan C.G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Surat, Gujarat, India Dhruti Amin C. G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Maliba Campus, Surat, Gujarat, India R.

Anandham Department of Agricultural Microbiology, TamilNadu Agricultural University, Coimbatore, Tamil Nadu, India

Harika Annapragada Division of Basic Sciences, Indian Institute of Pulses Research, Kanpur, Uttar Pradesh, India Juliana Soler Arango YPF Tecnología (Y-TEC) Laboratorio de Biocorrosión, La Plata, Argentina Thalita Suelen Avelar Monteiro Departamento de Fitopatologia, Universidade Federal de Viçosa, Viçosa, MG, Brazil Beatriz E. Baca Centro de Investigaciones en Ciencias Microbiológicas, Benemérita Universidad Autónoma de Puebla, Ciudad Universitaria, Puebla Pue, México Huarlen Marcio Balbino Departamento de Fitopatologia, Universidade Federal de Viçosa, Viçosa, MG, Brazil Claudia Guerrero Barajas Departamento de Bioprocesos, Unidad Profesional Interdisciplinaria de Biotecnología, Instituto Politécnico Nacional, Mexico City, Mexico Soma Barman Soil and Agro-Bioengineering Laboratory, Department of Environmental Science, Tezpur University, Tezpur, Assam, India Judá Ben-Hur de Oliveira Departamento de Ciências Biológicas, Universidade Federal do Espírito Santo, Vitória, Espírito Santo, Brazil

Pamela Romina Bernabeu Centro de Investigación y Desarrollo en Fermentaciones Industriales (CINDEFI), UNLP, CCT La Plata CONICET, Departamento de Química, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina Satya Sundar Bhattacharya Soil and Agro-Bioengineering Laboratory, Department of Environmental Science, Tezpur University, Tezpur, Assam, India Saskia Bindschedler Laboratory of Microbiology, University of Neuchatel, Neuchatel, Switzerland Hemant P. Borase C. G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Surat, Gujarat, India Rainer Borriss Institut für Biologie, Humboldt Universität, Berlin, Germany; Institute of Marine Biotechnology (IMaB), Greifswald, Germany Francesca Brescia Department of Sustainable Agro-ecosystems and Bioresources, Research and Innovation Centre, Fondazione Edmund Mach, San Michele all’Adige, Italy; Agricultural Science and Biotechnology, Department of Agricultural, Food, Environmental and Animal Sciences, University of Udine, Udine, Italy Marcela Candido Camara Bioprocess Engineering and Biotechnology Department, Federal University of Paraná-UFPR, Curitiba, PR, Brazil Luciano Pasqualoto Canellas Campos Goytacazes, Rio de Janeiro, Brazil

dos

Victoria Casajus Instituto de Fisiología Vegetal (INFIVE), Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, La Plata, Argentina

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CONTRIBUTORS

Paula Castro Universidade Católica Portuguesa, CBQF-Centro de Biotecnologia e Química Fina, Laboratório Associado, Escola Superior de Biotecnologia, Porto, Portugal Hillol Chakdar ICAR-National Bureau of Agriculturally Important Microorganisms (ICAR-NBAIM), Mau, Uttar Pradesh, India Narayan Chandra Mandal Mycology and Plant Pathology Laboratory, Department of Botany, Visva-Bharati, Santiniketan, West Bengal, India Sohini Chatterjee Mycology and Plant Pathology Laboratory, Department of Botany, Santiniketan, West Bengal, India M. Chaudhary Crop Production Division, ICAR-Indian Grassland and Fodder Research Institute, Jhansi, Uttar Pradesh, India Prassan Choudhary ICAR-National Bureau of Agriculturally Important Microorganisms (ICAR-NBAIM), Mau, Uttar Pradesh, India Ming Chu Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi, Xinjiang, China; Xinjiang Laboratory of Special Enviromental Microbiology, Urumqi, Xinjiang, China; Key Laboratory of Agriculture Environment in Northwest Oasis of Agriculture, Urumqi, Xinjiang, China Ana Corrêa CE3C - Center for Ecology, Evolution and Environmental Changes, Faculdade de Ciências da Universidade de Lisboa, Lisboa, Portugal Paula Roberta Costalonga Pereira Departamento de Ciências Biológicas, Universidade Federal do Espírito Santo, Vitória, Espírito Santo, Brazil Juan Antonio Cruz-Maya Unidad Profesional Interdisciplinaria en Ingeniería y Tecnologías Avanzadas, Instituto Politécnico Nacional, Mexico City, Mexico WenFang Dang Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi, Xinjiang, China; Xinjiang Laboratory of Special Enviromental Microbiology, Urumqi, Xinjiang, China; Key Laboratory of Agriculture Environment in Northwest Oasis of Agriculture, Urumqi, Xinjiang, China Surendra K. Dara University of California Cooperative Extension, Division of Agriculture and Natural Resources, San Luis Obispo, CA, United States

Krishnasis Das Division of Basic Sciences, Indian Institute of Pulses Research, Kanpur, Uttar Pradesh, India Deepesh ICAR-Central Arid Zone Research Institute, Jodhpur, Rajasthan, India Hemen Deka Department of Botany, Gauhati University, Guwahati, Assam, India D. Dhanasekaran Department of Microbiology, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India Mitesh Dwivedi C. G. Bhakta Institute of Biotechnology, Faculty of Science, Uka Tarsadia University, Surat, Gujarat, India Jean Moisés Ferreira Laboratório de Biologia Molecular e Expressão Gênica, Departamento de Ciências Biológicas, Universidade Federal de Alagoas, Arapiraca, Alagoas, Brazil Guilherme Grodzki Oliveira Figueiredo Department of Plant Science and Crop Protection, Federal University of Paraná-UFPR, Curitiba, PR, Brazil María P. Filippone Facultad de Agronomía y Zootecnia, Universidad Nacional de Tucumán, San Miguel de Tucumán, Tucumán, Argentina Cecilia Fontana Argentina

INTA EEA Famaillá, Tucumán,

María Lina Galar Centro de Investigación y Desarrollo en Fermentaciones Industriales (CINDEFI), UNLP, CCT La Plata CONICET, Departamento de Química, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina Yan Gao Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi, Xinjiang, China; Xinjiang Laboratory of Special Enviromental Microbiology, Urumqi, Xinjiang, China; Key Laboratory of Agriculture Environment in Northwest Oasis of Agriculture, Urumqi, Xinjiang, China Sabrina Soledad García Centro de Investigación y Desarrollo en Fermentaciones Industriales (CINDEFI), UNLP, CCT La Plata CONICET, Departamento de Química, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina

CONTRIBUTORS

xv

Ranjan Ghosh Department of Botany, Bankura Sammilani College, Kenduadihi, Bankura, West Bengal, India

Hardik Naik Jinal C.G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Surat, Gujarat, India

Surendra K. Gond Department of Botany, MMV, Banaras Hindu University, Varanasi, Uttar Pradesh, India

Pilar Junier Laboratory of Microbiology, University of Neuchatel, Neuchatel, Switzerland

S. Gopalakrishnan International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, Telangana, India Pralay Shankar Gorai Mycology and Plant Pathology Laboratory, Department of Botany, Visva-Bharati, Santiniketan, West Bengal, India

Nalini Katiyar Division of Basic Sciences, Indian Institute of Pulses Research, Kanpur, Uttar Pradesh, India T.R. Kavitha University of Agricutural Sciences, GKVK, Bangalore, Karnataka, India

Angélica Souza Gouveia Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, Viçosa, MG, Brazil

Ashraf Khalifa Department of Biological Sciences, College of Sciences, King Faisal University, Al-Ahsa, Saudi Arabia; Botany and Microbiology Department, Faculty of Sciences, Beni-Suef University, Beni-Suef, Egypt

Leandro Grassi de Freitas Departamento de Fitopatologia, Universidade Federal de Viçosa, Viçosa, MG, Brazil

Sunil H. Koli School of Life Sciences, Kavayitri Bahinabai Chaudhari North Maharashtra University, Jalgaon, Maharashtra, India

Isha Hashmi Laboratory of Microbiology, University of Neuchatel, Neuchatel, Switzerland

R. Krishnamoorthy Department of Crop Management, Vanavarayar Institute of Agriculture, Pollachi, Tamil Nadu, India

Shabiha Nudrat Hazarika Microbial Biotechnology Laboratory, Life Sciences Division, Institute of Advanced Study in Science and Technology, Guwahati, Assam, India; Department of Molecular Biology and Biotechnology, Cotton University, Guwahati, Assam, India Richard A. Humber Previously United States Department of Food and Agriculture-Agricultural Research Service, Ithaca, NY, United States XiangDong Huo Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi, Xinjiang, China; Xinjiang Laboratory of Special Enviromental Microbiology, Urumqi, Xinjiang, China; Key Laboratory of Agriculture Environment in Northwest Oasis of Agriculture, Urumqi, Xinjiang, China Anwar Hussain Department of Botany, Garden Campus, Abdul Wali Khan University Mardan, Mardan, Khyber Pakhtunkhwa, Pakistan Janet Jan-Roblero Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City, Mexico

Murugan Kumar ICAR-National Bureau of Agriculturally Important Microorganisms (ICAR-NBAIM), Mau, Uttar Pradesh, India Ashok Kumar Department of Genetics and Plant Breeding (Plant Biotechnology), Rajiv Gandhi South Campus, Banaras Hindu University, Mirzapur, Uttar Pradesh, India XueYan Li Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi, Xinjiang, China; Xinjiang Laboratory of Special Enviromental Microbiology, Urumqi, Xinjiang, China; Key Laboratory of Agriculture Environment in Northwest Oasis of Agriculture, Urumqi, Xinjiang, China Chun Li School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing, China YuGuo Li Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi, Xinjiang, China; Xinjiang Laboratory of Special Enviromental Microbiology, Urumqi, Xinjiang, China; Key Laboratory of Agriculture Environment in Northwest Oasis of Agriculture, Urumqi, Xinjiang, China

xvi

CONTRIBUTORS

Valéria Rosa Lopes Department of Plant Science and Crop Protection, Federal University of Paraná-UFPR, Curitiba, PR, Brazil Kai Lou Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi, Xinjiang, China; Xinjiang Laboratory of Special Enviromental Microbiology, Urumqi, Xinjiang, China; Key Laboratory of Agriculture Environment in Northwest Oasis of Agriculture, Urumqi, Xinjiang, China María Flavia Luna Centro de Investigación y Desarrollo en Fermentaciones Industriales (CINDEFI), UNLP, CCT La Plata CONICET, Departamento de Química, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina; Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC-PBA), La Plata, Argentina Paulo Victor Magalhães Pacheco Departamento de Fitopatologia, Universidade Federal de Lavras, Lavras, MG, Brazil G. Mahesh University of Agricultural Sciences, Bangalore, Karnataka, India; ICAR- Indian Institute of Pulses Research, Kanpur, Uttar Pradesh, India Narayan C. Mandal Mycology and Plant Pathology Laboratory, Department of Botany, Visva-Bharati, Santiniketan, West Bengal, India L. Manjunatha University of Agricultural Sciences, Bangalore, Karnataka, India; ICARIndian Institute of Pulses Research, Kanpur, Uttar Pradesh, India Tanu Mathur ICAR-Central Arid Zone Research Institute, Jodhpur, Rajasthan, India Filipe Pereira Matteoli Campos dos Goytacazes, Rio de Janeiro, Brazil Ritu Mawar ICAR-Central Arid Zone Research Institute, Jodhpur, Rajasthan, India M. Mishra Crop Production Division, ICARIndian Grassland and Fodder Research Institute, Jhansi, Uttar Pradesh, India Bhavana V. Mohite Department of Microbiology, Bajaj College of Science, Wardha, Maharashtra, India

Drielly Dayanne Monteiro dos Santos Baliza Food Engineering, Biodiversity and Biotechnology Graduate Program - BIONORTE, Universidade Federal do Tocantins, Palmas, Tocantins, Brazil Juliana Fonseca Moreira da Silva Microbiology, Medicine Course. Universidade Federal do Tocantins, Palmas, Tocantins, Brazil Túlio Morgan Programa de Pós-Graduação em Bioinformática, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil Lizzy A. Mwamburi Department of Biological Sciences, University of Eldoret, Eldoret, Uasin Gishu, Kenya G. Narasa Reddy University of Agricultural Sciences, Bangalore, Karnataka, India M. Narayanasamy Department of Microbiology, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India XinXiang Niu Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi, Xinjiang, China; Xinjiang Laboratory of Special Enviromental Microbiology, Urumqi, Xinjiang, China; Key Laboratory of Agriculture Environment in Northwest Oasis of Agriculture, Urumqi, Xinjiang, China Fabio Lopes Olivares Campos dos Goytacazes, Rio de Janeiro, Brazil Letícia Oliveira da Rocha Campos dos Goytacazes, Rio de Janeiro, Brazil KuEr Outi Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi, Xinjiang, China; Xinjiang Laboratory of Special Enviromental Microbiology, Urumqi, Xinjiang, China; Key Laboratory of Agriculture Environment in Northwest Oasis of Agriculture, Urumqi, Xinjiang, China Neha Pandey Department of Biotechnology, Guru Ghasidas Vishwavidyalaya (A Central University), Bilaspur, Chhattisgarh, India Tarun Kumar Patel Sant Guru Ghasidas Government P.G. College, Kurud, Dhamtari, India

CONTRIBUTORS

Nafisa Patel Department of Microbiology, Naran Lala College of Professional and Applied Sciences, Bhagwati Sankul, Navsari, Gujarat, India Satish V. Patil School of Life Sciences, Kavayitri Bahinabai Chaudhari North Maharashtra University, Jalgaon, Maharashtra, India; North Maharashtra Microbial Culture Collection Centre (NMCC), Kavayitri Bahinabai Chaudhari North Maharashtra University, Jalgaon, Maharashtra, India Chandrashekhar D. Patil PSL Université Paris: EPHE-UPVD-CNRS, USR 3278 CRIOBE, Université de Perpignan, Perpignan Cedex, France Vikas S. Patil University Institute of Chemical Technology, Kavayitri Bahinabai Chaudhari North Maharashtra University, Jalgaon, Maharashtra, India Rupshikha Patowary Institute of Advanced Study in Science and Technology, Guwahati, Assam, India Surinder Paul ICAR-National Bureau of Agriculturally Important Microorganisms (ICAR-NBAIM), Mau, Uttar Pradesh, India Raúl O. Pedraza Facultad de Agronomía y Zootecnia, Universidad Nacional de Tucumán, San Miguel de Tucumán, Tucumán, Argentina Ilaria Pertot Department of Sustainable Agroecosystems and Bioresources, Research and Innovation Centre, Fondazione Edmund Mach, San Michele all’Adige, Italy; Center Agriculture Food Environment, University of Trento, Trento, Italy Raphael Sanzio Pimenta Medicine course, Microbiology, Research and Graduate Studies of Universidade Federal do Tocantins, Palmas, Tocantins, Brazil G. Prabhu Crop Production Division, ICARIndian Grassland and Fodder Research Institute, Jhansi, Uttar Pradesh, India M. Prasad Crop Production Division, ICARIndian Grassland and Fodder Research Institute, Jhansi, Uttar Pradesh, India S.L. Prasanna International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, Telangana, India

xvii

R.U. Priya University of Agricutural Sciences, GKVK, Bangalore, Karnataka, India M. Priya University of Agricultural Sciences, Bangalore, Karnataka, India; ICAR- Indian Institute of Pulses Research, Kanpur, Uttar Pradesh, India Gerardo Puopolo Department of Sustainable Agro-ecosystems and Bioresources, Research and Innovation Centre, Fondazione Edmund Mach, San Michele all’Adige, Italy Ladhu Ram College of Horticulture Forestry, Jhalawad, Rajasthan, India

and

Alberto Ramírez-Mata Centro de Investigaciones en Ciencias Microbiológicas, Benemérita Universidad Autónoma de Puebla, Ciudad Universitaria, Puebla Pue, México Miguel Ramos Universidade Católica Portuguesa, CBQF-Centro de Biotecnologia e Química Fina, Laboratório Associado, Escola Superior de Biotecnologia, Porto, Portugal B.M. Ravikumara University of Agricultural Sciences, Bangalore, Karnataka, India Sanket Ray Department of Microbiology, Naran Lala College of Professional and Applied Sciences, Bhagwati Sankul, Navsari, Gujarat, India Narasa Reddy University of Agricultural Sciences, Bangalore, Karnataka, India; ICARIndian Institute of Pulses Research, Kanpur, Uttar Pradesh, India Kim Maria Rodrigues Department of Botany, Goa University, Taleigao Plateau, Goa, India Bernard Felinov Rodrigues Department of Botany, Goa University, Taleigao Plateau, Goa, India Tales Romano Department of Plant Science and Crop Protection, Federal University of ParanáUFPR, Curitiba, PR, Brazil Pratiti Roy Department of Genetics and Plant Breeding (Plant Biotechnology), Rajiv Gandhi South Campus, Banaras Hindu University, Mirzapur, Uttar Pradesh, India Sergio M. Salazar Facultad de Agronomía y Zootecnia, Universidad Nacional de Tucumán, San Miguel de Tucumán, Tucumán,

xviii

CONTRIBUTORS

Argentina; INTA EEA Famaillá, Tucumán, Argentina

Luiz Eduardo Souza da Silva Irineu Campos dos Goytacazes, Rio de Janeiro, Brazil Srinivas International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, Telangana, India

A. Sankaranarayanan C.G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Surat, Gujarat, India

V.

Aqib Sayyed Key Laboratory of Plant Cell Engineering and Germplasm Innovation, Ministry of Education, School of Life Sciences, Shandong University, Jinan, Shandong, China

R. Srinivasan Crop Production Division, ICARIndian Grassland and Fodder Research Institute, Jhansi, Uttar Pradesh, India

Mónica Sebastiana University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute, Lisboa, Portugal M. Senthilkumar Department of Agricultural Microbiology, TamilNadu Agricultural University, Coimbatore, Tamil Nadu, India M. Senthilkumar Division of Basic Sciences, Indian Institute of Pulses Research, Kanpur, Uttar Pradesh, India YingWu Shi Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi, Xinjiang, China; Xinjiang Laboratory of Special Enviromental Microbiology, Urumqi, Xinjiang, China; Key Laboratory of Agriculture Environment in Northwest Oasis of Agriculture, Urumqi, Xinjiang, China Makoto Shoda Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama, Japan Nalini Shrutika Division of Plant Biotechnology, Indian Institute of Pulses Research, Kanpur, Uttar Pradesh, India Daniel Sierra-Cacho Centro de Investigaciones en Ciencias Microbiológicas, Benemérita Universidad Autónoma de Puebla, Ciudad Universitaria, Puebla Pue, México Vanessa Silva dos Santos Departamento de Ciências Biológicas, Universidade Federal do Espírito Santo, Vitória, Espírito Santo, Brazil R.U. Shailendra Singh University of Agricultural Sciences, Bangalore, Karnataka, India; ICAR- Indian Institute of Pulses Research, Kanpur, Uttar Pradesh, India Mahendra Vikram Singh Rajawat ICARNational Bureau of Agriculturally Important Microorganisms (ICAR-NBAIM), Mau, Uttar Pradesh, India

R. Srivastava Crop Production Division, ICARIndian Grassland and Fodder Research Institute, Jhansi, Uttar Pradesh, India T.R. Sunitha University of Agricutural Sciences, GKVK, Bangalore, Karnataka, India N. Thajuddin Department of Microbiology, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India Debajit Thakur Microbial Biotechnology Laboratory, Life Sciences Division, Institute of Advanced Study in Science and Technology, Guwahati, Assam, India; Department of Molecular Biology and Biotechnology, Cotton University, Guwahati, Assam, India Thiago Motta Venancio Campos dos Goytacazes, Rio de Janeiro, Brazil Jean Carlos Vencioneck Dutra Departamento de Ciências Biológicas, Universidade Federal do Espírito Santo, Vitória, Espírito Santo, Brazil Renu Verma Division of Basic Sciences, Indian Institute of Pulses Research, Kanpur, Uttar Pradesh, India Santiago Adolfo Vio Centro de Investigación y Desarrollo en Fermentaciones Industriales (CINDEFI), UNLP, CCT La Plata CONICET, Departamento de Química, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina Hongmei Yang Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi, Xinjiang, China; Xinjiang Laboratory of Special Enviromental Microbiology, Urumqi, Xinjiang, China; Key Laboratory of Agriculture Environment in Northwest Oasis of Agriculture, Urumqi, Xinjiang, China Jun Zeng Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi,

CONTRIBUTORS

Xinjiang, China; Xinjiang Laboratory of Special Enviromental Microbiology, Urumqi, Xinjiang, China; Key Laboratory of Agriculture Environment in Northwest Oasis of Agriculture, Urumqi, Xinjiang, China Mohammad Tarique Zeyad ICAR-National Bureau of Agriculturally Important Microorganisms (ICAR-NBAIM), Mau, Uttar Pradesh, India

xix

Tao Zhang Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi, Xinjiang, China; Xinjiang Laboratory of Special Enviromental Microbiology, Urumqi, Xinjiang, China; Key Laboratory of Agriculture Environment in Northwest Oasis of Agriculture, Urumqi, Xinjiang, China

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Preface agriculture for the management of plant diseases, plant growth promotion, and so forth. Collectively, this book covers a total of 44 bacteria and fungi in single book. In addition, this book will help researchers plan and explore soil microflora for sustainable agricultural production technologies. This book presents the role of microbial genera in agricultural practices and provides recent taxonomical examples for the identification of respective genera. Beneficial Microbes in Agroecology: Bacteria and Fungi will benefit postgraduate students, research scholars, postdoctoral fellows, and teachers in plant microbiology, pathology, entomology, and nematology. The readers will find different microbial genera used in agroecology. We, the editors, welcome healthy criticism and comments from readers.

The continuous use of chemicals above the prescribed limits poses a negative impact on soil, environment, and human health. The indiscriminate use of fertilizers and pesticides not only destroy the beneficial microbes in soil, they also disturb the agroecology. The deterioration of soil productivity leads to the impairment of beneficial microbial diversity and may threaten ecosystem function and environmental quality. The use of conventional, ecofriendly agricultural practices is vital for sustainable agricultural development and food security. Beneficial Microbes in Agroecology: Bacteria and Fungi, describes almost all agriculturally important and beneficial bacteria and fungi used in agricultural production technologies. This book covers a wide range of bacteria and fungi on biocontrol, bioremediation, and plant growthepromoting technologies. Section I describes the beneficial bacteria used in agroecology. This section addresses 26 different bacterial genera and their roles in sustainability, various mechanisms, and beneficial natural processes that enhance soil fertility and plant growth. Section II represents 18 different fungal genera used in

N. Amaresan M. Senthil Kumar K. Annapurna Krishna Kumar A. Sankaranarayanan

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S E C T I O N I

Bacteria

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C H A P T E R

1 Arthrobacter Pratiti Roy, Ashok Kumar Department of Genetics and Plant Breeding (Plant Biotechnology), Rajiv Gandhi South Campus, Banaras Hindu University, Mirzapur, Uttar Pradesh, India

1. Introduction Arthrobacter is a group of pleomorphic bacteria, ubiquitous in origin with effective roles in agriculture. This group of bacteria is difficult for stringent classification as the members provide dynamism in shape due to nutrient availability and resources that they utilize for growth. A general classification can be made based on the 16S rRNA sequence phylogeny data by which Arthrobacter genus has been subdivided into 11 major groups as Arthrobacter aurescens, Arthrobacter globiformis, Arthrobacter pascens, Arthrobacter oryzae, Arthrobacter humicola, Arthrobacter oxydans, Arthrobacter protophormiae, Arthrobacter sulfureus, Arthrobacter citreus, Arthrobacter agilis, Arthrobacter psychrolactophilus, Arthrobacter pigmenti, Arthrobacter albus/cumminsii, and Sinomonas soli (Busse et al., 2012). A chemotaxonomic classification has been done based on the A3a or A4a peptidoglycan types, the former showing a quinone system and with MK9-2(H), whereas the latter bears unsaturated menaquinones (MK-8, MK-9, and MK-10) mostly in the cell walls (Busse et al., 2012; Schleifer and Kandler, 1972). The maximum species of this genus harbors the cellular fatty acids (Schleifer and Kandler, 1972). The bacteria may be rods changing to coccoid in the stationary phase from the growth (log) phase and coryneform (comma shaped) due to many reasons. These bacteria can effectively utilize organic and inorganic compounds as a substrate of metabolism, thus acting as a tool for bioremediation in agriculture. Arthrobacter has been also reported with plant growth-promoting activity, mainly the soil dwellers that form a greater group of rhizobacteria. Although, some groups of Arthrobacter have been isolated from the phyllosphere, as scientists predict them to be transported by rain splashes from soil or through any other means of contact to the phyllosphere (Scheublin and Leveau, 2013). Actinobacteria, members of the genus Arthrobacter, are of massive significance as they are among the most frequently isolated bacteria and are commonly isolated from soils and water contaminated with industrial chemicals and radioactive materials (Dsouza et al., 2015). It has also been reported that up till 2015, there have been complete and published genomes for just six species, namely A. aurescens TC1, Arthrobacter sp. FB24, Arthrobacter chlorophenolicus A6, A. phenanthrenivorans Sphe3, A. arilaitensis re117,

Beneficial Microbes in Agro-Ecology https://doi.org/10.1016/B978-0-12-823414-3.00001-0

3

© 2020 Elsevier Inc. All rights reserved.

4

1. Arthrobacter

and A. nitroguajacolicus Rue61a. The genome sequences of seven Antarctic Arthrobacter isolates have been compared with the available seven temperate isolates (Pagani et al., 2012). Fewer genes could be identified from the polar ones but the “final pan-genome” of Arthrobacter was found to comprise 14,902 sequences, indicating an array of accessory genes with great diversity. Several strategies for adapting in the colder climes was found in these species, reported by the presence of rpoE, fliA, rpoD, sodA, sodC, katE, katG, bcp, osmC, ohr, trx, trxB, trxA, soxR, glgA, glgB, glgC, rtB, crtI, crtEB, idi, crtE, glgP, glgX, otsA, otsB, treS, treY, treZ, proV, proW, proX, csd, capA, nusA, pnp, rbfA, infA, infB, crtYe, crtYf, and desA genes.

2. Taxonomy Conn and Dimmick (1947) had proposed the genus Arthrobacter that included Arthrobacter globiforme, also known as A. globiformis, A. tumescens, and Arthrobacter helvolum on the basis of morphology, cultural characteristics, and physiology of the bacteria. As per the description cited by Conn and Dimmick (1947) or Keddie (1974), 16 more species were reported by 1995: A. aurescens, Arthrobacter crystallopoietes Arthrobacter nicotianae, A. citreus, Arthrobacter histidinolovorans, Arthrobacter mysorens, Arthrobacter nicotinivorans, Arthrobacter polychromogenes, Arthrobacter ramosus, A. protophormiae, Arthrobacter uratoxydans, A. sulfureus, Arthrobacter ureafaciens, A. pascens, Arthrobacter oxydans, and Arthrobacter ilicis (Busse, 2016). A. agilis was included in Arthrobacter genus (Koch et al., 1995). Later, 51 more species were described, as of 2012, and novel species like Arthrobacter cryoconiti (Margesin et al., 2012), Arthrobacter siccitolerans (SantaCruz-Calvo et al., 2013), Arthrobacter gyeryongensis (Hoang et al., 2014), and Arthrobacter cupressi (Zhang et al., 2012) were added.

3. Isolation and identification of Arthrobacter genus Arthrobacter species are isolated from soil and water, from a range of varying climates in the tropics and temperate zones to even harsher places of Antarctica and metalaccumulated, polluted lands. Arthrobacter species has been isolated from the phyllosphere also, and their epiphytotic fitness has been demonstrated in comparison to bacterial epiphyte Pantoea agglomerans (Scheublin and Leveau, 2013). Phyllosphere bacterial isolation is done by collection of leaves from infected plants washed in phosphate-buffered saline and incubated in Brunner mineral media at 25
C with shaking conditions. After 2 weeks of enrichment, serial dilutions are performed, followed by plating in tryptone soya agar and resteaking twice for ensuring a pure culture. 16S rRNA gene amplification is done using suitable primers, and the new sequence is compared to the available Arthrobacter sequences from suitable databases. Unique sequences obtained are submitted as new reports, and older ones are conferred for further analysis. Isolation from soil is elaborate and needs solubilizing soil in sterile saline solution and performing serial dilutions to obtain colonies in liquid or solid media (R2A Agar and several other media are used). Again, 16S rRNA sequencing is done to confirm the bacterial strain, and further phylogenetic analysis is carried out to reveal the relationship among various bacterial species and strains. Basic tests are generally performed like cell morphology test, Gram Test, Catalase Test, Oxidase Test, sodium chloride (NaCl) tolerance, and pH tolerance I. Bacteria

5. Siderophore production and metal solubilization by Arthrobacter

5

tests, and various biochemical tests like hydrolysis of casein, Voges-Proskauer, Indole production test, hydrolysis of gelatine, starch, aesculin, DNA, cellulose, Tween 80, Tween 20, and urea tests, along with incubation in different types of media like MacConkey agar, tryptone soy agar, Luria Bertani agar, marine agar, yeast mold agar, and nutrient agar (Park et al., 2014). Any novel species reported is analyzed thoroughly before submission of data, and the DNA GþC content of the new strain is determined (Mesbah et al., 1989). Reversed phase HPLC analysis is done to analyze the nucleosides following DNA hydrolysis, and DNA-DNA relatedness is confirmed by hybridization experiments (Ezaki et al., 1989). Cell wall analysis, fatty acid profile, amino acid and peptide analysis, isoprenoid quinine analysis, and several other enzyme activity tests are noted (Park et al., 2014). Thus, to conclude, any bacterial isolate, whether from rhizospheric soil or the phyllosphere, is thoroughly evaluated before its role is judged in agricultural spheres.

4. Arthrobacter as a plant growth-promoting rhizobacteria The association of diverse strains of Arthrobacter with different plants reveal the beneficial implications of the latter in plant growth and yield. In plants that are grown in saline, polluted, drought prone, and low-nutritive agricultural soils, Arthrobacter species has played eminent roles in protecting plants from abiotic stresses and has also helped in improving plant health and yield, thus proving itself as a notable member of the rhizosphere microflora (Krishnan et al., 2016). The following Arthrobacter species were able to produce auxin: A. globiformis, A. pascens, A. aurescens, A. tumescens, A. citreus, and A. atrocyaneous. It was experimentally proven by conversion of tryptophan to indole 3-acetic acid. IAA, as we know, is responsible for the growth and development of higher plants, so Arthrobacter may act as a promising Plant Growth Promoting Rhizobacteria (PGPR) in agricultural land (Katznelson and Sirois, 1961). However, A. cupressi, A. bambusae, A. humicola, A. oryza, A. siccitolerans, and A. gyeryongensis, having been isolated from rhizospheric soil, did not show any plant growth-promoting properties, and it also was reported that the conglomeration of Arthrobacter species with various crops growing under stressful conditions and its significance are yet to be delved into in detail in spite of their immense roles toward maintenance of plant health (Krishnan et al., 2016). Scientists have recently discovered Arthrobacter pokkalii sp. nov from pokkali rice (a highly saline-tolerant rice variety) that was able to synthesize IAA (indole 3-acetic acid) by utilizing L-tryptophan and could grow on ACC (1-aminocyclopropane-1carboxylic acid), which proved the ability of the bacteria to utilize ACC under abiotic stresses to save the plant from the adverse effects of ethylene. This strain is also able to synthesize siderophores, form biofilm on the walls of the glass tube, effectively growing in an acid saline environment of pH 5.5 and 8% NaCl and also shows growth in polyethylene glycol (PEG) media, thereby proving its ability to sustain drought conditions. This strain, however, did not show any evidence of nitrogen utilization or for nif H gene amplification.

5. Siderophore production and metal solubilization by Arthrobacter Rare earth elements consist of the 14 lanthanides, including yttrium (Y) besides cerium, gadolinium, neodymium, samarium, ytterbium, holmium, lanthanum, and so on. I. Bacteria

6

1. Arthrobacter

Microorganisms have the ability to biosorp these metals and sequester them, in many cases, in their cell walls via novel pathways with the aid of their genes (Emmanuel et al., 2010; Andras et al., 2000). The microorganisms can chelate the various divalent and trivalent cations as the polycarboxylic acids and aminocarboxylic acids can form stable complexes. Metal siderophores are very specific in action. Specific membrane proteins actively transport the complexes, and Fe release occurs either at the membrane or within the cytoplasm (Urrutia et al., 1992).

6. Role of Arthrobacter in bioremediation The word bioremediation refers to the breaking down of environmental pollutants by a group of microbial consortiums, plants, or a combination of both (Bjerketorp et al., 2018). This chapter evokes the idea of bacterial degradation of toxic compounds from soil and conversion to lesser toxic intermediates to be used up by plants or other microbiota. Instances of bioremediation by the various species and strains of Arthrobacter have been tabulated in Table 1.1. The importance of each bacterial strain or species in an agri-ecosystem has been elucidated by mentioning its major degrading role. This has paved its path for sustainable agriculture and development in a global context. The latest researches have focused on the biodegrading ability of Arthrobacter strains owing to the need of less expensive and technically less challenging means of remediation (Table 1.2). Thus, studies have been conducted with the 4-chlorophenol degrading Arthrobacter cholophenolicus A6 in soil microcosms to check the degrading ability and its effectiveness in the environment (Bjerketorp et al., 2018). The A6 strain is also able to degrade a large proportion of 4-chlorophenol in soil at colder climates, as demonstrated (Bjerketorp et al., 2004). 4-Chlorophenol degradation via hydroxyquinol pathway is a novel path for aerobic degradation by Arthrobacter chlorophenolicus A6 and the group of cph gene cluster was identified showing cphA-I and cphA-II encoding functional hydroxyquinol 1, 2TABLE 1.1

Taxonomic hierarchy of Arthrobacter.

Rank

Name

Reference

Kingdom

Bacteria

Cavalier-Smith (2002)

Subkingdom

Posibacteria

Cavalier-Smith (2002)

Phylum

Actinobacteria

Cavalier-Smith (2002)

Class

Arthrobacteria

Cavalier-Smith (2002)

Subclass

Actinobacteridae

Stackebrandt et al. (1997)

Order

Actinomycetales

Oren (2017)

Suborder

Micrococcineae

Stackebrandt et al. (1997)

Family

Micrococcaceae

Pribram (1929)

Genus

Arthrobacter

Conn and Dimmick (1947), Koch et al. (1995)

I. Bacteria

7

6. Role of Arthrobacter in bioremediation

TABLE 1.2

Role of Arthrobacter strains on bioremediation.

Arthrobacter species

Role

References

Arthrobacter chlorophenolicus A6

Can remove high levels of toxic pollutant 4-chlorophenol from contaminated soil

Bjerketorp et al. (2018)

Arthrobacter sp.

Grow in presence of toxic hexavalent chromium and bioremediation

Megharaj et al. (2003)

Arthrobacter aurescens TC1

Atrazine utilization from industrial wastewater

Li et al. (2008)

Arthrobacter icotianae

Can degrade pentachloronitrobenzene (PCNB), dichlorodiphenyl Wang et al. trichloroethane, hexachlorocyclohexane, cypermethrin, and cyhalothrin (2015)

Arthrobacter crystallopoietes strain BAB-32

Complex hydrocarbon, herbicide, and pesticide degrading ability; iron Joshi et al. uptake and phenyl acetic acid degradation ability as revealed by its (2013) whole genome sequencing

Arthrobacter protophormiae RKJ100

Biodegradation of p-nitrophenol

Labana et al. (2005)

Arthrobacter luteolus

Accumulation of rare earth elements by production of siderophore

Emmanuel et al. (2012)

Arthrobacter nicotianae

Accumulation of higher amounts of Sm than other REEs from a solution containing 6 LREEs

Tsuruta (2006)

Arthrobacter sp.

Atrazine-degradation

Wang and Xie (2012)

Arthrobacter sp. strain B1B

Polychlorinated biphenyl degradation by carvone and salicylic acid

Singer et al. (2000)

Arthrobacter strain DAT1 Atrazine biodegradation

Xie et al. (2013)

Arthrobacter sp. strain MCM B-436

Atrazine degradation

Parag et al. (2007)

Arthrobacter strain SD325

Simazine biodegradation

Guo et al. (2014)

Arthrobacter sp. strain B1B

Polychlorinated biphenyl biodegradation

Gilbert and Crowley (1997)

Arthrobacter sp. WZ2

Chromium (VI) reduction

Xiao et al. (2017)

Arthrobacter sp. W1

Degradation of coking wastewater containing carbazole, dibenzofuran, Shi et al. (2014) dibenzothiophene, and naphthalene

dioxygenases, which are critical in 4-chlorophenol degradation (Nordin et al., 2005). Arthrobacter HB5, a notable atrazine degrading strain of Arthrobacter, isolated from industrial wastewater aids in the conversion of atrazine to hydroxyatrazine and the latter to cyanuric acid (Wang et al., 2015). This is how the atrazine-polluted soil may be cleared from its harmful effects, thus paving the way for the maintenance of soil sustainability. Arthrobacter AD26 is

I. Bacteria

8

1. Arthrobacter

also a potent atrazine degrader and helps in bioremediation of contaminated soils by 98% with the help of its trzN and atzBC genes, operative at a soil with 300 mg/kg levels of atrazine contamination at 26
C for 20 days, thus being a good candidate for bioremediation programs (Li et al., 2008). Among other atrazine degrading strains, Arthrobacter nicotinovorans HIM is able to degrade simazine, terbuthylazine, propazine, cyanazine, and prometryn besides atrazine to cyanuric acid. The atzABC genes have been elucidated in this strain to find out the underlying mechanisms of atrazine degradation by the bacterial isolate. PNP (p-nitrophenol), entailed as a hazardous substance, is utilized by Arthrobacter species strain JS443 (Perry and Zylstra, 2007) and is degraded to 4-nitrocatechol by uninduced cells and to nitrohydroquinone from m-nitrophenol by induced cells (Jain et al., 1994). The Npd family of genes were expressed in E. coli cells to identify the related gene products. NpdA2 was found to be p-nitrophenol monooxygenase and NpdA1 was a reductase,NpdB a hydroxyquinol 1, 2-dioxygenase, and npdC a putative maleylacetate reductase gene (Perry and Zylstra, 2007). Naphthalene, a carcinogenic benzenoid polycyclic aromatic compound, is a priority pollutant that needs to be kept in check in nature. 1- and 2-methylnapthalene degradation by Arthrobacter alpinus R3.8 genes for salicylate-1-monoxygenase, imidazole glycerol phosphate synthase cyclase has been elucidated. 1, 4-dichlorobenzene degradation is aided by enoyl-CA hydratase, aliphatic amidase amiE, nitrilotriacetate monooxygenase, and alkaline phosphatise genes of the same Arthrobacter strain (Too et al., 2017).

7. Future prospects Arthrobacter possesses a range of genes for heavy metal uptake and breaking down of complex organic and inorganic compounds that may damage the environment. Several such potential genes for different metal degradation may be integrated to form a transgene and may be used under a suitable promoter and terminator to construct synthetic bacteria with the potency to degrade different xenobiotics in different environments. Studies on Arthrobacter simplex have demonstrated the biotransformation of steroid drug cortisone acetate (CA) to prednisone acetate. Additional copies of ksdD gene, a gene for 3-ketosteroid-D1-dehydrogenase synthesis under the control of cat promoter, were transferred to the strain A. simplex 156, and a new recombinant strain M158 was formed that showed higher CA biotransforming abilities (Zhang et al., 2013).

8. Conclusion The ability of Arthrobacter to benefit plants under stressful conditions and various other isolates to degrade complex compounds and sequester heavy metals is always a burgeoning branch of research. Several pathways may be addressed to know the detailed mechanism of how the stress-responsive genes perform within the organism and how that may be exploited for human welfare. Agriculture is a sector of utmost importance and offers food security to all the countries of the world; hence, microbiota related to plant growth and environmental

I. Bacteria

References

9

management are indispensable for research. Arthrobacter sp. may be studied in depth for sustainable agriculture for human welfare.

References Andras, Y., Thousand, G., Boualam, M., Mergeay, M., 2000. Factors influencing the biosorption of gadolinium by microorganism and its mobilization from sand. Appl. Microbiol. Biotechnol. 54, 262e267. Bjerketorp, J., Röling, W.F.M., Feng, X., Garcia, A.H., Heipieper, H.J., 2018. Formulation and stabilization of an Arthobacter strain with good storage stability and 4-chlorophenol-degradation activity for bioremediation. Appl. Microbiol. Biotechnol. 102 (4), 2031e2040. Bjerketorp, J., Röling, W.F.M., Feng, X.M., Garcia, A.H., Heipieper, H.J., Håkansson, S., 2004. Impact of temperature on the physiological status of a potential bioremediation inoculant, Arthrobacter chlorophenolicus A6. Appl. Environ. Microbiol. 70 (5), 2952e2958. Busse, H.J., 2016. Review of the taxonomy of the genus Arthrobacter, emendation of the genus Arthrobacter sensu lato, proposal to reclassify selected species of the genus Arthrobacter in the novel genera Glutamicibacter gen. nov., Paeniglutamicibacter gen. nov., Pseudoglutamicibacter gen. nov., Paenarthrobacter gen. nov. and Pseudarthrobacter gen nov., and emended description of Arthrobacter roseus. Int. J. Syst. Evol. Microbiol. 66, 9e37. Busse, H.J., Wieser, M., Buczolits, S., 2012. Genus III Arthrobacter. In: Whitman, W.B., Parte, A., Goodfellow, M., Kamfer, P., Busee, H.J., et al. (Eds.), Bergey’s Manual of Syst Bacteriol, Vol. 5, pp. 578e625. Cavalier-Smith, T., 2002. The phagotrophic origin of eukaryotes and phylogenetic classification of Protozoa. Int. J. Syst. Evol. Microbiol. 52, 297e354. Conn, H.J., Dimmick, I., 1947. Soil bacteria similar in morphology to Mycobacterium and Corynebacterium. J. Bacteriol. 54, 291e303. Dsouza, M., Taylor, M.W., Turner, S.J., Aislabie, J., 2015. Genomic and phenotypic insights into the ecology of Arthrobacter from Antarctic soils. BMC Genom. 16, 36. Emmanuel, E.S., Ananthi, T., Anandkumar, B., Maruthamuthu, S., 2012. Accumulation of rare earth elements by siderophore eforming Arthrobacter luteolus isolated from rare earth environment of Chavara, India. J. Biosci. 37 (1), 25e31. Emmanuel, E.S., Vignesh, V., Anandkumar, B., Maruthamuthu, S., 2010. Bioaccumulation of cerium and neodymium by Bacillus cereus isolated from rare earth environments of Chavara and Manavalakurichi, India. Indian J. Microbiol. 51, 488e495. Ezaki, T., Hashimoto, Y., Yabuuchi, E., 1989. Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int. J. Syst. Bacteriol. 39, 224e229. Gilbert, E.S., Crowley, D.E., 1997. Plant compounds that induce polychlorinated biphenyl biodegradation by Arthrobacter sp. strain B1B. App. Environ. Microbiol. 63, 1933e1938. Guo, Q., Zhang, J., Wan, R., Xie, S., 2014. Impacts of carbon sources on simazine biodegradation by Arthrobacter strain SD3-25 in liquid culture and soil microcosm. Int. Biodeterior. Biodegrad. 89, 1e6. Hoang, V.A., Kim, Y.J., Nguyen, N.L., Yang, D.C., 2014. Arthrobacter gyeryongensis sp. nov., isolated from soil of a Gynostemma pentaphyllum field. Int. J. Syst. Evol. Microbiol. 64 (2), 420e425. Jain, R.K., Dreisbach, J.H., Spain, J.C., 1994. Biodegradation of p-nitrophenol via 1,2,4-benzenetriol by an Arthrobacter sp. Appl. Environ. Microbiol. 60, 3030e3032. Joshi, M.N., Pandit, A.S., Sharma, A., Pandya, R.V., Desai, S.M., Saxena, A.K., Bagatharia, S.B., 2013. Draft genome sequence of Arthrobacter crystallopoietes strain BAB-32, revealing genes for bioremediation. Genome Announc. 1 (4), 1e2. Katznelson, H., Sirois, J.C., 1961. Auxin production by species of Arthrobacter. Nature 19, 1324. Keddie, R.M., 1974. Genus II Arthrobacter. In: Buchanan, R.E., Gibbons Baltimore, N.E. (Eds.), Bergey’s Manual of Determinative Bacteriology, eighth ed. Williams Wilkins, pp. 618e625. Koch, C., Schumann, P., Stackebrandt, E., 1995. Reclassification of Micrococcus agilis (Ali-Cohen 1889) to the genus Arthrobacter as Arthrobacter agilis comb. Nov. and emendation of the genus Arthrobacter. Int. J. Syst. Bacteriol. 45, 837e839.

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1. Arthrobacter

Krishnan, R., Menon, R.R., Tanaka, N., Busse, H.J., Krisnamurthi, S., Rameshkumar, N., 2016. Arthrobacter pokkalii sp nov, a novel plant associated Actinobacterium with plant beneficial properties, isolated from saline tolerant pokkali rice,Kerala,India. PLoS One 11 (3). Labana, S., Singh, O.V., Basu, A., Pandey, G., Jain, R.K., 2005. A microcosm study on bioremediation of p-nitrophenol-contaminated soil using Arthrobacter protophormiae RKJ100. App. Microbiol. Biotechnnol. 68 (3), 417e424. Li, Q., Li, Y., Zhu, X., Cai, B., 2008. Isolation and Characterization of atrazine degrading Arthrobacter sp. AD26 and use of this strain in bioremediation of contaminated soil. J. Environ. Sci. (China) 20 (10), 1226e1230. Margesin, R., Schumann, P., Zhang, D.C., Redzic, M., Zhou, Y.G., Liu, H.C., Schinner, F., 2012. Arthrobacter cryoconiti sp. nov., a psychrophilic bacterium isolated from alpine glacier cryoconite. Int. J. Syst. Evol. Microbiol. 62, 397e402. Megharaj, M., Avudainayagam, S., Naidu, R., 2003. Toxicity of hexavalent chromium and its reduction by bacteria isolated from soil contaminated with tannery waste. Curr. Microbiol. 47 (1), 0051e0054. Mesbah, M., Premachandran, U., Whitman, W.B., 1989. Precise measurement of the GþC content of the deoxyribonucleic acid by high-performance liquid chromatography. Int. J. Syst. Bacteriol. 39, 159e167. Nordin, K., Unell, M., Jansson, J.K., 2005. Novel 4-chlorophenol degradation gene cluster and degradation route via hydroxyquinol in Arthrobacter chlorophenolicus A6. Appl. Environ. Microbiol. 71 (11), 6538e6544. Oren, A., 2017. Proposal to designate the order Actinomycetales Buchanan 1917, 162 (Approved Lists 1980) as the nomenclatural type of the class Actinobacteria. Request for an Opinion. Int. J. Syst. Evol. Microbiol. 67 (9), 10e1099. Pagani, I., Liolios, K., Jansson, J., Chen, I.M., Smirnova, T., Nosrat, B., Markowitz, V.M., Kyrpides, N.C., 2012. The Genomes OnLine Database (GOLD) v.4: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res. 40 (D1), 571e579. Parag, A., Vaishampayan, Kanekar, P.P., Dhakephalkar, P.K., 2007. Isolation and characterization of Arthrobacter sp. strain MCM B-436, an atrazine-degrading bacterium, from rhizospheric soil. Int. Biodeterior. Biodegrad. 60, 273e278. Perry, L.L., Zylstra, G.J., 2007. Cloning of a gene cluster involved in the catabolism ofnp-nitrophenol by Arthrobacter sp. strain JS443 and characterization of the p-nitrophenol monooxygenase. J. Bacteriol. 7563e7572. Pribram, E., 1929. Type genus: Micrococcus cohn 1872. J. Bacteriol. 18, 361e394. SantaCruz-Calvo, L., López, G., Manzanera, M., 2013. Arthrobacter siccitolerans sp. nov., a highly dessication-tolerant, xeroprotectant-producing strain isolated from dry soil. Int. J. Syst. Evol. Microbiol. 63, 4172e4180. Scheublin, T.R., Leveau, J.H.J., 2013. Isolation of Arthrobacter species from the phyllosphere and demonstration of their epiphytic fitness. Microbiol. Open 2 (1), 205e213. Schleifer, K.H., Kandler, O., 1972. Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol. Rev. 36, 407e477. Shi, S., Qu, Y., Ma, F., Zhou, J., 2014. Bioremediation of coking wastewater containing carbazole, dibenzofuran, dibenzothiophene and naphthalene by a naphthalene-cultivated Arthrobacter sp. W1. Bioresour. Technol. 164, 28e33. Singer, A.C., Gilbert, E.S., Luepromchai, E., Crowley, D.E., 2000. Bioremediation of polychlorinated biphenylcontaminated soil using carvone and surfactant-grown bacteria. Appl. Microbiol. Biotechnol. 54 (6), 838e843. Stackebrandt, E., Rainey, F.A., Ward-Rainey, N.L., 1997. Proposal for a new hierarchic classification system, Actinobacteria classis nov. Int. J. Syst. Bacteriol. 47 (2), 479e491. Too, W.S., Ee, R., Lim, Y.L., Convey, P., Pearce, D.A., Mohidin, T.B.M., Yin, W.F., Chan, K.G., 2017. Complete Genome of Arthrobacter alpines strain R3.8, bioremediation potential unraveled with genomic analysis. Stand Genomic Sci. 12, 52. Tsuruta, 2006. Selective accumulation of light or heavy earth elements using gram-positive bacteria. Colloids 52, 117e122. Urrutia, M.M., Kemper, M., Doyle, R., Beveridge, T.J., 1992. The membrane induced proton proton motive force influences the metal binding ability of Bacillus subtilis cell walls. Appl. Environ. Microbiol. 58, 3837e3844. Wang, Q., Xie, S., 2012. Isolation and characterization of a high-efficiency soil atrazine-degrading Arthrobacter sp. Strain. Int. Biodeterior. Biodegrad. 71, 61e66.

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Wang, Y., Wang, C., Li, A., Gao, J., 2015. Biodegradation of pentachloronitrobenzene by Arthrobacter nicotianae DH19. Lett. Appl. Microbiol. 61 (4). Xiao, W., Ye, X., Yang, X., Zhu, Z., Sun, C., Zhang, Q., Xu, P., 2017. Isolation and characterization of chromium (VI)-reducing Bacillus sp. FY1 and Arthrobacter sp. WZ2 and their bioremediation potential. Bioremed. J. 21 (2), 100e108. Xie, S., Wan, R., Wang, Z., Wang, Q., 2013. Atrazine biodegradation by Arthrobacter strain DAT1: effect of glucose supplementation and change of the soil microbial community. Environ. Sci. Pollut. Res. 20 (6), 4078e4084. Park, Y., Kook, M., Ngo, H.T., Kim, K., Park, S.Y., Mavlonov, G.T., Tae-Hoo, Y., 2014. Arthrobacter bambusae sp. Nov., isolated from soil of a bamboo grove. Int. J. Syst. Evol. Microbiol. 64, 3069e3074. Zhang, H., Tian, Y., Wang, J., Li, Y., Wang, H., Mao, S., Liu, X., Wang, C., Bie, S., Lu, F., 2013. Construction of engineered Arthrobacter simplex with improved performance for cortisone acetate transformation. Appl. Microbiol. Biotechnol. 97, 9503e9514. Zhang, J., Ma, Y., Yu, H., 2012. Arthrobacter cupressi sp. nov., an actinomycete isolated from the rhizosphere soil of Cupressus sempervirens. Int. J. Syst. Evol. Microbiol. 62 (11), 2731e2736.

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C H A P T E R

2 Alcaligenes Makoto Shoda Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama, Japan

1. Taxonomy Members of the genus Alcaligenes are common, apparently saprophytic, inhabitants of the intestinal tract of vertebrates. They occur in dairy products, rotting eggs, and other foods and in freshwater, marine, and terrestrial environments in which they are involved in decomposition and mineralization processes. They are not known to enter into either pathogenic or symbiotic associations with plants or animals. They are respiratory and never fermentative. Molecular oxygen is the final electron accepter. Although most of them are strict aerobes, some strains are capable of anaerobic respiration in the presence of nitrate or nitrite, which act as electron accepters. The generic description is similar fundamentally to that of certain other bacteria that enter into special association with plants or animals. Most strains have simple nitrogenous nutritional requirements and produce turbid growth in liquid media with ammonium or nitrate salts as the sole nitrogen source. They do not fix gaseous nitrogen and are not actively proteolytic in casein or gelatin media. Alcaligenes faecalis utilizes as sole carbon and energy source acetate, propionate, butyrate, and other organic acids, as well as aspartic acids, asparagine, histidine, glutathione, and other organic nitrogenous compounds. This strain does not utilize carbohydrates and does not show chemolithotropic growth. Some strains are able to denitrify, by respiring anaerobically in the presence of nitrite or nitrate to produce nitrogen gas. However, they lose this ability on prolonged aerobic cultivation and conduct heterotrophic nitrification and aerobic denitrification as shown in A. faecalis No. 4. In the process of screening effective microorganisms against plant pathogens, a Gramnegative A. faecalis was isolated (Honda et al., 1999). There are few reports on the inhibitory effect of Alcaligenes sp. against plant pathogens and Alcaligenes sp. against Fusarium oxysporum f. sp. dianthi via production of siderophores (Martinetti and Loper, 1992). An iron-chelating substance (Leong, 1986), alcaligin, was reported as a siderophore (Nishio et al., 1988). However, experiments using plants are rare. Here, the newly isolated A. faecalis strain No. 4 (No. 4) was characterized, and its suppression of damping-off caused by Rhizoctonia solani was intensively investigated.

Beneficial Microbes in Agro-Ecology https://doi.org/10.1016/B978-0-12-823414-3.00002-2

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© 2020 Elsevier Inc. All rights reserved.

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2. Alcaligenes

2. Isolation of A. faecalis No. 4 Strain No. 4 was isolated from a sewage sludge. As a plant pathogen, Rhizoctonia solani K-1 was used to determine the suppressive microorganisms in an in vitro test. R. solani K-1 was isolated from cockscomb (Colosiacristata L) at Kanagawa Horticultural Experiment Station, Japan, and fell under the praticola type as a severe damping-off pathogen of many plants (Ushiyama et al., 1987). This test was carried out on MM-agar medium. MM-agar medium consisted of 14 g K2HPO4, 6 g KH2PO4, 2 g (NH4)2SO4, 0.2 g MgSO4$7H2O, 1 g trisodium citrate dehydrate, and 15 g agar (per liter) (pH ¼ 7.0). The cells of the fungal plant pathogen were grown on PDA medium (200 g potato infusion, 20 g glucose, and 15 g agar (per liter)) and suspended in 50 mM Tris-HCl buffer (pH 7.0), and the suspension was passed through a glass wool filter to obtain a spore suspension. This suspension was plated onto the MM-agar medium. Then, about 0.5 g of the sludge was placed onto the center of the plates, and they were incubated at 27 C. Changes of the surface were observed by the naked eye or using an observatory microscope, for 20e30 days. From the outer vicinity of the suppressive sludge placed on the agar plates, a small amount of sludge was picked up by a sterilized platinum string and suspended into the MM medium. The colonies that appeared on the plates were purified.

3. Identification of A. faecalis No. 4 Table 2.1 shows the characteristics of the isolated one strain No. 4. The result of the first check items, shape, Gram stain, spores, mobility, anaerobic growth, oxidase, catalase, and gas from glucose indicated that this bacterium belongs to Alcaligenes sp. The rest of the check confirmed that this strain is A. faecalis subsp. faecalis. This strain was named A. faecalis No. 4.

4. Beneficial properties of A. faecalis No. 4 4.1 In vitro test of A. faecalis No. 4 against plant pathogens Thirteen kinds of plant pathogens were used to determine the suppressive spectrum of A. faecalis No. 4 with an in vitro test. This test was carried out on two media, MM-agar medium and L-agar medium. L-agar medium contained (per liter) 10 g Polypepton (Nippon Pharmaceutical Co., Tokyo), 5 g yeast extract, 5 g NaCl, and 5 g agar (pH ¼ 7.0). The cells of each fungal plant pathogen were grown on PDA medium (200 g potato infusion, 20 g glucose, and 15 g agar (per liter)) and suspended in 50 mM Tris-HCl buffer (pH 7.0), and the suspension was passed through a glass wool filter to obtain a spore suspension. This suspension was plated onto the MM-agar and L-agar media. A sterilized paper disc (8 mm in diameter; 1 mm in thickness) was placed at the center of each plate, and 5 mL of the culture broth of A. faecalis No. 4 grown in MM and L media was allowed to soak into the paper disc. Plates with discs were cultivated at 30 C for 7 days, and the diameter of the inhibitory zone formed around the paper disc was measured. Table 2.2 shows the diameters of the inhibitory zones formed on the two media (Honda et al., 1999). It is clear that A. faecalis No. 4 suppressed the growth of 13 kinds of plant pathogens, either on synthetic MM-agar medium or on complex L-agar medium. I. Bacteria

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4. Beneficial properties of A. faecalis No. 4

TABLE 2.1

Characteristics of strain No. 4.

Shape

Coccal rods

Gram stain



Spores



Mobility

þ

Anaerobic growth

þ

Oxidase

þ

Catalase

þ

Gas from glucose



Arginine dihydrolase



Gelatin decomposition



Sodium citrate

þ

dl-Malic acid

þ

n-Capric acid

þ

Growth in 0.5% phenol



Growth at 42 C

þ

Growth at 7% NaCl

þ

Starch decomposition



Lipase



4.2 Product analysis and denitrification by A. faecalis No. 4 4.2.1 Growth and products of A. faecalis No. 4 To clarify the suppressive mechanism of A. faecalis No. 4, the growth and product analysis of A. faecalis No. 4 were conducted. A. faecalis No. 4 was cultivated in L medium at 30 C on a shaker at 120 strokes per minute, and the growth measurement of A. faecalis No. 4 at an optical density of 660 nm and product analysis were periodically performed. The culture broth of A. faecalis No. 4 was centrifuged at 12,000  g for 10 min, and the supernatant was filtered through a 0.2-mm polytetrafluoroethylene (PTFE) membrane (Advantec, Tokyo). The concentration of hydroxylamine (NH2OH) was determined by a colorimetric method (Frear and Burrell, 1995). The concentrations of nitrite (NO2) and nitrate (NO3) were determined by ion chromatography (HIC-6A, Shimadzu, Kyoto). Fig. 2.1 shows the changes in growth and concentrations of nitrogenous compounds produced by A. faecalis No. 4 in L medium. The detection of hydroxylamine, NO2, and NO3 indicates that A. faecalis No. 4 is a heterotrophic nitrifying bacterium. Similar nitrification production occurred in No. 4 even on the synthetic MM medium, where (NH4)2SO4 is the sole nitrogen source (data not shown).

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2. Alcaligenes

Suppressive effect by culture broth of A. faecalis No. 4 on plant pathogens in vitro test. Diameter of inhibitory zone (mm) MM-agar medium

Sourcea

8

12

1

MAFF305117

7

20

2

Fusarium oxysporum f. sp. radicis-lycopersici

KEF2R-1

6

12

3

Rhizoctonia solani

MAFF305219

11

18

2

Rhizoctonia solani

MAFF305223

15

32

2

Rhizoctonia solani

K-1

14

23

3

Cercospora kikuchii

MAFF305039

13

24

2

Botrytis cinerea

Bot 1

9

24

3

Cochliobolus miyabeanus

MAFF305425

7

22

2

Alternaria mali

IFO 8984

14

23

4

Verticillium dahliae

Klebahn V-3

0

25

3

Phomopsis sp.

4e2

0

16

3

Xanthomonas oryzae

IFO 3998

33

0

4

Plant pathogen

Isolate

Fusarium oxysporum f. sp. lycopersici race J1

SUF119

Fusarium oxysporum f. sp. cucumerinum

L-agar medium

a 1. Faculty of Textile Science and Technology, Shinshu Univ., Nagano, Japan. 2. National Institute of Agrobiological Resources, Ministry of Agriculture, Forestry and Fisheries, Ibaraki, Japan. 3. Kanagawa Institute of Agriculture Science, Kanagawa, Japan. 4. Institute for Fermentation, Osaka, Japan.

FIGURE 2.1 Growth (C,B) and production of hydroxylamine (NH2OH) (-,,), nitrite (NO 2 ) (:), and nitrate

(NO 3 ) (A) when A. faecalis No. 4 and a nonhydroxylamine-producing mutant, No. 4-1 were grown in L medium. Symbols: closed symbols are for No. 4 and open symbols are for No. 4-1.

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4. Beneficial properties of A. faecalis No. 4

4.2.2 Aerobic denitrification by A. faecalis No. 4 As it was found that the denitrification ability of A. faecalis No. 4 from nitrite and nitrate was significantly low (data not shown), the denitrification from ammonium was speculated in detail. A 15N tracer experiment using (15NH4)2SO4 (50% by atomic fraction, Nippon Sanso Co.,Ltd., Japan) was carried out to confirm the production of N2 from ammonium by A. faecalis No. 4 in an aerated batch culture in MM medium at 30 C (Joo et al., 2005). The exhaust gas was directly introduced into the GC/MS (GC 6850, Agilent Technologies Japan, Ltd.). The change in nitrogen isotope ratio was measured, and N2 production by A. faecalis No. 4 was calculated from the difference between output 29N2 and input 29N2 (Joo et al., 2005). Fig. 2.2 shows temporal changes in N2 and N2O concentrations. It was confirmed that A. faecalis No. 4 can convert NHþ 4 -N to N2 gas and that the N2 production ratio among denitrified products was approximately 90%. This result will be used to harvest A. faecalis No. 4 cells after the treatment of high-strength ammonium wastewater, as described in Section 4.6. 4.2.3 In vitro tests of the products

NO, N2O and N2 concentrations (on volume basis ppm)

To prove the suppressive effect in vitro by the nitrogenous compounds produced by A. faecalis No. 4, R. solani K-1 was used. Five-millimeter plugs were taken from a PDA Petri dish culture of R. solani K-1 and placed in the center of a fresh PDA plate. Sterile stainlesssteel cylinders (8 mm in diameter  10 mm in height) were placed on the surface of the plates; then, 100 mL of each of three solutions, hydroxylamine 100 mg/L, nitrite 100 mg/L, and nitrate 100 mg/L, was added to each cylinder, and the formation of inhibitory zones was observed after incubation for 7 days. Fig. 2.3 shows that only hydroxylamine exhibited an inhibitory zone on the PDA plates, suggesting that the suppression of growth of R. solani K-1 and other plant pathogens in the in vitro test (Table 2.2) was caused by hydroxylamine produced by A. faecalis No. 4 during heterotrophic nitrification.

100 N2=90%

75

NO+N2O=10%

50 25

0

0

10

20 30 Time (h)

40

50

FIGURE 2.2 Denitrification characteristics of A. faecalis No. 4 detected using (15NH4)2SO4. Symbols: circles, NO; triangles, N2O; squares, N2.

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2. Alcaligenes

 FIGURE 2.3 Effect of hydroxylamine (NH2OH), nitrite (NO 2 ), and nitrate (NO3 ) solutions on the growth suppression of the plant pathogen R. solani K-1 on PDA medium.

It has previously been reported that A. faecalis is capable of heterotrophic nitrification (Otte et al., 1996; Papen et al., 1989), but neither the detection of hydroxylamine nor its effect on plant pathogens has been reported, either in vitro or in vivo. 4.2.4 Production of a nonhydroxylamine-producing mutant of A. faecalis No. 4 and in vitro tests To confirm the suppressive effect by hydroxylamine, nonhydroxylamine-producing mutants. Were produced and compared with a parent strain on the suppressive effect against a plant pathogen (Honda et al., 1999). A. faecalis No. 4 was subjected to transposon Tn5 mutagenesis according to the method previously reported (Simon et al., 1983; Rella et al., 1985) to produce a nonhydroxylamineproducing mutant of A. faecalis No. 4. The plasmid pSUP2021 carrying Tn5 was kindly provided by Dr. A. Puhler, University of Bielefeld, Germany. This plasmid confers neomycin resistance. Escherichia coli S17-1, the host for pSUP2021, was grown in L medium for 12 h, then mixed with an equal volume of A. faecalis No. 4 that was also grown in L medium. The mixture was incubated for 12 h at 30 C, and then the cell suspension was spread onto L-agar medium containing 100 mg/L neomycin and 100 mg/L streptomycin, and colonies that showed resistance to streptomycin and neomycin were spread onto PDA medium containing R. solani K-1. Colonies that lost their suppressive effect on R. solani K-1 were selected. All the mutants were grown in L medium, and high performance liquid chromatography (HPLC) was used to confirm that the mutants did not produce hydroxylamine. Among the mutants, one mutant, A. faecalis No. 4-1 was used in further experiments. The growth of the mutant A. faecalis No. 4-1 is also shown in Fig. 2.1. The growth of A. faecalis No. 4-1 was similar to that of A. faecalis No. 4, but no hydroxylamine, nitrite, or nitrate were produced. Using a similar procedure to that explained in Fig. 2.3, the inhibitory

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4. Beneficial properties of A. faecalis No. 4

19

FIGURE 2.4 Effect of A. faecalis No. 4 and a nonhydroxylamine-producing mutant, No. 4-1 on the growth suppression of the plant pathogen R. solani K-1 on PDA medium.

zones against R. solani K-1 were compared between the culture of A. faecalis No. 4 and that of A. faecalis No. 4-1, as shown in Fig. 2.4. No inhibitory zone was formed by A. faecalis No. 4-1, which also verifies that hydroxylamine suppresses the growth of R. solani K-1.

4.3 Plant test using A. faecalis No. 4 and nonhydroxylamine-producing mutant, A. faecalis No. 4-1 The plant test using nonsterilized and sterilized soils and R. solani K-1 was performed for A. faecalis No. 4 and A. faecalis No. 4-1 (Honda et al., 1999). The soil used was a low-humic andosol taken from a field at the Kanagawa Horticultural Station, Japan. The soil was sieved through a No. 8 mesh screen and air dried. The main characteristics of the soil thus prepared were as follows: moisture content (%), 12.7; maximum water holding capacity (g/100 g dry soil), 137; pH, 5.9; and bulk density (g/cm3), 0.522. The soil was divided into 150-g portions and placed in plastic pots with a diameter of 90 mm. The soil and vermiculite were mixed in a ratio of 4:1 (w/w), and nutrients were enriched, so the final concentrations of N, P2O5, and K2O were 70 mg, 240 mg, and 70 mg per 100 g dry soil, respectively. The sterilized soil was prepared by autoclaving for 60 min at 121 C four times at 12-h intervals. R. solani K-1 was cultivated in 50 mL of sterile PDP medium in a flask, and the mycelia mats formed on the surface of the medium were homogenized for 2 min and added to the soil 5 days before planting germinated tomato seeds. Preparation of culture broth, cell suspension, and the supernatant was carried out as follows: 50 mL of culture broth of A. faecalis No. 4 were obtained after cultivation in L medium for 24 h at 30 C, and the whole culture broth was mixed with 150 g of soil in a pot. The culture broth was centrifuged at 8000  g for 10 min at 4 C, and the sedimented cells were washed with 0.85% NaCl (pH 7), and the washed cells were suspended in 50 mL sterile distilled water as a cell suspension. This suspension was mixed with 150 g of soil in a pot. The culture broth was centrifuged, and 50 mL of the supernatant was mixed with 150 g of

I. Bacteria

20

2. Alcaligenes

soil in a pot. The treatment of soil with these three culture samples was carried out 3 days prior to planting germinated tomato seeds. Method of plant growth test of A. faecalis No. 4 is as follows. For each treatment, three pots were prepared. Tomato (Lycopersicon esculentum) seeds (cv. Ponderosa, Takii Seeds Co., Kyoto, Japan) were surface disinfected with 70% ethanol and 0.5% sodium hypochlorite and germinated on 2% agar plates at 30 C for 2 days. Nine germinated seeds were planted in each pot and incubated in a growth chamber at 30 C with 90% relative humidity under 16 h light (approximately 12,000 lux). The moisture content of the soil was maintained at 60% of the maximum water-holding capacity. After 2 weeks, the percentage of diseased seedlings per pot was recorded, and the dry weight of the shoots and the leaf length were measured. The results are shown in Table 2.3 (Honda et al., 1999). In pots that were infested with only R. solani, the percentage of diseased plants was 78%e82%, and the shoot weight and leaf length were markedly decreased in both soils. However, when the culture broth was introduced into the sterilized soil, the percentage of diseased plants was reduced to 17%, and the leaf length and shoot weights were significantly higher than those of plants grown in the sterilized soil treated with R. solani alone. When the culture broth was introduced into nonsterilized soil, the percentage of diseased plants was reduced to 46%. One example of effect of A. faecalis No. 4 culture on the suppression of damping-off of tomato caused by R. solani K-1 in sterilized soil is shown in Fig. 2.5.

TABLE 2.3

Effect of different treatments with A. faecalis No. 4 cultures in sterilized and nonsterilized soils on the suppression of damping-off of tomato seedlings caused by R. solani 14 days after planting. Treatment

Soil

R. solani

No .4

Leaf length (mm)

Shoot weight (mg dry/pot)

Diseased plants (%)

Sterilized

þ

e

11.0a

100a

81.6a

Sterilized

þ

Culture broth

70.8c

450c

16.5c

Sterilized

þ

Cell suspension

80.2d

572d

22.0c

Sterilized



e

81.8d

659e

0d

Nonsterilized

þ

e

25.7a

76.5a

77.8a

Nonsterilized

þ

Culture broth

58.1b

208b

46.3b

Nonsterilized

þ

Cell suspension

79.8b

422c

35.2b

Nonsterilized

þ

Supernatant of culture broth

33.0a

152b

74.1a

Nonsterilized



e

122c

560d

0c

For each treatment, each datum is an average of results from three pots (which contain nine seeds per pot) from experiments repeated five times. Means in all columns with different letters are significantly different at P ¼ .05 according to Fisher’s protected least significant difference (LSD) analysis.

I. Bacteria

4. Beneficial properties of A. faecalis No. 4

21

FIGURE 2.5 Effect of A. faecalis No. 4 culture on the suppression of damping-off of tomato caused by R. solani K-1 in sterilized soil. ① control (without R. solani K-1 and A. faecalis No. 4), ② added with R. solani K-1 and A. faecalis No. 4, and ③ added with only R. solani K-1.

When the cell suspension was applied to sterilized or nonsterilized soils, the percentage of diseased plants in each soil type was similar to that of soil treated with A. faecalis No. 4 culture broth. The leaf length and the shoot weight were greater than those from the culture broth treatment for the two soils. As the cell suspension contained only A. faecalis No. 4 cells, the data indicate that A. faecalis No. 4 cells suppressed the infection of R. solani by producing suppressive substances in the two soils. Because A. faecalis No. 4 produced hydroxylamine in liquid medium, as shown in Fig. 2.1, it was attempted to measure the hydroxylamine concentration by extracting the soil, but no hydroxylamine was detected. When the supernatant that contained almost no A. faecalis No. 4 cells was introduced to the two soils, the results were different from the previous cases. In the sterilized soil, a suppressive effect of the supernatant was observed, although the effect was much weaker than that of the culture broth or the cell suspension. In the nonsterilized soil, the percentage of the diseased plants was almost the same as that of the R. solani control treatment. Plant test of nonhydroxylamine-producing mutant, A. faecalis No. 4-1 was conducted in a similar way described before. The nonhydroxylamine-producing mutant, A. faecalis No. 4-1 was grown in L medium, and 50 mL of the culture broth was added to the two soils described earlier. The results of the plant test are given in Table 2.4. The data obtained after A. faecalis No. 4-1treatment were similar to those of the R. solani control treatment, and they were significantly different from the data obtained after A. faecalis No. 4 treatment. This result indicates that A. faecalis No. 4-1does not have the ability to suppress the disease in soil.

4.4 Effect of hydroxylamine solution on plant disease To investigate the effect of hydroxylamine solution on the growth of tomato seedlings and on the suppression of damping-off, hydroxylamine solution (50 mL) of different

I. Bacteria

22 TABLE 2.4

2. Alcaligenes

Effect of A. faecalis No. 4, and its nonhydroxylamine-producing mutant, No. 4-1, on the suppression of damping-off of tomato seedlings caused by R. solani 14 days after planting in sterilized and nonsterilized soil. Treatment

Soil

R. solani

Bacteria

Leaf length (mm)

Shoot weight (mg dry/pot)

Diseased ratio (%)

Sterilized

þ

e

42.6a

230a

65.0a

Sterilized

þ

No. 4

71.0b

394b

33.0b

Sterilized 65.0a

þ

Mutant, No. 4-1

37.0a

162a

Sterilized



e

121c

632c

0c

Nonsterilized

þ

e

72.5a

406a

45.8a

Nonsterilized

þ

No. 4

112b

543b

11.0b

Nonsterilized

þ

Mutant, No. 4-1

72.6a

352a

38.6a

Nonsterilized



e

122b

657b

0b

For each treatment, each datum is an average of results from three pots (which contain nine seeds per pot) from experiments repeated five times. Means in all columns with different letters are significantly different at P ¼ .05 according to Fisher’s protected least significant difference (LSD) analysis.

concentrations was applied to the soil, and plant testing was conducted in a manner similar to that described earlier. Different concentrations of hydroxylamine were added to the sterilized or nonsterilized soils, and the disease occurrence was compared, as shown in Table 2.5. In the sterilized soil containing 10 mg/L hydroxylamine solution, the percentage of diseased plants was 61%, which was less than the 85% obtained for the R. solani control treatment. When 50 mg/L of hydroxylamine solution was added, a further reduction in the percentage of diseased plants was observed, and the shoot weight and leaf length significantly increased. However, when a similar experiment was conducted in nonsterilized soil, no significant hydroxylamine effect was observed. From the results in Tables 2.3 and 2.5, the introduction of hydroxylamine solution in nonsterilized soil was not effective for disease suppression.

4.5 Additional experiment Neither 10 mg/L hydroxylamine solution nor the supernatant of A. faecalis No. 4 were effective in nonsterilized soil, indicating that hydroxylamine is easily decomposed in soil or is partly oxidized to nitrate by soil microorganisms. The results that the culture broth or cell suspension were effective for suppressing the plant pathogen suggest that No. 4 can produce hydroxylamine in soil. To demonstrate this indirectly, 150 g of soil was shaken in 150 mL of sterile water, and the supernatant was obtained by filtration of the soil mixture. Then, the cell suspension was added to the supernatant and cultivated at 30 C for 3 days. The growth of A. faecalis No. 4 and approximately 1 mg/L of hydroxylamine were confirmed, indicating the possibility of production of hydroxylamine by A. faecalis No. 4 in soil.

I. Bacteria

23

4. Beneficial properties of A. faecalis No. 4

TABLE 2.5

Effect of hydroxylamine solution on the suppression of damping-off of tomato seedlings in sterilized and nonsterilized soils caused by R. solani K-1 14 days after planting. Treatment

Soil

R. solani Hydroxylamine Leaf length (mm) Shoot weight (mg dry/pot) Diseased plants (%)

Sterilized

þ

e

15.8a

47.1a

84.7a

Sterilized

þ

0.5 mg/pot*

56.2b

371b

61.1b

Sterilized

þ

2.5 mg/pot**

80.7c

635c

33.2c

Sterilized



e

120d

772d

0d

Nonsterilized þ

e

67.1a

424a

50.0a

Nonsterilized þ

0.5 mg/pot**

8a

485b

42.6a

Nonsterilized þ

1.0 mg/pot***

63.2a

460b

44.4a

Nonsterilized 

e

119b

638c

0b

For each treatment, each datum is an average of results from three pots (which contain nine seeds per pot) from experiments repeated five times. Means in all columns with different letters are significantly different at P ¼ .05 according to Fisher’s protected least significant difference (LSD) analysis. * 50 mL of 10 mg/L hydroxylamine solution was added. ** 50 mL of 50 mg/L hydroxylamine solution was added. *** 50 mL of 20 mg/L hydroxylamine solution was added.

The rhizosphere where the plant roots are growing under nutrient-rich conditions may provide the appropriate nutritional conditions for A. faecalis No. 4 to produce hydroxylamine. Hydroxylamine was reported to show adverse effects on the plant cells of Vicia faba (Cohn, 1964) at a concentration of 330 mg/L. In our experiment, the production of hydroxylamine was approximately 10 mg/L in liquid medium and 1 mg/L in soil-extract solution. These levels were much lower than those required to observe adverse effects. Furthermore, hydroxylamine was not detected in soil, mainly due to its rapid degradation in soil. Therefore, the persistence of hydroxylamine in soil and its adverse effect on the plant cells will not need to be taken into account for application of A. faecalis No. 4 to soil. The finding that the effect of the cell suspension on disease suppression is comparable to that of the culture broth suggests that harvesting of A. faecalis No. 4 cells is the key to utilizing No. 4 in agricultural fields. The next section will show the possibility of harvesting massive numbers of A. faecalis No. 4 cells after wastewater treatment.

4.6 Harvesting of massive numbers of A. faecalis No. 4 from wastewater treatment Livestock waste, municipal garbage, excess sludge in wastewater treatment, and waste from the food industry are used for the digestion materials in anaerobic digestion to produce methane as nonfossil energy. This digestion inevitably leads to the production of wastewater containing a high concentration of ammonium (1000e2000 mg-N/L). It is extremely difficult to treat such high concentrations of ammonium by a conventional nitrification-denitrification

I. Bacteria

24

2. Alcaligenes

method. Therefore, the development of more effective methods of wastewater treatment than the conventional method is a crucial factor enabling the practical production of methane. Thus, A. faecalis No. 4 was utilized to remove high-strength ammonium from digested sludge generated in a municipal anaerobic digestion plant to assess the possibility of efficient biologic treatment of the wastewater and to harvest massive numbers of A. faecalis No. 4 cells. The treatment was carried out in a laboratory-scale jar fermenter (total volume 1 L, working volume 300 mL). Dissolved oxygen (DO) concentrations and pH values were monitored with a DO sensor and a pH sensor inserted into the fermenter. The temperature was controlled at 30 C. The digested sludge was supplied by Yokohama Municipal Sewage Center (Yokohama, Japan) where the excess municipal dehydrated activated sludge was digested at 37 C in an 8000-ton-scale anaerobic digester. The main characteristics of the digested sludge were as follows: pH 7.3, 24 mg/L volatile fatty acids, 1200 mg/L ammonium nitrogen, and 1000 mg/L total biochemical oxygen demand. Repeated batch operation was conducted. Fifty milliliters of the preculture of No. 4 using MM medium, 250 mL of the digested sludge, and 20 g of trisodium citrate dihydrate as additional carbon source were mixed in the fermenter, and the treatment of the ammonium was conducted. The concentration of ammonium was determined using an ammonium sensor. After the ammonium concentration was confirmed to be reduced by more than 90% of the initial concentration, 250 mL of the culture was removed from the jar fermenter, and 50 mL of the culture was used for the subsequence treatment by adding a fresh 250 mL of digested sludge and 20 g of trisodium citrate dihydrate. The cell number of No. 4 was determined at the start and at the end of each cycle of batch cultivation. To determine the number of A. faecalis No. 4 cells, the sampled culture was diluted and plated on MM-agar medium plates, and then the plates were incubated at 30 C for 2 days. The colonies that appeared on the plates after 2 days were counted as No. 4 cells, and the cell concentration was expressed as cells/mL. Fig. 2.6 shows the change in ammonium concentration over time in a repeated batch at 30 C, and Fig. 2.7 shows the change in the number of A. faecalis No. 4 cells during the same experiment (Shoda and Ishikawa, 2014). More than 90% of ammonium was removed within 10e20 h, and the number of A. faecalis No. 4 cells

FIGURE 2.6 Change in ammonium concentrations of the digested sludge in repeated batch treatment by

A. faecalis No. 4.

I. Bacteria

References

FIGURE 2.7

25

Change in the cell number of A. faecalis No. 4 in the same experiment as shown in Fig. 2 6.

varied between 108 and 109 cells/mL. The average ammonium removal rate during the experimental period was 2.9 kg-N/m3/day. This value was approximately 100 times higher than that in the conventional nitrification-denitrification processes. In this process, the A. faecalis No. 4 cells grown after ammonium treatment were harvested as excess sludge that can be reutilized in agricultural fields as a potential biocontrol agent because 1 g of the initial cell mass increased to 160 g after 22 days of operation. Because A. faecalis No. 4 was shown to be effective for removing high concentrations of ammonium from different wastewaters (Shoda and Ishikawa, 2014, 2015, 2016a,b, 2017), massive supplies of A. faecalis No. 4 cells are possible from various areas.

5. Conclusion A. faecalis No. 4 (No. 4) showed a broad suppressive spectrum against 13 kinds of plant pathogens in vitro. The culture broth and cell suspension of A. faecalis No. 4 exhibited a significantly suppressive effect on the damping-off of tomato caused by R. solani. The suppressive factor was determined to be hydroxylamine, which is an intermediate produced by the nitrification process of A. faecalis No. 4. Hydroxylamine may be produced by A. faecalis No. 4 in the rhizosphere of roots in soil. Because massive numbers of A. faecalis No. 4 cells are required to apply A. faecalis No. 4 as a biocontrol agent in agricultural fields, the excess A. faecalis No. 4 sludge obtained from wastewater treatment plants, where high concentrations of ammonium are denitrified as nitrogen gas, can be reused for this purpose.

References Cohn, N.S., 1964. The effect of hydroxylamine on the rejoining of X-ray-induced chromatid breaks in Vicia faba. Mutat. Res. 1, 409e413. Frear, D.S., Burrell, R.C., 1995. Spectrophotometric method for determining hydroxylamine reductase activity in higher plants. Anal. Chem. 27, 1664e1665.

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26

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Honda, N., Hirai, M., Ano, T., Shoda, M., 1999. Control of tomato damping-off caused by Rhizoctonia solani by the heterotrophic nitrifier Alcaligenes faecalis and its product, hydroxylamine. Ann. Phytopathol. Soc. Jpn. 65, 153e162. Joo, H.S., Hirai, M., Shoda, M., 2005. Characteristics of ammonium removal by heterotrophic nitrification-aerobic denitrification by Alcaligenes faecalis No.4. J. Biosci. Bioeng. 100, 184e191. Leong, J., 1986. Siderophores: their biochemistry and possible role in the biocontrol of plant pathogen. Annu. Rev. Phytopathol. 24, 187e209. Martinetti, G., Loper, J.E., 1992. Mutational analysis of genes determining antagonism of Alcaligenes sp.strain MFAl against the phytopathogenic fungus Fusarium oxysporum. Can. J. Microbiol. 38, 241e247. Nishio, T., Tanaka, N., Hiratake, J., Katsube, Y., Ishida, Y., Oda, J., 1988. Isolation and structure of the novel dihydroxamate siderophore Alcaligin. J. Am. Chem. Soc. 110, 8733e8734. Otte, S., Grobben, N.G., Robertson, L.A., Jetten, M.S.M., Kuenen, J.G., 1996. Nitrous oxide production by Alcaligenes faecalis under transient and dynamic aerobic and anaerobic conditions. Appl. Environ. Microbiol. 62, 2421e2426. Papen, H., von Berg, R., Hinkel, I., Thone, B., Rennenberg, H., 1989. Heterotrophic nitrification by Alcaligenes faecalis:NO-2, NO-3, N2O and NO production in exponentially growing cultures. Appl. Environ. Microbiol. 55, 2068e2072. Rella, M., Mercenier, A., Hass, D., 1985. Transposon insertion mutagenesis of Pseudomonas aeroginosa with a Tn5 derivative: application to physical mapping of the arc gene cluster. Gene 33, 293e303. Shoda, M., Ishikawa, Y., 2014. Heterotrophic nitrification and aerobic denitrification of high-strength ammonium in anaerobically digested sludge by Alcaligenes faecalis No.4. J. Biosci. Bioeng. 117, 737e741. Shoda, M., Ishikawa, Y., 2015. Heterotrophic nitrification and aerobic denitrificationof a wastewater from a chemical company by Alcaligenes faecalis No.4. Int. J. Water Wastewater Treat 1 (2). https://doi.org/10.16966/23815299.111. Shoda, M., Ishikawa, Y., 2016a. Removal of high-strength of ammonium and phenol from coking wastewater by Alcaligenes faecalis No.4. J. Appl. Biotechnol. Bioeng. 1 (3). https://doi.org/10.15406/jabb.2016.01.00014, 00014. Shoda, M., Ishikawa, Y., 2016b. Utilization of organic acid solution prepared for high-strength ammonium treatment using Alcaligenes faecalis No.4. Int. J. Water Wastewater Treat 2 (5). https://doi.org/10.16966/2381-5299.131. Shoda, M., Ishikawa, Y., 2017. Simultaneous removal of high-strength ammonium and phosphorus by Alcaligenes faecalis No.4. Int. J. Water Wastewater Treat 3 (3), 47. https://doi.org/10.16966/2381-5299.147. Simon, R., Priefer, U., Puhler, A., 1983. A broad host range mobilization system for in vitro genetic engineering: transposon mutagenesis in gram-negative bacteria. Bio. Technol. 1, 784e791. Ushiyama, K., Nishimura, J., Aono, N., 1987. Damping-off of feather cockscomb (Celosia argentea L.Var. cristata O. Kuntze) caused by Rhizoctonia solani Kuhn. Bull. Kanagawa Hortic. Exp. Stn. 34, 33e37.

I. Bacteria

C H A P T E R

3 Serratia Soma Barman1, Satya Sundar Bhattacharya1, Narayan Chandra Mandal2 Soil and Agro-Bioengineering Laboratory, Department of Environmental Science, Tezpur University, Tezpur, Assam, India; 2Mycology and Plant Pathology Laboratory, Department of Botany, Visva-Bharati, Santiniketan, West Bengal, India 1

1. Introduction Plants are colonized by a number of (micro) organisms that can reach cell densities greater than the number of plant cells. To date, the interaction between plants and microbes has been studied in depth for diverse symbiotic rhizobia and mycorrhizal fungi. The rhizosphere, that is, the narrow region surrounding plant roots, is influenced by the activities of several microorganisms and is considered one of the most composite ecosystems on Earth (Pierret et al., 2007; Jones and Hinsinger, 2008; Raaijmakers et al., 2009). A widespread research in the previous few decades has emphasized the role of both Gram-positive as well as Gram-negative bacterial strains as promising biological control agents (Kumar et al., 2005; Ge et al., 2007; HoÈfte and Altier, 2010; Selim et al., 2010). Rhizospheric bacteria can overall plant growth and development (Philippot et al., 2013; Latz et al., 2015). Bacteria-induced plant growth promotion is accomplished by several plant growth-promoting traits, viz., nitrogen fixation, solubilization of minerals, production of hormones, siderophores, and several antimicrobial substances to combat pathogenic attack (Kloepper et al., 1993). Cook et al. (1995) suggested that plants may transform the microbiome of rhizosphere to their own benefit by selectively stimulating microorganisms with PGP attributes that are beneficial to plant growth and health. Serratia sp. is a Gram-negative bacterium belonging to the family Enterobacteriaceae. It has been widely known as an insect pathogen (Flyg and Xanthopoulos, 1983; Bahar and Demirbag, 2007) and as a food-spoilage microorganism (Abdour, 2003). Different species of Serratia were isolated from soil, water, air, and are considered opportunistic pathogens, viz., Serratia marcescens, S. plymuthica, S. liquefaciens, S. fonticola, S. rubidaea, S. Serratia odorifera, S. marnorubra, and S. proteamaculans. They secrete several virulence factors like

Beneficial Microbes in Agro-Ecology https://doi.org/10.1016/B978-0-12-823414-3.00003-4

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© 2020 Elsevier Inc. All rights reserved.

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3. Serratia

chitinase, protease, DNase, lipase, hemolysin, gelatinase, chloroperoxidase, and a red pigment prodigiosin (Kuehnert and Basavaraju, 2015). Apart from the harmful activities, it has also been reported to stimulate plant growth by inducing resistance against plant pathogens (Kloepper et al., 1993), production of antimicrobial substances (de Queiroz and de Melo, 2006), and solubilization of insoluble phosphates (Tripura et al., 2007). Phytosphere isolates of Serratia belong to the “Serratia liquefacienseproteamaculansegrimesii’” complex (Nakamura, 1981). It also called S. liquefaciens-like species (Grimont et al., 1982). Several strains of these species secrete plant-growth promoting substances, antifungal metabolites, and promote the establishment of nitrogen-fixing symbionts. Plant-associated Serratia consist of both endophytic and free-living species in the rhizosphere. The production of indole-3acetic acid (IAA) by S. plymuthica AS12 and S. plymuthica AS13 may be involved in plant growth promotion. Similarly, S. plymuthica HRO-C48 has been used as a successful biocontrol agent against soil-borne fungal diseases in strawberries and rapeseed (Muller et al., 2009). S. marcescens strain 90e166 was used as potential biocontrol agent against Rhizoctonia solani on cotton. It elicits induced-systemic resistance against diverse plant pathogens, such as Colletotrichum orbiculare, Erwinia tracheiphila, Fusarium oxysporum, cucumber mosaic virus, and Pseudomonas syringae pv. lachrymans (Khan et al., 2017).

2. Taxonomy of the genus Serratia The taxonomy of the genus Serratia is still in confusion. More than 42 different species have been associated with the genus Serratia. According to Bergey’s Manual (from the first edition to the seventh editions), the number of different species of Serratia dropped from 23 to 5. These five species were S. marcescens, S. plymuthicum, S. kilensis (sic), S. indica, and S. piscatorum. Following the early work of Hefferan (1903), several taxonomists like Ewing et al. (1959a,b), Martinec and Kocur (1960, 1961a,b,c,d), and Ewing et al. (1962) have tried to simplify the taxonomy of red-pigmented microorganisms. The greatest simplification was achieved by Ewing et al. (1959a,b), Ewing et al. (1962), and Martinec and Kocur (1960, 1961a,b,c,d). After simplification, Serratia became a monospecific genus. Another species, i.e., S. marcescens var. kiliensis, was retained only because of its negative result for Voges-Proskauer test. However, S. marcescens was the only species accepted in the eighth edition of Bergey’s Manual of Determinative Bacteriology (Buchanan and Gibbons, 1974). Apart from the monospecific nature of the genus, there were some evidence of other species of Serratia that produce red pigment (Grimont, 1969; Bascomb et al., 1971; Grimont and Dulong de Rosnay, 1972). Ewing et al. (1972, 1973) documented two species of redpigmented Serratia, S. marcescens and S. rubidaea. Serratia and Enterobacter ziquefaciens were closely related, as they were sensitive to the same bacteriocins (Hamon et al., 1970). Bascomb et al. (1971) separated Enterobacter liquefaciens to the genus Serratia. Molecular techniques may give some evidences on the phylogenetic relationships among enteric bacteria and may help to stipulate a particular position for the genus Serratia at a suprageneric level. The mol% GþC content of DNAs of Serratia is usually given as 54%e60% (Colwell and Mandel, 1965; Hill, 1966). Mandel and Rownd (1964) considered mol% GþC content of Serratia strains too heterogeneous and proposed three provisional

I. Bacteria

3. Isolation of the genus

29

species: Serratia I (58.0e58.4), Serratia II (56.4e57), and Serratia III (53.4e55.4). Serratia III DNA was estimated to differ from Serratia I DNA by an extra amount of 200,000 A-T nucleotide pairs. Serratia I corresponds to S. marcescens and identified all strains of Serratia III as S. plymuthica (Grimont et al., 1977). Serratia II represents one strain of S. marinorubra and some other strains of S. marcescens. The DNA of S. marcescens has the maximum GþC content among the enteric bacteria (Mandel and Rownd, 1964). The genome size of only one strain of S. marcescens has been determined (Gillis et al., 1970) as 3.57  109 Da. The criterion of DNA relatedness clearly explains that the genus Serratia is different from all known genera of the family Enterobacteriaceae (Steigerwalt et al., 1976).

3. Isolation of the genus Serratia appears to be a ubiquitous genus in nature. Overall, 10 species are currently recognized (Grimont and Grimont, 1992). It has been isolated from soil, water, animals (including man), as well as from plant surfaces (Grimont and Grimont, 1992). Typical phytospheric isolates belong to the “Serratia liquefaciens, S. grimesii” and S. proteamaculans complex (Grimont et al., 1977), also called S. liquefaciens-like species (Grimont et al., 1982). For isolation of the bacteria, samples were collected from the site. In the case of soil isolates, the roots along with adherent soil part were collected from healthy plants aseptically in sterilized containers and transported to the laboratory within an hour. Isolation of bacteria was done following standard protocols (Purkayastha et al., 2010; Saha et al., 2012) in nutrient agar (NA)/Luriabertaini (LA) medium. After 72 h incubation at 30 C in inverted position, single colonies were picked up from spread plates and pure cultures were obtained on NA slants. The strains were subsequently maintained in NA/LA slants with periodic transfers to fresh medium. For selective isolation of the genus Serratia, Serratia-selective medium (Ashelford et al., 2000) were used apart from NA/LA media (Figs. 3.1 and 3.2).

FIGURE 3.1

Scanning electron micrograph of Serratia marcessens.

I. Bacteria

30

3. Serratia

FIGURE 3.2 A schematic representation of the isolation, characterization, and plant growth-promoting traits of Serratia.

4. Characterization and identification of the genus After isolation, the strains were characterized by different morphologic as well as biochemical methods. Then the strains were identified according to Bergey’s Manual of Determinative Bacteriology (Holt et al., 1994). The techniques for the various biochemical tests performed followed the Benson’s Microbiological Applications, Laboratory Manual in General Microbiology (Brown and Benson, 2007). Serratia can be differentiated from the other bacterial genera by its production of three special enzymes: DNase, gelatinase, and lipase. It grows well on ordinary microbiologic media under both anaerobic and aerobic conditions. Moreover, it grows well on different synthetic media by modifying the carbon source. It grows well at pH 9 and temperatures from 20 to 37 C (Giri et al., 2004). Another remarkable characteristic trait of Serratia is the production of cell-associated red color pigment, prodigiosin. The production of the pigment, prodigiosin, is highly inconsistent among different species and is dependent on different strains as well as the time of incubation. Prodigiosin were associated with extracellular vesicles or present in intracellular granules (Matsuyama et al., 1986; Kobayashi and Ichikawa, 1991). Nonpigmented strains played a significant role in causing infections (Carbonell et al., 2000). Someya et al. (2001) observed a synergistic interaction of prodigiosin and chitinolytic enzymes against spore germination of Botrytis cinerea. Nowadays, many types of differential and selective growth media have been developed for the particular isolation and probable testing of Serratia. For example, caprylate thallous agar was used as a selective media for Serratia. The medium contains caprylate as a carbon source and thallous salts as inhibitors for other microbes (Starr et al., 1976). Apart from that, the regular liquid media currently being used for prodigiosin biosynthesis are nutrient broth (Pryce and Terry, 2000), peptone

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glycerol broth (Montaner et al., 2000), etc. According to Nakamura, the particular economically cheap media patented by him contains 2% sodium oleate and has used only triolein as substrate. Another media contains sesame seed powder in water. The media supplemented with sesame seed was found to be better in terms of growth and prodigiosin biosynthesis. Moreover, other readily available cheaper sources are used in media like peanut and coconut (Giri et al., 2004). Sesame seed triggered the cell density in the medium, which resulted in a higher accumulation of the intracellular positive pigment regulator by triggering excessive pigment production. The powdered peanut seed medium supported the biosynthesis of prodigiosin even at 37 C. Pigment production was not detected in nutrient or peptone glycerol broth in presence or absence of sugars (Giri et al., 2004).

5. Plant growth-promoting attributes of Serratia spp. 5.1 Biological control potential Various rhizospheric bacteria, including B subtilis, Stenotrophomonas maltophilia, Serratia plymuthica, Pseudomonas fluorescens, P. trivialis, and Burkholderia cepacia produced volatile organic compounds that inhibit mycelial growth of different fungal plant pathogens. Moreover, quorum-sensing molecules from rhizobacteria can aggravate a range of intrinsic plant responses, including the activation of different defense-related genes (Mendes et al., 2013). Over the last 20 years, there have been remarkable increases in the use of Serratia as biocontrol agent of plant diseases and therefore used as a biofertilizer (Barman et al., 2019). It was reported that the pigment of Serratia takes part in biocontrol activity. The biosynthesis of prodigiosin was related to pig operon of S. marcescens ATCC274 in the SMU genome composed of 14 genes (Harris et al., 2004). Prodigiosin has been studied to suppress growth of various bacteria, fungi, protozoans, and also in viruses (Matteoli et al., 2018). Some isolates of S. marcescens, S. plymuthica, S. liquefaciens, and S. entomophila have been used against different plant pathogens. Among them, S. plymuthica seems to be the most promising biocontrol agent (Czajkowski et al. 2012a,b). Some biocontrol strains of S. plymuthica were isolated both from the rhizosphere as well as from the internal tissues of many crop plants (Benhamou et al., 2000; Czajkowski et al. 2012a,b). S. plymuthica A30 was helpful in controlling blackleg and soft rot disease in Solanum tuberosum caused by Dickeya sp. IPO3012. Blackleg incidence was reduced from 55% in the control Dickeya sp.-inoculated treatment to 0%. The disease incidence was reduced by 100% and the prevalence of stem colonization was cut down by 97% (Czajkowski et al. 2012a,b) when seed tubers were vacuum coinfiltrated with Dickeya spp. and high concentration of S. plymuthica A30 and then planted in the compost. Some strains of S. plymuthica were reported to induce systemic resistance in several crops: S. plymuthica R1GC4 stimulated defense mechanisms against fungal pathogens in Cucumis sativus (Benhamou et al., 2000), and S. plymuthica IC270 induced defenses of Oryza sativa against the blast pathogen Magnaporthe oryzae (De Vleesschauwer et al., 2009). S. plymuthica IC1270, isolated from the rhizospheric soil of Vitis vinifera, controlled the pathogenic attack of Rhizoctonia solani on bean and cotton and Pythium aphanidermatum in cucumber (De Vleesschauwer et al., 2009). S. plymuthica 3Re4-18 was used for preservation of potato sprouts against the effect of R. solani. S. plymuthica HRO-C48 was effective against R. solani, Sclerotinia

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sclerotiorum, and Verticillium dahliae (Muller et al., 2009). Some strains of S. plymuthica effectively controlled air-borne fungal plant pathogens; S. plymuthica G15 showed antifungal activity against Botrytis cinerea and S. plymuthica strains IC14 and IC1270 against Penicillium italicum and P. digitatum (Muller et al., 2009). Apart from that, some strains of S. plymuthica was found to control bacterial plant pathogens. S. plymuthica IC1270 successfully inhibited crown gall of Lycopersicum esculentum caused by Agrobacterium tumefaciens and A vitis (Muèller and Berg, 2008).

6. Phytoremediation Nowadays, microbe-mediated phytoremediation has emerged as a more successful approach for the remediation of heavy metalecontaminated soils. Many Serratia sp. exhibit tolerance to heavy metals and have phytoremedial consequences on host plants (Khan et al., 2015; Luo et al., 2011; Wan et al., 2012). An endophytic strain of Serratia nematodiphila LRE07 has been isolated from Solanum nigrum (Luo et al., 2011). S. marcescens RSC-14 was isolated from the same plant rhizosphere as the hyperaccumulator of Cd as it harbors Cd-tolerant genes. The Cd and other heavy metaleresistant genes in the genome of RSC14 might be engaged in Cd uptake, accumulation, and detoxification within the cell. Low-level resistance to heavy metals is accomplished by binding the metal ions in the inactive form nonspecifically to the cell wall to prevent toxic effects in the cell. Several antioxidant enzymes, viz., catalase, superoxide dismutase, and glutathione peroxidase, and reduced glutathione produced by the strain may allow the plants to tolerate detoxification and alleviate oxidative stress caused by intracellular Cd accumulation (Khan et al., 2015, 2017). Serratia sp. SY5 increased the biomass of root under Cd-contaminated environments. So the strain SY5 could be used as an effective inoculant for phytoremediation in toxic heavy metalepolluted soil (So-Yeon and Cho, 2009).

6.1 Other plant growth-promoting activities Apart from the biologic control potential and phytoremediation, several other PGP activities were reported by several workers. The other PGP activities include P- and Zn solubilization, secretion of lytic enzymes, siderophore production, IAA production, etc. Some of the PGP activities by different species of Serratia are now described. The expression and secretion of lytic enzymes like chitinase, cellulase, protease, and DNase produced by soil bacteria also results in direct suppression of pathogenic activities by hydrolyzing several polymeric compounds (Kumar et al., 2005; Someya et al., 2001; Muller et al., 2009). Chitinase from soil microorganisms and some plants takes part in a defense mechanism (Liu et al., 2003). It was very proficient in depolymerization of chitin because of its ability to produce different chitinolytic enzymes (Brurberg et al., 1995). Although, chitinase production and its activity depends on several physical and growth conditions, viz., culture, pH, temperature, etc. Furthermore, bacterial siderophores play a major role in plant disease suppression by iron sequestration since fungal siderophores have lower affinity toward iron available in soil (Compant et al., 2005; Berg, 2009). S. marcescens strain ETR17 isolated from tea rhizosphere was found to produce lytic enzymes, HCN, siderophores, and IAA (Dhar

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Purkayastha et al., 2018). The strain also produced some amount of IAA, thereby promoting plant growth. Serratia is one of the most potent producers of chitinases, whose production, gene regulation, and activity were well characterized (Gutierrez-Roman et al., 2014). Purified chitinases from S. proteamaculans (Mehmood et al., 2009), S. plymuthica (Frankowski et al., 2001), and S. marcescens (Wang et al., 2013) exhibited antifungal activity against different pathogenic fungi. S. marcescens SR1 produced chitinase and thus is used as a biocontrol agent. The one ml cell free supernatant of the strain was effective against F. oxysporum, Sclerotium rolfsii, Rhizoctonia solanii, and Alternaria alternata up to 60.0%e73.3% (Parani et al., 2011). Nitrogen phosphate (P) is considered one of the major macroelements for growth and development of crop plants. But most of the soil P is unavailable to the plants because it is complexed with several cations like Ca, Mg, Fe, and Al soil for its low solubility (Ghosh et al., 2019). P is mostly present in poorly soluble mineral phosphates that are not readily available for plant uptake (Ghosh et al., 2016). Soil microbes played a key role in phosphate solubilization. Microbial conversion of insoluble mineral P forms into soluble ionic phosphate (H2PO 4 ) is a key mechanism of release of the bound P (Alori et al., 2017). Moreover, formation of biofilm on the P granules plays a major role in P solubilization processes (Ghosh et al., 2019). S. marcescens UENF-22GI (SMU) was able to solubilize inorganic P and Zn. The strain also forms biofilms and thereby helps in P and Zn solubilization processes. Biofilm-forming capability of the strain inhibits two strains of phytopathogenic Fusarium species (Matteoli et al., 2018).

Acknowledgment Authors were thankful to SERB- National Post-Doctoral Fellowship (File Number: PDF/2017/002639) for financial assistance.

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4 Rhizobium Renu Verma1, Harika Annapragada1, Nalini Katiyar1, Nalini Shrutika2, Krishnasis Das1, Senthilkumar Murugesan1 Division of Basic Sciences, Indian Institute of Pulses Research, Kanpur, Uttar Pradesh, India; Division of Plant Biotechnology, Indian Institute of Pulses Research, Kanpur, Uttar Pradesh, India

1 2

1. Introduction Soil contains different bacterial genera as vital components. These bacteria carry out various biotic activities in soil and make the ecosystem dynamic for nutrient turnover and sustainable for crop production. They mobilize plant nutrients in the soil, stimulate plant growth by producing phytohormones, and control phytopathogens with the help of various secondary metabolites. The rhizosphere consists of plant growth-promoting (PGP) rhizobacteria that are heterogeneous in nature. Depending on the closeness with the roots, rhizobacteria are categorized as (i) bacteria present in soil close to the roots (rhizosphere), (ii) bacteria present at the root surface (rhizoplane), (iii) bacteria inhabiting inside the root tissue (endophytes, inhabiting spaces between cortical cells), and (iv) bacteria residing within cells of specific structures called root nodules. Rhizobia are Gram-negative bacteria that can form root nodules in leguminous plants and establish a symbiosis with a host to reduce atmospheric nitrogen into ammonium. The use of chemical fertilizers in agriculture is extensively reduced with the nitrogen fixation ability of rhizobia. Moreover legumeerhizobium symbiosis has many advantages such as enhanced crop productivity and soil fertility maintenance and restoration, so it has become the key component for ecologic and economic function in agricultural soil. In addition to its essential role in symbiotic nitrogen fixation (SNF), rhizobia also play a significant role in PGP activities such as secretion of growth hormones like indole acetic acid (IAA), which shows a positive response on plant growth, and also perform a chief function in the root nodule formation and development. Potential Rhizobium species with P solubilization trait have double advantageous for plants and reduce the cost of fertilizer and potentially improve the yield of plants through the release of phosphate from inorganic and organic pools of total soil P. Rhizobia also possess several other PGP traits such as phosphate solubilization and utilization of plant ethylene precursor molecule, i.e., 1-aminocyclopropane-1-carboxylate (ACC) to protect plants from ethylene stress. The breakdown of ACC to ammonia and a-ketobutyrate

Beneficial Microbes in Agro-Ecology https://doi.org/10.1016/B978-0-12-823414-3.00004-6

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is mediated by the enzyme ACC deaminase, and this process reduces the ethylene production in plants under a stressed environment. ACC deaminase-producing rhizobia have better nodulation and N2 fixation potentials compared to other rhizobial strains. This book chapter deals with (i) the diversity and taxonomy of rhizobia, (ii) the physiologic aspects, and (iii) the beneficial effect on the agro-ecosystem for sustainability of soil health and crop production.

2. Diversity and taxonomy of rhizobia Hermann Hellriegel and Hermann Wilfarth of German recognized the potential of legume root nodules to reduce atmospheric nitrogen into ammonia and make it available to plants. Symbiotic microorganisms from root nodules of different legumes were first isolated by Martinus Beijerinck of Holland and further termed Rhizobium (rhiza ¼ root; bios ¼ life). The term legume-nodulating bacteria (LNB) was proposed by Zakhia et al. (2004) to avoid confusion between the genus Rhizobium and a common term rhizobia used for nodulating bacteria. Berrada and Fikri-Benbrahim (2014) reported that there are 98 species of LNB belonging to 14 genera via Rhizobium, Bradyrhizobium, Mesorhizobium, Azorhizobium, Ensifer (formerly Sinorhizobium) Methylobacterium, Devosia, Microvirga, Ochrobactrum, Phyllobacterium, and Shinella, which are classified under a-Proteobacteria, while Burkholderia and Cupriavidus (formerly Ralstonia) belong to b-Proteobacteria, and finally, one genus Pseudomonas is classified under subclass g-Proteobacteria. A review on LNB classification described that legume symbionts belong to 238 species of 18 genera (Table 4.1) under three distinct phylogenetic classes: a, b, and g-Proteobacteria (Shamseldin et al., 2017).

3. Physiologic aspects of rhizobia Root-nodulating bacteria are aerobic, chemoorganotrophic organisms that have oxidative metabolism. The optimum temperature for the growth of rhizobia is 25e30 C. Rhizobia commonly use EntnereDoudoroff and pentose phosphate pathway for sugar metabolism with a fully functional TCA cycle. Rhizobia use a variety of nitrogen sources and ammonia assimilation through GS/GOGAT system. Rhizobia utilize a wide array of carbon compounds and use iron from a wide range of sources with the help of siderophores (Poole et al., 2008). Some strains of rhizobia need biotin, thiamine, and pantothenate for their growth (Sullivan et al., 2001; Watson et al., 2001). Slow-growing rhizobia are generally heterotrophic in nature. Genes for ribulose-bis-phosphate carboxylase metabolism are not conserved, and autotrophic growth in soil may be possible. Both slow- and fast-growing rhizobial groups utilize C5 and C6 sugars and sugar alcohols. The EntnereDoudoroff pathway is the main sugar degrading pathway. Fast-growing rhizobia metabolize C12 sugars (sucrose) and result in the acidification of media. Metabolic pathways for the C5 sugar metabolism are different among fast- and slow-growing groups. C4-dicarboxylic acids (malate, fumarate, and succinate) are utilized by both fast- and slow-growing groups. Growth on organic substrates results in the alkalinization of solid or liquid media. Rhizobia are strain specific for growth on amino acids, so the growth of particular strains has to be checked individually. Some amino acids cause marked changes in cell morphology when used as the sole carbon source, as the growth of Rhizobium leguminosarum on histidine produces almost coccoid cells. I. Bacteria

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TABLE 4.1

Taxonomy of legume-nodulating bacteria.

Phylum

Class

Order

Family

Genus

No. of species

Proteobacteria

a-proteobacteria

Rhizobiales

Rhizobiaceae

Rhizobium

98

Ensifer

18

Pararhizobium

1

Allorhizobium

1

Shinella Bradyrhizobiaceae

1

Photorhizobium

1

Mesorhizobium

40

Phyllobacterium

8

Aminobacter

1

Azorhizobium

3

Devosia

1

Methylobacterium

3

Microvirga

4

Brucellaceae

Shinella

1

Burkholderiaceae

Burkholderia

17

Cupriavidus

2

Pseudomonas

1

Hyphomicrobiaceae

Methylobacteriaceae

g-proteobacteria

Burkholderiales

Pseudomonadales

1 36

Blastobacter

Phyllobacteriaceae

b-proteobacteria

Bradyrhizobium

Pseudomonadaceae

Legume plants supply reduced carbon to rhizobia in nodules, which they utilize to fix atmospheric N2 to NH3. Symbiotic rhizobia may sequester some of this carbon in bacterial storage polymers, such as the lipid poly-3-hydroxybutyrate (PHB). PHB accumulation often exceeds 50% of cell dry weight in rhizobia (Bergersen and Turner, 1990; Tavernier et al., 1997). There are various ways through which rhizobia use PHB to benefit the host plant and to protect nitrogenase from O2 inactivation. Alternatively, rhizobia may use PHB in ways that enhance their own fitness. Rhizobia use PHB as energy and as a carbon source for reproduction and stress tolerance. PHB synthesis directly competes with N2 fixation. Thus a tradeoff occurs between N2 fixation and rhizobia. Peralta et al. (2004) demonstrated that a PHB mutant of Rhizobium etli fixed significantly more nitrogen than the isogenic PHB wild type. Rhizobia with disturbed PHB synthesis are shown to be less competitive for nodulation (Aneja et al., 2005) and reproduce less under starvation (Cai et al., 2000; Povolo and Casella, 2004) than wild-type cells. The ability to synthesize and degrade PHB may improve Rhizobium fitness by stabilizing cellular redox and relieving TCA cycle inhibition under low oxygen condition.

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The ability to use particular compounds as nitrogen sources varies widely among rhizobia. Many strains utilize urea, ammonium salts, nitrates, or amino acids as N sources, but some do not. Growth may also be faster with a particular form, and this can only be determined experimentally. Growth on sugars and alcohols results in the acidification of media and pH falls into the range of 4e5. Similarly, growth on organic acids results in the alkalization of media and pH rises to 8.5 or higher. Growth on amino acids (e.g., histidine or glutamate) as the sole source of carbon results in alkalinization, partly from hydroxide exchange, as with the organic acids, and partly from release of NH3 into the medium. Since the quantitative demand for C is much greater than that for N, very high concentrations of NH3 may be released; for an N-rich amino acid like histidine, growth on this amino acid alone can result in up to 100 mM ammonia release. Nutritional requirement studies for nodulating bacteria require the use of purified defined media (Abreu et al., 2012) and specialized culture techniques (Cassman et al., 1981; Smart et al., 1984). In particular, the uses of specific anionic or cationic resins to purify media components of the nutrient being studied, and the addition of metal chelators, such as ethylene diamine tetra-acetic acid (EDTA) and nitrile-triacetate to remove residual nutrient contaminants from media and solutions, are essential for studies determining roles of micronutrients in nodule bacteria.

4. Beneficial effect of rhizobia 4.1 Nitrogen fixation Nitrogen (N) is a vital element for plant growth and essential for the synthesis of nucleic acids, enzymes, proteins, and chlorophyll. Although 78% nitrogen is available in the atmospheric air in gaseous form, plants cannot directly assimilate it. The energetically expensive process of converting atmospheric nitrogen into plant-useable organic form is called biologic nitrogen fixation (BNF). BNF, discovered by Beijerinck in 1901, is carried out by a specialized group of prokaryotes that utilize nitrogenase for the conversion of atmospheric nitrogen (N2) to ammonia (NH3). Plants can readily assimilate NH3 to produce the aforementioned nitrogenous biomolecules. Annually, 2.5  1011 kg NH3 are fixed from the atmosphere through BNF, and approximately 8  1010 kg NH3 are manufactured chemically. Further, lightning may also contribute 1010 kg NH3 per year. Globally, BNF in natural terrestrial ecosystems contributes about 107 Tg of nitrogen (1 Tg ¼ 1 million tons), while marine BNF contributes 121 Tg of nitrogen each year (Galloway et al., 2004). Cultivationinduced BNF in agricultural crops and fields adds 33 Tg per year (Smil, 1999). Symbiotic BNF by Rhizobium associated with seed legumes is 10 Tg (range 8e12 Tg), leguminous cover crops (forages and green manures) is 12 Tg/yr, nonrhizobium N fixing species is 4 Tg/yr, cyanobacteria in wet rice fields 4-66 Tg, and entophytic N fixing organisms in sugarcane is 1e3 Tg. Thus, total terrestrial nitrogen fixation is 140 Tg N/year. It is estimated that cultivation-induced BNF may have risen to 40 Tg/yr due to an increase in soybean production (Galloway et al., 2008). Relative to cultivation-induced BNF, about three times as much N was fixed as ammonia by the HabereBosch process, about 100 Tg N per year of ammonia in 1995 of which about 86% was used to make fertilizers (Kramer, 1999). The industrial fixation of nitrogen is increasing each year with the setting up of more plants, and it reached

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121 Tg by 2006 (FAO, 2006) with the worldwide manufacture of N fertilizer at 105 Tg. Cultivation of pulses contributed 2.95 Tg of fixed N, while the same for oilseed legumes was 18 Tg. Soybean is the dominant crop legume, representing 50% of the global crop legume area, and able to fix 16.4 Tg N annually. In the agriculture production system, 50% of the total N used is delivered by the legume crops (Graham and Vance, 2003). So, the biologically fixed N is enough to provide the required nitrogen for plant growth (Sorensen and Sessitsch, 2007; Buragohain et al., 2017). Approximately 200e300 kg of N ha/year is produced by various legume crops and pasture species. Rhizobia play a very important role in symbiotic nitrogen fixation. Presently, about 700 genera and 1300 species of leguminous plants, which can develop root nodules and fix atmospheric nitrogen, are identified. Nitrogenase, a major enzyme involved in nitrogen fixation, has two components: (1) dinitrogenase reductase, the iron protein, and (2) dinitrogenase (metal cofactor). The iron protein provides the electrons with a high reducing power to dinitrogenase, which in turn reduces N2 to NH3. Depending on the availability of metal cofactor, three types of N fixing systems have been identified: (1) Mo-nitrogenase, (2) V-nitrogenase, and (3) Fe-nitrogenase. Complexity of nitrogen fixation can be clearly understood only by investigating the contribution of several gene clusters involved in the following processes: (1) nitrogen fixation (nif HDKdnitrogenase, nifA, fixLJ, fixKdtranscriptional regulator, nif BENdbiosynthesis of the Fe-Mo cofactor, fixABCXdelectron transport chain to nitrogenase, fix- NOPQdcytochrome oxidase, fixGHISdcopper uptake and metabolism, fdxNdferredoxin); (2) nodulation (nodAdacyltransferase, nodBdchitooligosaccharide deacetylase, nodCdN-acetylglucosaminyltransferase, nodDdtranscriptional regulator of common nod genes, nodIJdNod factors transport, nodPQ, nodX, nofEF, NOEdsynthesis of Nod factors substituents, nol genesdseveral functions in synthesis of Nod factors substituents and secretion); and also (3) the genes involved in the transport of other essential elements (exod exopolyssacharide production, hupdhydrogen uptake, glndglutamine synthase, dctd dicarboxylate transport, nfednodulation efficiency and competitiveness, ndvdb-1,2 glucan synthesis, plsd lipopolysaccharide production) (Laranjo et al., 2014). However, many environmental factors can affect the nitrogen fixation, such as extremes of temperature, soil salinity, soil acidity, alkalinity, soil moisture deficiency, osmotic stress, nutrient deficiency, and overdoses of fertilizers and pesticides; all these biotic factors have an effect on the survival and infection rate of rhizobia (Zahran, 1999).

4.2 Abiotic stress tolerance Rhizobacteria and vesicularearbuscular mycorrhiza confer stress tolerance to the host plants through a process called induced systemic tolerance. However, the impact of root nodule bacteria on legume drought stress tolerance is still poorly understood. Inoculation of Sinorhizobium medicae or Sinorhizobium meliloti, that differ in the performance of N fixation significantly delayed drought-induced leaf senescence in nodulated plants of Medicago truncatula relative to nonnodulated plants, so the process is independent of rhizobial strain efficiency for N fixation and uncoupled from initial leaf N content. Consequently, nodulated plants recovered more effectively from drought, relative to nonnodulated M. Truncatula (Staudinger et al., 2016). The major mechanisms involved are increased concentrations of

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potassium and shifts in the carbon partitioning between starch and sugars under well-watered conditions, as well as the enhanced allocation of reserves to osmolytes during drought. Senescence-inhibiting hormones such as cytokinin might play an important since rhizobia-derived cytokinin in the xylem of legumes was reported (Upadhyaya et al., 1991). Furthermore, decreased abundances of proteins involved in ethylene synthesis resulted in symbiosis-induced stay-green phenotype with delayed leaf senescence. Several ribosomal leaf proteins, involved in protein synthesis and general drought stress response, exhibited induced levels in nodulated (NOD) plants under well-watered conditions compared to nonnodulated (NN) plants and resulted in priming effect with reduced leaf senescence in NOD plants. Hence, nodulation, independent of the N fixation efficiency of the microsymbiont, induces abundance increases in drought stresserelevant proteins. Leaves of NOD alfalfa plants were less sensitive to decreasing leaf relative water content, since they maintained higher net photosynthesis and chlorophyll content at moderate stress than N fertilizer applied NN plants (Antolin et al., 1995). In a cyclic drought experiment, NOD alfalfa accumulated more biomass as a result of altered leaf ABA/cytokinin balance relative to NN plants (Goicoechea et al., 1997). Others also observed an enhanced drought tolerance in nodulated Phaseolus vulgaris and Pisum sativum on the basis of pod yield or biomass accumulation relative to NN, nitrate-fed plants (Lodeiro et al., 2000; Frechilla et al., 2000). In both, NN and NOD(e) plants the drought stress treatment led to the accumulation of glucose and fructose, as also reported by other researchers (Sanchez et al., 2012; Zhang et al., 2014). Pinitol is a major carbohydrate in legumes that also acts as an osmolyte (Streeter et al., 2001; Reddy et al., 2004). The concentration of pinitol, however, only increased in nodulating NOD plants but not in NN plants (Staudinger et al., 2012). In another study, pinitol increased only at very low water potentials (w3 MPa, day 7) in NN M. truncatula (Zhang et al., 2014). Ethylene is a stress hormone released during stress condition such as heavy metals, drought, water logging, salinity, etc. Plant-associated bacteria have evolved intricate mechanisms to modulate plant ethylene levels, either through the production of ACC deaminase (Glick et al., 1998) or vinyl-glycine compound rhizobitoxine (RTX) (Sugawara et al., 2006). ACC deaminase, encoded by acdS, degrades the ethylene precursor called 1aminocyclopropane-1-carboxylic acid (ACC) into ammonia and alpha-ketobutyrate. Several bacterial genera such as Azospirillum, Acinetobacter, Bacillus, Burkholderia, Enterobacter, Pseudomonas, Achromobacter, Agrobacterium, Alcaligenes, Ralstonia, Serratia, and Rhizobium, are known to produce ACC deaminase. Some leguminous plants possess a relatively large number of genetic elements involved in ethylene production compared to other plants (Desbrosses and Stougaard, 2011), reflected in overall production and response to ACC and ethylene (Miyata et al., 2013). Glycine max possesses 14 ACC synthase genes, while most other leguminous plants possess only six ACC synthase genes. Hence, leguminous plant hosts subjected to stress may produce increased ACC and ethylene levels and induce the selection of ACC-degrading and RtxC-producing rhizobia. Research works on stress-induced selection of ACC deaminase-producing bacteria were reviewed (Nascimento et al., 2018). ACC deaminase promotes plant growth and development by reducing drought stress and inducing salt tolerance in plants (Nadeem et al., 2007; Zahir et al., 2008). Ethylene is a central regulator of the nodulation process (Berrabah et al., 2018; Guinel, 2015; Larrainzar et al., 2015). Rhizobia expressing ACC deaminase reduce the negative

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effects of ethylene in the nodulation process (Nascimento et al., 2016). Ethylene content in legume tissues and the rhizosphere is decreased by inoculation of ACC deaminaseproducing R. leguminosarum bv. viciae, R. hedysari, R. japonicum, R. gallicum, Bradyrhizobium japonicum, Bradyrhizobium elkani, Mesorhizobium loti, and Sinorhizobium meliloti (Duan et al., 2009; Hafeez et al., 2008; Kaneko et al., 2000; Ma et al., 2003; Madhaiyan et al., 2006; Okazaki et al., 2004; Uchiumi et al., 2004). Rhizobia have the capability to uptake ACC and break it down into a-ketobutyrate and NH3. Its breakdown product is used as a source of carbon and nitrogen. Overexpression of ACC deaminase gene in several rhizobial species enhanced the nodule number and its competitiveness (Conforte et al., 2010). Moreover, environmental stress tolerance (such as salinity) in legumes was enhanced through ACC deaminase (Brigido et al., 2013). Inoculation of ACC deaminase minus mutants of rhizobial strain produced fewer nodules and were less competitive than their wild-type counterparts (Ma et al., 2003; Uchiumi et al., 2004); ACC deaminase genes are highly prevalent and are stably vertically transmitted in Bradyrhizobium spp. and Paraburkholderia spp. (Nascimento et al., 2014). However, the genes were acquired by horizontal gene transfer with a positive selection in other rhizobial groups. Symbiotic Paraburkholderia strains possess two acdS genes; one of these is present in the symbiotic plasmid and is a result of a gene duplication event (Nascimento et al., 2014), indicating a strong selective pressure to maintain this gene. Stable existence of acdS might be due to previous ancestral selection relating to the ability of those bacteria for internal colonization and regulation of plant ethylene levels and basal plant defense response (Nascimento et al., 2018). This was evidenced by the endophytic nature of Bradyrhizobium spp. and Paraburkholderia spp. with several nonleguminous plant species (OnofreLemus et al., 2009).

4.3 Growth regulators Growth regulators such as indole-3-acetic acid (IAA), gibberellins, cytokinins, and abscisic acid are the phytohormones that stimulate plant growth at different micromolar concentrations. 4.3.1 Indole-3-acetic acid Rhizobia are considered the most excellent candidate for plant growth promotion compared to rhizobacteria due to their nitrogen fixing ability and endophytic nature (Asghar et al., 2004). Apart from several plant beneficial traits, they also produce auxins that play an important role in plantemicrobe interactions and plant growth (Boivin et al., 2016). IAA is physiologically the most active phytohormone that stimulates rapid increase in cell elongation, cell division, differentiation, and vascular bundle formation in plants and finally results in enhanced plant growth and development. On inoculation of R. leguminosarum bv. viciae, a 60-fold increase in IAA was observed in the nodules of vetch roots (Camerini et al., 2008). One of the highest productions of IAA had been reported with the inoculation with B. japonicum SB1 with Bacillus thuringiensis KR1 (Mishra et al., 2006). IAA-producing efficiency of Rhizobium sp. Br5 from barseem helped to enhance the growth and yield of cotton in comparison to other isolates (Hussain et al., 2014; Parthiban et al., 2016). Coinoculation of Pseudomonas with Rhizobium galegae bv. orientalis resulted in enhanced production of IAA and further increased

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the nodule number, shoot and root growth, and nitrogen content. Environmental stress factors via acidic pH, osmotic and matrix stress, and carbon limitation and genetic factors via auxin biosynthesis genes and the mode of expression were shown to influence the biosynthesis of IAA (Spaepen et al., 2007; Spaepen and Vanderleyden, 2011). Biosynthesis of IAA in Rhizobium cells requires transamination and decarboxylation reaction of L-tryptophan, which is commonly found in root exudates. Moreover, IAA production was not detected in Ltryptophan-free medium (Shoukry et al., 2018). It is reported that a higher amount of auxin produced by Rhizobium isolates from nodules of berseem, chickpea, lentil, vegetable pea, and mung bean (Br5, Cp5, L3, Vp2, and M3) in concentrations of 4.48, 3.81, 4.47, 3.67, and 4.24 mg/mL and its values is more enhanced with the addition of L-tryptophan, i.e., 6.54, 5.95, 5.39, 4.76, and 4.91, in that order (Qureshi et al., 2019). Soil bacteria can use tryptophan as a nitrogen source, which may be the reason for the increased production of IAA in some bacteria challenged with tryptophan (Monteiro et al., 1988). Rhizobial isolates have different abilities for carbon source utilization, such as isolates from Vicia faba and Lens culinaris producing 48.31 mg/mL and 33.99 mg/mL of IAA from mannitol source. In contrast, IAA production from lactose as a source of carbon is 32.93 mg/mL (Shoukry et al., 2018). The maximum production of IAA by Rhizobium sp. from Dalbergial anceolaria and Rhizobium undicola from Neptunia oleracea required mannitol as a carbon source (Ghosh et al., 2015). 4.3.2 Gibberellins The first evidence for the production of GA by rhizobia was obtained from R. etli bv. phaseoli 8002 using modern techniques such as GC-MS and HPLC. This bacterium produced GA9, GA20, GA4, and GA1 in aerobic culture. Putative GA operons possess the genes for the biosynthesis of (i) isoprenoid diphosphate synthases, (ii) diterpene cyclases, (iii) cytochrome p450 monooxygenases, (iv) ferredoxins, and (v) short chain dehydrogenases/reductases. As a part of the initial discovery and analysis of the putative GA operon, expression of CYP112 and CYP114 proteins of B. japonicum were specifically reported in soybean root nodules and with a lower level of expression during anaerobic culture growth (Tully and Keister, 1993). Similarly, upregulation of GA operon gene expression during symbiosis has been confirmed in M. loti, R. etli, and Sinorhizobium fredii (Perret et al., 1999; Vercruysse et al., 2011). In some plant species like Pisum sativum and Medicago truncatula, GA plays a negative role in nodule senescence (Van de Velde et al., 2006). Genomic analysis has revealed that GA operon is invariably located within the symbiotic island or plasmid of rhizobial species. This operon has been controlled by NifA-RpoN promoters (Hershey et al., 2014; Salazar, et al., 2010; Dombrecht, et al., 2002; Hauser, et al., 2007), which are chiefly involved in nitrogen fixation. It has been found that this GA operon plays a role in a rhizobial species symbiotic interaction with its host legume. 4.3.3 Cytokinins Cytokinins regulate cell proliferation and differentiation in plant development. Exogenous application of a high concentration of cytokinins (6-benzylaminopurine, N6-(D2-isopentenyl)-adenine and trans-zeatin) via either root drenching or a petiole feeding technique reduced the number of nodules, while the same with lower concentration enhanced the nodulation. Cytokinins act as regulators of cell division during nodulation, and further cytokinin-response genes are activated in either inner or outer cortex cells during nodule

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initiation in indeterminate and determinate nodules, respectively (Held et al., 2014). Approximately 90% of rhizospheric microorganisms are able to produce cytokinins and about 30 well-known growth-promoting compounds of the cytokinin group are from microbial origin (Nieto and Frankenberger, 1990, 1991). Rhizobium strains are also reported as the effective producers of cytokinins (Caba et al., 2000; Senthilkumar et al., 2009). Production of zeatins from B. japonicum was first determined 4 decades ago. S. meliloti was also found to synthesize zeatins. These zeatins are probably derived from tRNA molecules, as indicated by the fact that no gene homologous to ipt was found in the genomes of symbiotic Rhizobium species. Cytokinin isolated from Rhizobium japonicum promotes cell proliferation in a cytokinin-requiring soybean callus tissue and produced polyploid divisions with diplochromosomes in root nodule primordium (Phillips and Torrey, 2006). Sinorhizobium strains engineered for expressing ipt gene of Agrobacterium overproduced zeatins compared to the control strain and confer stress tolerance to alfalfa without any apparent change in nodulation and nitrogenase activity under severe drought stress (Xu et al., 2012).

4.4 Abscisic acid Abscisic acid (ABA) mediates the regulation of plant water balance and osmotic stress tolerance. It synchronizes the expression of stress-responsive proteins and results in accumulation of compatible osmolytes, dehydrins, and other protective proteins. Transcription factors via DREB2A/2B, AREB1, RD22BP1, and MYC/MYB regulate the ABA-responsive gene expression by interacting with their corresponding cis-acting elements, viz., DRE/CRT(A/GCCGAC), ABRE (PyACGTGGC), and MYCRS(CANNTG)/MYBRS (C/TAACNA/G), respectively. ABA-responsive element (ABRE) is present upstream of the ABA-dependent stress responsive genes. The ABRE-binding (AREB) proteins or ABRE-binding factors (ABFs) encode bZIP transcription factors, among which AREB1/ABF2, AREB2/ABF4, and ABF3 are induced by dehydration, high salinity, or ABA treatment in vegetative tissues and are involved in enhanced drought stress tolerance (Dar et al., 2017). Rhizobial isolates indirectly contribute to plant stress tolerance by producing ABA. Production of ABA at minimum concentration together with high synthesis of GA and auxin by Rhizobium leguminosarum was correlated with higher plant biomass under drought conditions (Bano et al., 2010). Exogenous application of ABA showed inhibitory effect on nodulation with decreased nodule number being established (Ferguson and Mathesius, 2003). However, concentration of ABA in nodules was higher than any other adjacent root tissues, suggesting the possible role of ABA in development and/or functioning of root nodules. It was further supported by an increased number of lateral roots in ABA-treated legumes compared with nonlegume species (Liang and Harris, 2005).

4.5 Phosphate solubilization Phosphorus (P) is the second most important nutrient after nitrogen required for plant growth. It is present in soil in both inorganic and organic forms, and its concentration varies from 140 to 1000 ppm. P availability to plants is influenced by several factors like pH, temperature, moisture, organic content of soil, soil microbes, etc. P content in plant tissues is 0.2%e0.8% on dry weight basis, but only 0.1% is available for plants from soil. Available P range of soil phosphate solution lies between 0.01 and 3.0 mg-P/L, which represents a I. Bacteria

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small portion of plant need. The rest of the required phosphate must be obtained either through phosphatic fertilizers or microbial P solubilization (Sharma et al., 2013). Numerous P fertilizers are available on the market, but they are more expensive, and excessive use leads to eutrophication effect and harms the environment. P use efficiency of field crops is between 15% and 20%, and the remaining phosphatic fertilizer is fixed in the soil and becomes unavailable to plants. Several microorganisms are capable of solubilizing the fixed P by producing organic acids. In addition to BNF, rhizobia can also enhance soil-available P in the legume rhizosphere. Rhizobia, including R. leguminosarum, R. meliloti, M. Mediterranean, Bradyrhizobium sp., and B. japonicum (Afzal and Bano, 2008; Rodrigues et al., 2006; Egamberdiyeva et al., 2004) are the potential P solubilizers. Secretion of low molecular weight organic acids like 2-ketogluconic acid with a phosphatesolubilizing ability has been identified in R. leguminosarum (Halder et al., 1990) and R. meliloti (Halder and Chakrabarty, 1993). Mineralization of organic phosphates takes place by several enzymes including phosphatases, phospho-hydrolases, phytase, D-a-glycerophosphatase, and CeP lyases, while some of the bacterial strains had both mineralization and solubilization capacity (Abd-Alla, 1994). Rhizobia from Iran soils solubilized the mineral phosphate with the formation of visible dissolution halos on agar plates. Among the tested rhizobial isolates, 198 (44%) and 341 (76%) isolates were reported to solubilize Ca3(PO4)2 (TCP) and inositol hexaphosphate (IHP), respectively. Rhizobium leguminosarum bv. viciae mobilized a significantly higher amount of P (197.1 mg-P/L/360 h) in liquid TCP-Sperber medium compared to other rhizobia. The release of soluble P was significantly correlated with a drop in the pH of the culture filtrates, indicating the importance of acid production in the mobilization process. However, none of the 70 Bradyrhizobium isolates was able to solubilize TCP (Alikhani et al., 2007). Around 80% of rhizobial isolates from Sesbania grandiflora root nodules were found to solubilize mineral phosphate with a P solubilization index (PSI) that ranged from 1.96 to 4.85 (Singh and Gera, 2018). Phosphate solubilization ability of rhizobial isolates from root nodules of faba bean (Vicia faba L.) in Morocco was evaluated and found that PSR19, PSR26, and PSR29 were more resistant to extreme stresses and have shown a significant rate of solubilization on Sperber’s basal mediums and enhanced symbiotic effectiveness parameters with the highest number of nodules (34.333  1.527/plant), nodule dry weight (53.077  3.434 mg/plant), and shoot dry weight (1.039  0.051 g/plant) (Hajjam et al., 2016). Rhizobial isolates from faba bean grown in acidic soil of Wollega, Ethiopia, showed PSI ranges from 1.25 to 2.10 (Kenasa et al., 2014).

4.6 Siderophore production Iron is an essential plant micronutrient present in soils. Its concentration varies from 0.2% to 55%. Iron occurs in both divalent and trivalent forms. Under aerobic conditions, it exists as ferric ions (Fe3þ), which are not accessible directly by plants (Rajkumar et al., 2010). Bacteria are able to synthesize low molecular weight compounds called siderophores that chelate iron, and the complex is made available to plants. Rhizobial species, such as R. meliloti, R. tropici, R. leguminosarum bv. Viciae, R. leguminosarum bv. trifolii, and R. leguminosarum bv. phaseoli and S. meliloti and Bradyrhizobium sp. are known to produce siderophores (Antoun et al., 1998; Arora et al., 2001; Carson et al., 2000; Chabot et al., 1996). Symbiotic nitrogen fixation is highly dependent on iron because protein components of I. Bacteria

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functional nitrogenase require the metal. Iron-responsive regulation in rhizobia is mediated by the Irr and RirA regulatory proteins that sense iron availability indirectly (Johnston et al., 2007; O’Brian, 2015). Irr was first described in the soybean symbiont B. japonicum (Hamza et al., 1998) and later found its existence in all rhizobia. However, it is the only ironresponsive regulator in Bradyrhizobiaceae to represses genes encoding proteins that function under iron-sufficient conditions. The second regulatory protein, RirA (rhizobial iron regulator A), was reported in rhizobia distinct from Bradyrhizobiaceae, which helps to achieve iron homeostasis. RirA has been functionally characterized only in R. leguminosarum and S. meliloti. In R. leguminosarum, RirA regulates several ironresponsive genes such as genes for the synthesis and uptake of the siderophore vicibactin (vbs), genes involved in heme uptake (hmu and tonB), genes for the synthesis of ironesulfur clusters (suf), the irrA gene, as well as its own expression (Todd et al., 2002, 2005). In S. meliloti, the RirA regulon comprises several iron-responsive genes, which includes genes involved in iron uptake, energy metabolism, exopolysaccharide synthesis, or iron storage, but also genes that are not iron responsive (Chao et al., 2005; Viguier et al., 2005; Costa et al., 2017). Inactivation of rirA led to constitutive expression of siderophores in R. leguminosarum and S. meliloti. Mutations in rirA of the aforementioned rhizobial species did not impair symbiotic nitrogen fixation. However, a rirA mutant of S. fredii HH103 produced poorly infected nodules in soybean and finally impaired symbiotic nitrogen fixation compared to wild-type (Crespo-Rivas et al., 2019). The Rhizobium nepotum LT560376 isolated from stem nodules of Aeschynomene indica is able to produce siderophores after 4 h of incubation, and maximum siderophore production was observed after 48 h incubation (Ghorpade and Gupta, 2016). Strains isolated from groundnut via. Rhizobium sp. strain R1 and Rhizobium tropici strain R2 produced higher amounts of siderophores than Rhizobium taibaishanense strain R4 and Ensifer meliloti strain R5 in succinic acid broth (Igiehon et al., 2019). Rhizobium laguerreae PEPV40 isolated from an effective nodule of Phaseolus vulgaris grew on chrome azurol S (CAS) indicator medium, and the color change from greenish-blue to clear light yellow suggested that PEPV40 could produce carboxylate-type siderophores (Jimenez-Gómez et al., 2018). Rhizobium radiobacter produced catechol-type siderophores (Ferreira et al., 2019). A study with 31 bradyrhizobial and rhizobial strains infecting pigeon pea indicated that a majority of rhizobial strains, i.e., 21 strains among 23 siderophore-positive strains, showed the production of hydroxamate, while six strains showed the presence of catechol type of siderophore with the production range of 1.03e3.73 mg hydroxamate N per mg protein and 0.19e3.43 mmol/L of catechol per mg protein (Duhme et al., 1998). Two of the 12 Rhizobium meliloti strains (RMP3 and RMP5) isolated from Mucuna pruriens produced siderophores and inhibited a widely occurring plant pathogen, Macrophomina phaseolina, which causes charcoal rot in groundnut. Further, there was a marked enhancement in percentage seed germination, seedling biomass, nodule number, and nodule fresh weight of M. phaseolina-infected groundnut plants inoculated with the strains RMP3 and RMP5, compared to uninoculated and uninfected controls (Arora et al., 2001).

4.7 Biocontrol abilities of rhizobia The process through which a living organism limits the growth or propagation of undesired organisms or pathogens is called biocontrol. Several rhizobial strains were I. Bacteria

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reported to show the biocontrol properties. Therefore, usage of these strains against soilborne pathogens and pests can lead to eco-friendly management of pest and diseases in agricultural fields. Biocontrol mechanisms by rhizobia include competition for nutrients (Arora et al., 2001), production of antibiotics (Bardin et al., 2004; Chandra et al., 2007; Deshwal et al., 2003), production of enzymes to degrade cell walls (Ozkoc and Deliveli, 2001), and production of siderophores (Carson et al., 2000). The production of metabolites such as HCN, phenazines, pyrrolnitrin, viscoinamide and tensin by rhizobia are also reported as other mechanisms involved in biologic control of phytopathogens. R. leguminosarum bv. trifolii, R. leguminosarum bv. Viciae, R. meliloti, R. trifolii, S. meliloti, and B. japonicum have been reported to secrete antibiotics and cell walledegrading enzymes that can inhibit the phytopathogens (Bardin et al., 2004; Chandra et al., 2007; Shaukat and Siddqui, 2003; Siddiqui and Mahmood, 2001; Siddiqui et al., 2000). Seed treatment with appropriate rhizobial strains for biologic control of phytopathogens resulted in accumulation of phenolic compounds, iso-flavonoid phytoalexins, and activation of enzymes like L-phenylalanine ammonia lyase, chalcone synthase, peroxidase, polyphenol oxidase, and others involved in phenylpropanoid and isoflavonoid pathways (Das et al., 2017). Rhizobia starve the pathogens by producing high affinity siderophores and thereby limit the growth of the pathogen (Arora et al., 2001). Study on root colonization behavior of Pseudomonas fluorescens and S. meliloti in the alfalfa rhizosphere had sufficiently demonstrated the usage of biocontrol agents to suppress pathogens (Villacieros et al., 2003). Pathogens that infect okra and sunflower, such as Macrophomina phaseolina, Rhizoctonia solani, and Fusarium solani were shown to be controlled with the usage of B. japonicum, R. meliloti, and R. Leguminosarum (Shaukat and Siddqui, 2003). Biologic control of potatocyst nematode by R. etli strain G12 (Reitz et al., 2000), Pythium root rot of sugar beet by R. leguminosarum viciae (Bardin et al., 2004), white rot disease in Brassica campestris by M. loti (Chandra et al., 2007), sheath blight of rice by R. leguminosarum bv. phaseoli strain RRE6 and R. leguminosarum bv. Trifolii strain ANU843 (Mishra et al., 2006) were well established. Certain Rhizobium strains in combination with arbuscular mycorrhizal significantly reduced foot and root rot incidence in grasspea (Rahman et al., 2017). Xanthomonas maltophilia in combination with Mesorhizobium had been shown to enhance nodule number, nodule biomass, nodule occupancy, plant growth, and productivity in chickpea (Pathak et al., 2007). Biocontrol potential of rhizobial isolates against black root rot of faba bean (Vicia faba L) caused by Fusarium solani was recently reported by Tamiru and Muleta (2018). Nonpathogenic Rhizobium vitis strain ARK-1 effectively inhibited the tumor formation in grapevine under greenhouse as well as field trials (Kawaguchi et al., 2019). R. leguminosarum RPN5, isolated from root nodules of common bean, successfully inhibited the growth of phytopathogens like M. phaseolina, F. oxysporum, F. solani, Sclerotinia sclerotiorum, R. solani, and Colletotrichum sp. Bradyrhizobium sp. have been shown to control the infection of M. phaseolina in peanut with enhanced seed germination, nodule number, and grain yield (Deshwal et al., 2003).

5. Conclusion The best sustainable source of nitrogen input for the agricultural system is BNF. The contribution of BNF toward the global N budget is enormous. Since potential candidates of I. Bacteria

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rhizobia with high nitrogen fixation capacity play critical roles, lots of commercial rhizobial strains have been employed for enhancing agricultural production of food legumes and oilseeds. However, almost none of the commercial rhizobial strains could meet the requirements in field conditions. High priority should be given for selecting superior rhizobial strains that function in an efficient manner in a farmer’s field. The hurdle to achieve this is the missing link between basic and applied research that can be evidenced by comparing current selection criteria with the one available a hundred years before. Knowledge and information generated during past several decades on better survival and functionality of rhizobia should be effectively used for developing criteria to select potential candidates. We need to develop a high-throughput screening technique to handle the large number of nodulating bacteria in any research program. A majority of the research projects on BNF being carried out rarely aim to understand the nature and ecology of BNF process flawlessly. Understanding host genotype and phenotypic traits that support high N fixation and their molecular network and developing strategies on breeding for BNF are also equally important. BNF capacity is also determined by water stress and soil characteristics including availability of nitrate and phosphorus. Hence, quantum of nitrogen fixed through BNF is the result of ecosystem functioning in the rhizosphere of legumes that could be improved in the agricultural field through scientific interventions.

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Kenasa, G., Jida, M., Assefa, F., 2014. Characterization of phosphate solubilizing faba bean (Vicia faba L.) nodulating rhizobia isolated from acidic soils of Wollega, Ethiopia. Sci. Technol. Arts Res. J. 3 (3), 11e17. Kramer, D.A., 1999. Minerals Yearbook. Nitrogen. US Geological Survey Minerals Information. http://minerals.usgs. gov/minerals/pubs/commodity/nitrogen. Laranjo, M., Alexandre, A., Oliveira, S., 2014. Legume growth-promoting rhizobia: an overview on the Mesorhizobium genus. Microbiol. Res. 169 (1), 2e17. Larrainzar, E., Riely, B.K., Kim, S.C., Carrasquilla-Garcia, N., Yu, H.J., Hwang, H.J., Oh, M., Kim, G.B., Surendrarao, A.K., Chasman, D., Siahpirani, A.F., Penmetsa, R.V., Lee, G.S., Kim, N., Roy, S., Mun, J.H., Cook, D.R., 2015. Deep sequencing of the Medicago truncatula root transcriptome reveals a massive and early interaction between nodulation factor and ethylene signals. Plant Physiol. 169 (1), 233e265. Liang, Y., Harris, J.M., 2005. Response of root branching to abscisic acid is correlated with nodule formation both in legumes and non-legumes. Am. J. Bot. 92 (10), 1675e1683. Lodeiro, A.R., González, P., Hernández, A., Balagué, L.J., Favelukes, G., 2000. Comparison of drought tolerance in nitrogen-fixing and inorganic nitrogen-grown common beans. Plant Sci. 154 (1), 31e41. Ma, W., Guinel, F.C., Glick, B.R., 2003. Rhizobium leguminosarum biovar viciae 1-aminocyclopropane-1-carboxylate deaminase promotes nodulation of pea plants. Appl. Environ. Microbiol. 69 (8), 4396e4402. Madhaiyan, M., Poonguzhali, S., Ryu, J., Sa, T., 2006. Regulation of ethylene levels in canola (Brassica campestris) by 1aminocyclopropane-1-carboxylate deaminase-containing Methylobacterium fujisawaense. Planta 224 (2), 268e278. Mishra, R.P., Singh, R.K., Jaiswal, H.K., Kumar, V., Maurya, S., 2006. Rhizobium-mediated induction of phenolics and plant growth promotion in rice (Oryza sativa L.). Curr. Microbiol. 52 (5), 383e389. Miyata, K., Kawaguchi, M., Nakagawa, T., 2013. Two distinct EIN2 genes cooperatively regulate ethylene signalling in Lotus japonicus. Plant Cell Physiol. 54 (9), 1469e1477. Monteiro, A.M., Crozier, A., Sandberg, G., 1988. The biosynthesis and conjugation of indole-3-acetic acid in germinating seed and seedlings of Dalbergia dolichopetala. Planta 174 (4), 561e568. Nadeem, S.M., Zahir, Z.A., Naveed, M., Arshad, M., 2007. Preliminary investigations on inducing salt tolerance in maize through inoculation with rhizobacteria containing ACC deaminase activity. Can. J. Microbiol. 53 (10), 1141e1149. Nascimento, F.X., Rossi, M.J., Soares, C.R., McConkey, B.J., Glick, B.R., 2014. New insights into 1-aminocyclopropane1-carboxylate (ACC) deaminase phylogeny, evolution and ecological significance. PLoS One 9 (6), e99168. Nascimento, F.X., Brígido, C., Glick, B.R., Rossi, M.J., 2016. The role of rhizobial ACC deaminase in the nodulation process of leguminous plants. International Journal of Agronomy. https://doi.org/10.1155/2016/1369472. Article ID 1369472. Nascimento, F.X., Rossi, M.J., Glick, B.R., 2018. Ethylene and 1-Aminocyclopropane-1-carboxylate (ACC) in plante bacterial interactions. Front. Plant Sci. 9, 114. Nieto, K.F., Frankenberger, W.T., 1990. Influence of adenine, isopentyl alcohol and Azotobacter chroococcum on the growth of Raphanus sativus. Plant Soil 127 (2), 147e156. Nieto, K.F., Frankenberger, W.T., 1991. Influence of adenine, isopentyl alcohol and Azotobacter chroococcum on the vegetative growth of Zea mays. Plant Soil 135 (2), 213e221. O’Brian, M.R., 2015. Perception and homeostatic control of iron in the rhizobia and related bacteria. Ann. Rev. Microbiol. 69, 229e245. Okazaki, S., Nukui, N., Sugawara, M., Minamisawa, K., 2004. Rhizobial strategies to enhance symbiotic interactions: rhizobitoxine and 1-aminocyclopropane-1-carboxylate deaminase. Microb. Environ. 19 (2), 99e111. Onofre-Lemus, J., Hernández-Lucas, I., Girard, L., Caballero-Mellado, J., 2009. ACC (1-aminocyclopropane-1carboxylate) deaminase activity, a widespread trait in Burkholderia species, and its growth-promoting effect on tomato plants. Appl. Environ. Microbiol. 75 (20), 6581e6590. Ozkoc, I., Deliveli, M.H., 2001. In vitro inhibition of the mycelial growth of some root rot fungi by Rhizobium leguminosarum biovar phaseoli isolates. Turk. J. Biol. 25 (4), 435e445. Parthiban, P., Shijila-Rani, A.S., Mahesh, V., Ambikapathy, V., 2016. Studies on biosynthesis of auxin in rhizobium and their impact on growth of Vigna mungo L. Pharmaceutical and Biological Evaluations 3 (3), 371e376. Pathak, D.V., Kumar, M., Sharma, S.K., Kumar, N., Sharma, P.K., 2007. Crop improvement and root rot suppression by seed bacterization in chickpea. Arch. Agron Soil Sci. 53 (3), 287e292. Peralta, H., Mora, Y., Salazar, E., Encarnación, S., Palacios, R., Mora, J., 2004. Engineering the nifH promoter region and abolishing poly-b-hydroxy-butyrate accumulation in Rhizobium etli enhance nitrogen fixation in symbiosis with Phaseolus vulgaris. Appl. Environ. Microbiol. 70 (6), 3272e3281. Perret, X., Freiberg, C., Rosenthal, A., Broughton, W.J., Fellay, R., 1999. High resolution transcriptional analysis of the symbiotic plasmid of Rhizobium sp. NGR234. Mol. Microbiol. 32, 415e425. I. Bacteria

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Staudinger, C., Mehmeti-Tershani, V., Gil-Quintana, E., Gonzalez, E.M., Hofhansl, F., Bachmann, G., Wienkoop, S., 2016. Evidence for a rhizobia-induced drought stress response strategy in Medicago truncatula. J. proteom. 136, 202e213. Streeter, J.G., Lohnes, D.G., Fioritto, R.J., 2001. Patterns of pinitol accumulation in soybean plants and relationships to drought tolerance. Plant Cell Environ. 24 (4), 429e438. Sugawara, M., Okazaki, S., Nukui, N., Ezura, H., Mitsui, H., Minamisawa, K., 2006. Rhizobitoxine modulates plante microbe interactions by ethylene inhibition. Biotechnol. Adv. 24 (4), 382e388. Sullivan, J.T., Brown, S.D., Yocum, R.R., Ronson, C.W., 2001. The bio operon on the acquired symbiosis island of Mesorhizobium sp. strain R7A includes a novel gene involved in pimeloyl-CoA synthesis. Microbiol. 147 (5), 1315e1322. Tamiru, G., Muleta, D., 2018. The effect of rhizobia isolates against black root rot disease of faba bean (Vicia faba L) caused by Fusarium solani. Open Agric. J. 12 (1). Tavernier, P., Portais, J., Nava, S., Courtois, J.O., Courtois, B.E., Barbotin, J., 1997. Exopolysaccharide and poly-(beta)hydroxy-butyrate coproduction in two Rhizobium meliloti strains. Appl. Environ. Microbiol. 63 (1), 21e26. Todd, J.D., Wexler, M., Sawers, G., Yeoman, K.H., Poole, P.S., Johnston, A.W., 2002. RirA, an iron-responsive regulator in the symbiotic bacterium Rhizobium leguminosarum. Microbiol. 148 (12), 4059e4071. Todd, J.D., Sawers, G., Johnston, A.W., 2005. Proteomic analysis reveals the wide-ranging effects of the novel, ironresponsive regulator RirA in Rhizobium leguminosarum bv. viciae. Mol. Genet. Genom. 273 (2), 197e206. Tully, R.E., Keister, D.L., 1993. Cloning and mutagenesis of a cytochrome P-450 locus from Bradyrhizobium japonicum that is expressed anaerobically and symbiotically. Appl. Environ. Microbiol. 59, 4136e4142. Uchiumi, T., Ohwada, T., Itakura, M., Mitsui, H., Nukui, N., Dawadi, P., Saeki, K., 2004. Expression islands clustered on the symbiosis island of the Mesorhizobium loti genome. J. Bacteriol. 186 (8), 2439e2448. Upadhyaya, N.M., Parker, C.W., Letham, D.S., Scott, K.F., Dart, P.J., 1991. Evidence for cytokinin involvement in Rhizobium (IC3342)-induced leaf curl syndrome of pigeonpea (Cajanus cajan Millsp.). Plant Physiol. 95 (4), 1019e1025. Van de Velde, W., Guerra, J.C.P., De Keyser, A., De Rycke, R., Rombauts, S., Maunoury, N., Mergaert, P., Kondorosi, E., Holsters, M., Goormachtig, S., 2006. Aging in legume symbiosis. A molecular view on nodule senescence in Medicago truncatula. Plant Physiol. 141, 711e720. Vercruysse, M., Fauvart, M., Beullens, S., Braeken, K., Cloots, L., Engelen, K., Marchal, K., Michiels, J., 2011. A comparative transcriptome analysis of Rhizobium etli bacteroids: specific gene expression during symbiotic non-growth. Mol. Plant Microbe Interact. 24, 1553e1561. Viguier, C., Ó Cuív, P., Clarke, P., O’Connell, M., 2005. RirA is the iron response regulator of the rhizobactin 1021 biosynthesis and transport genes in Sinorhizobium meliloti 2011. FEMS Microbiol. Letters 246 (2), 235e242. Villacieros, M., Power, B., Sánchez-Contreras, M., Lloret, J., Oruezabal, R.I., Martín, M., Rivilla, R., 2003. Colonization behaviour of Pseudomonas fluorescens and Sinorhizobium meliloti in the alfalfa (Medicago sativa) rhizosphere. Plant Soil 251 (1), 47e54. Watson, R.J., Heys, R., Martin, T., Savard, M., 2001. Sinorhizobium meliloti cells require biotin and either cobalt or methionine for growth. Appl. Environ. Microbiol. 67 (8), 3767e3770. Xu, J., Li, X.L., Luo, L., 2012. Effects of engineered sinorhizobium meliloti on cytokinin synthesis and tolerance of alfalfa to extreme drought stress. Appl. Environ. Microbiol. 78 (22), 8056e8061. Zahir, Z.A., Munir, A., Asghar, H.N., Shaharoona, B., Arshad, M., 2008. Effectiveness of rhizobacteria containing ACC deaminase for growth promotion of peas (Pisum sativum) under drought conditions. J. Microbiol. Biotechnol. 18 (5), 958e963. Zahran, H.H., 1999. Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in an arid climate. Microbiol. Mol. Biol. Rev. 63 (4), 968e989. Zakhia, F., Jeder, H., Domergue, O., Willems, A., Cleyet-Marel, J.C., Gillis, M., Dreyfus, B., de Lajudie, P., 2004. Characterization of wild legume nodulating bacteria (LNB) in the infra-arid zone of Tunisia. Syst. Appl. Microbiol. 27, 380e395. Zhang, J.Y., Cruz, D.E., Carvalho, M.H., Torres-Jerez, Kang, Y., Allen, S.N., Huhman, D.V., Tang, Y., Murray, J., Sumner, L.W., Udvardi, M.K., 2014. Global reprogramming of transcription and metabolism in Medicago truncatula during progressive drought and after re-watering. Plant Cell Environ. 37 (11), 2553e2576.

I. Bacteria

C H A P T E R

5 Streptomyces S. Gopalakrishnan, V. Srinivas, S.L. Prasanna International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, Telangana, India

1. Introduction Streptomyces is a Gram-positive bacterium, with a high guanine þ cytosine (G þ C) content, belonging to the family Streptomycetaceae and order Actinomycetales. It is found commonly in marine and fresh water, rhizosphere soil, compost, and vermicompost. Streptomyces plays an important role in the plant growth promotion (PGP), plant health promotion (crop protection), degradation of organic residues, and production of byproducts (secondary metabolites) of commercial interest in agriculture and medical fields. Streptomyces, in the rhizosphere and rhizoplane, help crops in enhancing shoot and root growth, grain and stover yield, biologic nitrogen fixation, solubilization of minerals (such as phosphorus and zinc), and biocontrol of insect pests and plant pathogens. There is a growing interest in the use of secondary metabolites produced by Streptomyces such as blasticidin-s, kusagamycin, streptomycin, oxytetracycline, validamycin, polyoxins, natamycin, actinovate, mycostop, abamectin/avermectins, emamectin benzoate, polynactins and milbemycin for the control of insect pests and plant pathogens as these are highly specific, readily degradable, and less toxic to environment (Aggarwal et al., 2016). The PGP potential of Streptomyces is well documented in tomato, wheat, rice, bean, chickpea, pigeonpea, and pea. This chapter emphasizes the usefulness of Streptomyces in PGP, grain and stover yields, soil fertility, and plant health promotion.

2. Taxonomy of Streptomyces Streptomyces is Gram-positive aerobic actinobacteria with high G þ C DNA content of 69e78 mol % (Korn-Wendisch and Kutzner, 1992). The cell wall of Streptomyces is similar to that of any other Gram-positive bacteria as it contains a simple peptidoglycan mesh surrounding the cytoplasmic membrane (Gago et al., 2011). Streptomyces is the predominant genus among the actinobacteria followed by Micromonospora, Mycobacterium, Actinomadura,

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5. Streptomyces

Saccharopolyspora, Microbispora, Nonomurea, Frankia, Actinoplanes, Verrucosispora, and Nocardia (Martinez-Hidalgo et al., 2014). Streptomyces represents 50% of the total population of soil actinobacteria (Sathya et al., 2017). It is a filamentous bacteria representing the family Streptomycetaceae and order Actinomycetales that includes more than 500 species occurring in soil and water (Barca et al., 2016). Streptomyces was proposed by Waksman and Henricia (1943) and classified initially on the basis of morphology (color of hyphae and spores), chemotype, whole-cell sugars, fatty acid and phospholipid profiles, and composition of cell wall (peptidoglycan type) (Kroppenstedt et al., 1990) and later on the basis of phenotypic and genotypic traits (Anderson and Wellington, 2001). Previously, the taxonomic systems utilized phenotypic traits that helped to resolve the intergeneric relationships within the family Streptomycetaceae. However, the genotypic traits enabled considerable advances for genus delimitation within the Streptomyces (Stackebrandt et al., 1997). For instance, earlier Streptomyces and Streptoverticillium were considered two different genera; however, with the intervention of immunodiffusion and 16S and 23S rRNA analysis, these two genera were made synonyms of Streptomyces (Stackebrandt and Woese, 1981; Logan, 1994). Classification of Streptomyces based on morphologic, chemotaxonomic, and molecular studies is summarized in Table 5.1. TABLE 5.1

Classification of genus Streptomyces based on morphologic, chemotaxonomic, and molecular studies.

Classification

Parameters Characteristics

References

Morphologica classification

Mycelial Permanent and highly differentiated branched morphology mycelia

Atlas (1997)

Spore chain Spores grow out from the aerial mycelium morphology

Cross and Goodfellow (1973)

Spore chain Produce very long chains up to 100 spores. Spore Pridham et al. (1958) length chains classified as straight to flexuous, open loops, and open or closed spirals or verticillate Melanoid pigments

Produce brown melanin

Lechevalier and Lechevalier (1965)

Contains LL-DAP, glycine, no sugar and chemo type 1

Lechevalier and Lechevalier. (1980)

Taxonomic markers

Contains L-diaminopimelic acid, xylose, and madurose

Labeda (1987)

Fatty acids

Contains Iso- and ante iso-branched chain fatty acids,

Hofheinz and Grisebach (1965), Lechevalier (1977), Kroppenstedt (1985)

Chemotaxonomic Cell wall classification type

Biochemical Using fluorogenic probe (targets hydrolysis tests enzyme)

Molecular classification

Goodfellow et al. (1987)

Whole-cell analysis

Curie-point pyrolysis mass spectrometry (PyMS) Sanglier et al. (1992)

Genome sequencing

16S and 23S r RNA analysis, DNA-RNA pairing Gladek et al. (1985), Witt and Stackebrandt (1990)

I. Bacteria

3. Isolation of Streptomyces

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3. Isolation of Streptomyces Streptomyces is well known for its prolific production of useful bioactive compounds (Kekuda et al., 2014). More than 75% of all known naturally occurring antibiotics (such as chloramphenicol, cypemycin, grisemycin, neomycin, and bottromycins) and a wide range of structurally diverse compounds with various pharmaceutical applications were isolated from Streptomyces (Sathya et al., 2016a). Streptomyces has been the most preferred source of microbes for all types of bioactive secondary metabolites that have important applications in both human medicine and agriculture (Watve et al., 2001). Hence, isolation and identification of novel Streptomyces species is very important and the need of the hour. For preferential isolation of Streptomyces from soil, various methods have been reported, which include a variety of selective media formulations, where Streptomyces colonies can be increased and other bacterial colonies effectively decreased. For instance, Streptomyces can be grown exclusively on Benedict’s modification of the Lindenbein medium (Porter et al., 1959); pretreatment of soil suspension with yeast extract and sodium dodecyl sulfate followed by heat shock (dried in a hot air oven at 45 C for 1 h, this activated spores of Streptomyces and inhibited other soil bacteria; Williams et al., 1972); nalidixic acid (10e20 mg/L) has been proven effective when combined with humic acid vitamin agar, such as starch casein nitrate agar and glycerol arginine agar (Masayuki and Nideo, 1989); soil supplemented with sodium chloride and rifampicin enhanced the recovery of uncommon Streptomyces species (Duangmal et al., 2005); and pretreatment of the soil samples reduced the growth of ubiquitous microbial species (Singh et al., 2016a). Pretreatment of the soil suspension with 1.5% phenol (30 C for 30 min) denatures the proteins or disrupts the cell membrane of bacteria, fungi, and other common actinomycetes there by lowering their number but enhances the number of Streptomyces (Hayakawa et al., 1991). Hayakawa et al. (1991) reported a protocol for isolation of Streptomyces from rhizosphere soil. In brief, 1 g of soil sample was suspended in 10 mL of physiologic saline (0.85% of NaCl) and distributed in aliquots. One aliquot of the soil sample was treated with heat for 1 h at 120 C, and the other was treated with 1.5% phenol for 30 min at 30 C. The physicochemically treated soil samples were vortexed and left at room temperature for 30 min. The soil samples were serially diluted up to 105, and 0.1 mL of each dilution was plated on actinomycetes isolation agar (AIA), yeast malt glucose agar (M6), starch casein agar (SCA) and Czapek Dox agar. The prominent Streptomyces colonies were checked for purity and stored for characterization as desired. Gopalakrishnan et al. (2011a) reported a much simpler protocol for isolation of Streptomyces from soil/compost/vermicompost. In brief, 10 g of rhizosphere soil/compost/vermicompost were suspended in 90 mL of saline in a flask and placed on an orbital shaker (at 100e120 rpm) at room temperature (28  2 C) for 45e60 min. At the end of incubation, the soil/compost/vermicompost samples were serially diluted up to 106 dilutions with saline. Dilutions 103e106 were plated (0.1 mL) on AIA, SCA, and/or Kenknight and Munaiers agar (KMA) by spread plate techniques. The plates were normally incubated at 28  2 C for 5e15 days. The most prominent colonies (the ones that produce pigments, found abundantly in the media plate and inhibiting the adjacent colonies) were isolated and maintained on AIA/SCA/KMA slants at 4 C for further studies.

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5. Streptomyces

4. Identification of Streptomyces Streptomyces colonies can be easily identified by their small opaque, rough, nonspreading morphology and are usually embedded in agar medium (Fig. 5.1). Streptomyces is aerobic, slow growing, chalky or glabrous, and heaped. It’s aerial and substrate mycelia are of different colors, and the spore mass color may vary from white, gray, red, yellow, green, and blue to violet (Pridham, 1965). It was also observed that some of the strains produced highly colored diffusible pigments on surrounding medium (Fig. 5.1). Color of the aerial mycelium is one of the prominent identification characters of Streptomyces isolates at the species level (Pridham and Tresner, 1974). An earthy odor is the typical characteristic feature of Streptomyces species. Earthy odor is due to the production of a secondary metabolite called, “geosmin” by Streptomyces (Zuo et al., 2010). A usual confirmatory identification of Streptomyces genus is long filamentous, Gram positive, and acid-fast negative (Fig. 5.1). Streptomyces degrade substrates including casein, tyrosine, and xanthine, but the rate varies with individual isolate. Some of them produce antibiotics, reflected by growth inhibition among other inhabitants of soil samples. Pridham and Gottlieb (1948) and Shirling and Gottlieb (1966) characterized the Streptomyces for production of acid and utilization of different carbon sources including adonitol, sorbitol, dextrose, fructose, inositol, lactose, maltose, raffinose, rhamnose, sucrose, and xylose. Biochemical characterization of Streptomyces was done in detail by Hossain and Rahman (2014). According to this report, Streptomyces are nonmotile, catalase positive, oxidase positive, urease positive, and hydrogen sulfide positive. Detailed results of biochemical tests of Streptomyces are shown in Table 5.2. Molecular identification of the Streptomyces is usually done by 16S rDNA analysis as per the methods explained by Gopalakrishnan et al. (2011a). In brief, pure cultures of Streptomyces were grown in starch casein broth until log phase (4 days), and genomic DNA was isolated according to the protocols of Bazzicalupo and Fani (1995). The amplification of 16S rDNA gene was done by using universal primer 1492R (50 -TAC GGY TAC CTT GTT ACG ACT T-30 ) and 27F (50 - AGA GTT TGA TCM TGG CTC AG-30 ) (Pandey et al., 2005). The PCR product was sequenced. The sequences obtained were compared with those from the GenBank

Different morphology of Streptomyces

FIGURE 5.1

Gram staining

Morphology of Streptomyces on starch casein agar and in Gram’s staining.

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5. Beneficial role of Streptomyces in ago-ecology: in vitro PGP and biocontrol traits of the Streptomyces

TABLE 5.2

Biochemical characteristics of Streptomyces.

Traits

Reaction

Melanin pigment

D

Catalase

þ

Oxidase

þ

Urease

þ

Hydrogen sulfide

þ

Nitrate reduction



Methyl red



Voges-Proskauer



Citrate utilization

þ

Hydrolysis of casein

þ

Starch

D

Lipid

þ

Utilization of carbon source

þ

D-mannitol

þ

Fructose

þ

Sucrose

D

Utilization of nitrogen source D-alanine

þ

L-arginine

þ

L-tyrosine

þ

D-glucose

þ, positive reaction; , negative reaction; D, different isolates gave different reaction.

using the BLAST program, aligned using the Clustal W software and phylogenetic trees inferred using the neighbor-joining method in the MEGA version 4 program (Saitou and Nei, 1987; Alschul et al., 1990; Thompson et al., 1997; Tamura et al., 2007).

5. Beneficial role of Streptomyces in ago-ecology: in vitro PGP and biocontrol traits of the Streptomyces Streptomyces are known to produce PGP and biocontrol traits including cellulase (on cellulose congo red agar; Bhattacharya et al., 2009), lipase (on tween 80 agar; Hendricks et al., 1995), protease (casein agar; Hendricks et al., 1995), chitinase (minimal media with 5% colloidal chitin; Hirano and Nagao, 1988), b-1,3-glucanase (on tryptic soy broth supplemented with 1% colloidal chitin; Singh et al., 1999), indole acetic acid (IAA; on starch casein

I. Bacteria

60

5. Streptomyces

HCN

Siderophore

IAA

Cellulase

Lipase

Chitinase

FIGURE 5.2

In vitro PGP and biocontrol traits of Streptomyces.

broth supplemented with L-tryptophan; Patten and Glick, 2002), siderophore (on King’s B broth; Schwyn and Neilands, 1987), hydrocyanic acid (HCN; on starch casein broth amended with glycine by sulfocyanate method; Lorck, 1948) and 1-aminocyclopropane-1-carboxylate (ACC) deaminase (on starch casein agar using ACC as the sole nitrogen source; Penrose and Glick, 2003) under in vitro conditions (Fig. 5.2; Gopalakrishnan et al., 2011a, 2014; Vijayabharathi et al., 2018a, 2018b). The ability of the Streptomyces to produce hormones and enzymes is reported to play a significant role in PGP and plant health, particularly against insect pests and plant pathogens. For instance, protease- and cellulase-producing bacteria play an important role in the mineralization of major nutrients (NPK) and PGP (Lima et al., 1998); b-1,3-glucanase and HCN-producing bacteria plays a role in disease suppression (Haas et al., 1991; Singh et al., 1999); IAA-producing bacteria stimulate seed germination and root formation, so the host plant gets greater access to water and soil nutrients (Ahemad and Kibret, 2014); siderophore-producing bacteria solubilize iron from minerals under conditions of iron limitation (Indiragandhi et al., 2008).

6. In vitro physiologic traits of the Streptomyces Streptomyces is an alkaline lover, growing well in alkaline soil conditions. They were widely reported to grow well with pH 5e11, NaCl concentration of 10%, and temperatures

I. Bacteria

8. In planta biocontrol traits of the Streptomyces

61

between 20 and 40 C. Streptomyces were also found highly tolerant to fungicide bavistin, slightly tolerant to thiram and captan, but highly sensitive to radonil, benomyl, and benlate (Gopalakrishnan et al., 2012, 2014; Sadeghi et al., 2012). Streptomyces were also found highly resistant to ampicillin and trimethoprim (>800 ppm) and sensitive to chloramphenicol, kanamycin, and nalidixic acid (50e100 ppm) (Gopalakrishnan et al., 2012). It is concluded that Streptomyces may have the ability to survive in harsh environments including saline and acidic to alkaline pH soils and thus can be part of integrated disease management programs. Further, antibiotics such as ampicillin and trimethoprim (highly resistant; >800 ppm) and streptomycin and tetracycline (highly sensitive; 60%) are produced by Streptomyces (Bérdy, 2005; Sharma, 2014). Secondary metabolites produced by Streptomyces include blasticidin-S (produced by S. griseochromogenes), kusagamycin (by S. kasugaensis), streptomycin (by S. griseus), oxytetracycline (by S. rimosus), validamycin (by S. hygroscopicus), polyoxins (by S. cacaoi var asoensis), natamycin (by S. natalensis and S. chattanoogensis), actinovate (by S. lydicus WYEC 108), mycostop (by Streptomyces sp. K61), abamectin/avermectins (by S. avermitilis), emamectin benzoate (by S. avermitilis), polynactins (by S. aureus), and milbemycin (by S. hygroscopicus subsp. aureolacrimosus), which are used commercially as crop protection agents (Aggarwal et al., 2016). Owing to their high specificity, these novel secondary metabolites of microbial origin are much superior in safety for beneficial insect pests, mammalian animals, and humans. Therefore, Streptomyces can be a good alternative for the management of insect pest and plant pathogens of agriculturally important crops.

10. Streptomyces research at ICRISAT International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), located at Patancheru, Hyderabad, Telangana, India, has been working on PGP and biocontrol research using Streptomyces since 2008 on crops including rice, sorghum, chickpea, and pigeonpea. A total of 19 Streptomyces strains such as CAI-13 (isolated from the foliage compost of Allium sativum), CAI-17 (from the foliage compost of Chrysanthemum morifolium), CAI-21 (from the

I. Bacteria

10. Streptomyces research at ICRISAT

63

foliage compost of C. morifolium), CAI-24 (from the foliage compost of Momordica charantia), CAI-26 (from the foliage compost of A. sativum), CAI-68 (from the foliage compost of Nerium indicum), CAI-78 (from the foliage compost of Parthenium hysterophorus), CAI-85 (from the foliage compost of Pongamia pinnata), CAI-93 (from the foliage compost of Azadirchta indica), CAI-121(from foliage compost of C. morifolium), CAI-127 (from foliage compost of A. sativum), CAI-140 (from foliage compost of N. indicum), CAI-155 (from the foliage compost of Thevetia peruviana), KAI-26 (from the foliage compost of Oryza sativa), KAI-27 (from the foliage compost of O. sativa), KAI-32 (from the foliage compost of O. sativa), KAI-90 (from the foliage compost of O. sativa), KAI-180 (from the foliage compost of O. sativa), and MMA-32 (from live soil) were reported (Gopalakrishnan et al., 2011b). The majority of these Streptomyces strains were found to produce PGP and biocontrol traits including cellulase, lipase, protease, chitinase, b-1,3-glucanase, IAA, siderophore, HCN, and ACC deaminase under in vitro conditions (Table 5.3). The 19 Streptomyces strains were also found to grow well with pH 5e11, NaCl concentration up to 10%, temperatures between 20 and 40 C, and tolerant to fungicides bavistin, thiram, and captan and sensitive to radonil, benomyl, and benlate (Table 5.4). Under field conditions, the 19 Streptomyces strains were demonstrated to enhance tiller and panicle numbers (for rice in particular), root length, root volume, root weight, shoot weight, nodule number and weight (for chickpea and pigeonpea in particular), grain yield, and stover yield on rice, sorghum, chickpea, and pigeonpea on multiple years. These strains were found to enhance available P, total N, organic carbon (%), microbial biomass nitrogen and carbon, and dehydrogenase activity in the rhizosphere. The majority of these 19 strains were also demonstrated to enhance biofortification traits including iron and zinc under field conditions (Table 5.5), so these strains can be exploited in biofortification breeding programs. Colonization of these Streptomyces were demonstrated on chickpea roots by scanning electron microscopy analysis. Chickpea plants inoculated with Streptomyces exhibited significant surface colonization (Fig. 5.3). The data for PGP traits including shoot and root, grain and stover yields, the biologic activities of the rhizosphere soil, along with their root colonization under field conditions clearly demonstrate the PGP effects of the 19 Streptomyces strains. More details of the 19 strains can be found in these literatures (Gopalakrishnan et al., 2012, 2014, 2015, 2016a; Sathya et al., 2016a, 2017; Sreevidya et al., 2016). Apart from their PGP traits, the 19 Streptomyces strains were also shown to have antagonistic traits against important pathogens of chickpea and sorghum, particularly against Fusarium wilt in chickpea and charcoal rot in sorghum (Fig. 5.4). A set of five Streptomyces strains (CAI-24, CAI-121, CAI-127, KAI-32, and KAI-90) were demonstrated to control Fusarium wilt in chickpea under field conditions (Gopalakrishnan et al., 2011a), and another set of eight strains of Streptomyces (such as CAI-17, CAI-21, CAI-26, CAI-68, CAI-78, KAI-26, KAI-27, and MMA-32) were shown to have biocontrol traits against charcoal rot in sorghum (Gopalakrishnan et al., 2011b). Vijayabharathi et al. (2018a,b) also reported a set of three strains of Streptomyces and endophytic Streptomyces and their consortia to have biocontrol potential against Botrytis gray mold disease in chickpea. In addition to the aforementioned Streptomyces strains, ICRISAT also identified another series of 16 Streptomyces strains (BCA-508, BCA-546, BCA-659, BCA-667, BCA-689, BCA-698, CAI-8, CAI-13, CAI-70, CAI-85, CAI-87, CAI-132, CAI-133, CAI-155, and SAI-25) having the potential to control insect pests of chickpea and sorghum including Helicoverpa armigera, Spodoptera litura, and Chilo partellus (Vijayabharathi et al., 2014). A novel insecticidal

I. Bacteria

PGP properties

Biocontrol properties

64

TABLE 5.3 In vitro enzymatic and antagonistic activities and secondary metabolite production by the 19 Streptomyces strains of ICRISAT. Antagonistic activity

Scientific name

NCBI No.

IAA

Sid

HCN

Cel

Lip

Pro

Chi

b L1,3-

Foc

MP

RB-6

RB-24

RB-115

Bot

Scl

CAI-13

Streptomyces sp.

KF770891

25.4

2

2

þ

þ



þ

0.25

þ

þ

þ

þ



þ



CAI-17

Streptomyces sp.

JQ682619

0.34

2

3

þ

þ



þ

0.66

þ

þ

þ

þ

þ

þ



CAI-21

Streptomyces sp.

JQ682620

1.13

1

3

þ







0

þ

þ









þ

CAI-24

Streptomyces sp.

JN400112

5.9

3

3

þ

þ



þ

0

þ



þ





þ



CAI-26

Streptomyces sp.

JQ682621

1.17

2

2







þ

0

þ

þ

þ









CAI-68

Streptomyces sp.

JQ682622

0.22

3

3

þ

þ





0.66











þ



CAI-78

Streptomyces sp.

JQ682623

0.95

0

2

þ

þ





2.92

þ



þ

þ



þ



CAI-85

Streptomyces sp.

KF770897

43.6

1

2

þ







1.21

þ

þ

þ

þ



þ



CAI-93

S. fungicidicus

KF742498

33.6

2

2

þ



þ

þ

0

þ

þ

þ

þ

þ

þ



CAI-121

Streptomyces sp.

JN400113

43.7

3

2

þ





þ

0

þ









þ



CAI-127

Streptomyces sp.

JN400114

3.5

4

3

þ

þ



þ

0

þ









þ



CAI-140

S. coelicolor

KF742497

15.4

1.3

3

þ

þ

þ



0.353

þ

þ

þ

þ



þ



CAI-155

Streptomyces sp.

KF770896

12.6

2

3

þ

þ

þ

þ

0.76

þ

þ

þ

þ



þ



KAI-26

Streptomyces sp.

JQ682624

0.4

3

1

þ

þ

þ

þ

0.35

þ

þ

þ

þ



þ



KAI-27

Streptomyces sp.

JQ682625

0.74

1

2

3



þ

þ

0.2

þ

þ

þ

þ



þ



KAI-32

Streptomyces sp.

JN400115

2.3

3

3

þ





þ

0

þ

þ

þ

þ



þ



KAI-90

Streptomyces sp.

JN400116

0

3

3

þ

þ



þ

0

þ



þ

þ

þ

þ



KAI-180

Streptomyces sp.

KF742499

30.1

0

2

þ

þ

þ

þ

0

þ

þ

þ

þ

þ

þ



MMA-32

S. roseoviolaceus

JQ682626

4.66

3

2

þ





þ

0

þ

þ

þ





þ

þ

IAA (mg/mL), Bot, Botrytis cinerea; Cel, celluase; Chi, chitinase; Foc, Fusarium oxysporum; HCN, hydrocyanic acid; Lip, lipase; MP, Macrophomina phaseolina; Pro, proteae; RB6, 24 and 115, Rhizotonia bataticola; Scl, Sclerotia rolfsii; Sid, siderophore; b, 1e3, b, 1e3,glucanse.

5. Streptomyces

I. Bacteria

Isolate

65

10. Streptomyces research at ICRISAT

TABLE 5.4

Effect of pH, temperature, salinity and fungicides on the growth by the 19 Streptomyces strains of ICRISAT. Physiologic 

Fungicide

Isolate

pH

T C

S (%)

Bav

Ben

Cap

Rid

Thi

CAI-13

7e11

20e40

8

þ



þ

þ

þ

CAI-17

7e13

20e40

10

þ

þ

þ

þ

þ

CAI-21

7e11

20e40

12

þ

þ

þ

þ

þ

CAI-24

7e11

20e40

6

þ

þ

þ

þ

þ

CAI-26

7e13

20e40

10

þ

þ

þ

þ

þ

CAI-68

7e11

20e40

8

þ

þ

þ

þ

þ

CAI-78

7e13

20e40

10

þ

þ

þ

þ

þ

CAI-85

5e13

20e40

6

þ

þ

þ

þ

þ

CAI-93

7e11

20e40

8

þ

þ

þ

þ

þ

CAI-121

5e13

20e40

8

þ

þ

þ

þ

þ

CAI-127

7e11

20e40

8

þ

þ

þ

þ

þ

CAI-140

7e11

20e40

10

þ

þ

þ

þ

þ

CAI-155

7e13

20e40

6

þ

þ

þ

þ

þ

KAI-26

7e11

20e40

10

þ

þ

þ

þ

þ

KAI-27

7e11

20e40

10

þ

þ

þ

þ

þ

KAI-32

5e13

20e40

8

þ

þ

þ

þ

þ

KAI-90

5e13

20e40

12

þ

þ

þ

þ

þ

KAI-180

711

20e40

6

þ

þ

þ

þ

þ

MMA-32

711

20e40

6

þ

þ

þ

þ

þ

Bav, Bavistin; Ben, Benlate; Cap, Captan; Rid, Ridomil; S, Salinity; T, Temperature; Thi, Thiram.

TABLE 5.5

Biofortification potentials of the 19 Streptomyces strains of ICRISAT on chickpea.

Isolate

Fe

Zn

Ca

Cu

Mn

Mg

CAI-13

þ

þ

þ

þ

þ

þ

CAI-17

þ

þ

þ

þ

þ

þ

CAI-21

þ

þ

þ

þ

þ

þ

CAI-24

þ

þ

þ

þ

þ

þ

CAI-26

þ

þ

þ

þ

þ

þ

CAI-68

þ

þ

þ

þ

þ

þ (Continued)

I. Bacteria

66 TABLE 5.5

5. Streptomyces

Biofortification potentials of the 19 Streptomyces strains of ICRISAT on chickpea.dcont’d

Isolate

Fe

Zn

Ca

Cu

Mn

Mg

CAI-78

þ

þ

þ

þ

þ

þ

CAI-85

þ

þ

þ

þ

þ

þ

CAI-93

þ

þ

þ

þ

þ

þ

CAI-121

þ

þ

þ

þ

þ

þ

CAI-127

þ

þ

þ

þ

þ

þ

CAI-140

þ

þ

þ

þ

þ

þ

CAI-155

þ

þ

þ

þ

þ

þ

KAI-26

þ

þ

þ

þ

þ

þ

KAI-27

þ

þ

þ

þ

þ

þ

KAI-32

þ

þ

þ

þ

þ

þ

KAI-90

þ

þ

þ

þ

þ

þ

KAI-180

þ

þ

þ

þ

þ

þ

MMA-32

þ

þ

þ

þ

þ

þ

Ca, calcium; Cu, copper; Fe, iron; Mg, magnesium; Mn, manganese; Zn, zinc.

Streptomyces

Control

FIGURE 5.3 of chickpea.

Scanning electron microscopy (SEM) photographs of Streptomyces showing colonization on the roots

metabolite (against H. armigera), N-(1-(2,2-dimethyl-5-undecyl-1,3-dioxolan-4-yl)-2hydroxyethyl) stearamide, was purified from Streptomyces sp., CAI-155. The second insecticidal metabolite called cyclo(Trp-Phe) was purified from S. griseoplanus SAI-25. Both the metabolites were found to have antifeedant, larvicidal, and pupicidal properties against H. armigera (Vijayabharathi et al., 2014); Gopalakrishnan et al. (2016b), Sathya et al. (2016b).

I. Bacteria

11. Conclusion

(A)

67

(B)

Streptomyces showing inhibition against F. oxysporum (A) and M. phaseolina (B)

Metabolite production assay-against M. phaseolina

FIGURE 5.4

Antagonistic activity against fungal pathogens of chickpea and sorghum.

11. Conclusion This chapter was focused on the taxonomy, isolation, identification, and beneficial role of Streptomyces and their secondary metabolites in the field of agriculture with more emphasis on their usefulness in PGP and biocontrol against both plant pathogens and insect pests of agriculturally important crops of semiarid tropics. It is concluded that Streptomyces have been an important source not only for enhancing PGP and biocontrol traits as inoculants but also for isolation and identification of potent compounds having both insecticidal and fungicidal properties. Hence, this important group of actinobacteria can be a useful component for integrated pest management and integrated nutrition management programs.

Acknowledgments The authors wish to acknowledge CGIAR Research Program on Grain Legumes and Dryland Cereals (GLDC) for their generous support for preparing this chapter. ICRISAT is a member of CGIAR Consortium. We thank Mr. PVS Prasad for his enthusiastic technical assistance.

I. Bacteria

68

5. Streptomyces

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Gopalakrishnan, S., Vijayabharathi, R., Sathya, A., Sharma, H.C., Srinivas, V., Bhimineni, R.K., Gonzalez, S.V., Melø, T.M., Simic, N., 2016b. Insecticidal activity of a novel fatty acid amide derivative from Streptomyces species against Helicoverpa armigera. Nat. Prod. Res. 30, 2760e2769. Goodfellow, M., Lonsdale, C., James, A.L., MacNamara, O.C., 1987. Rapid biochemical tests for the characterization of Streptomycetes. FEMS Microbiol. Lett. 43, 39e44. Haas, D., Keel, C., Laville, J., Maurhofer, M., Oberhansli, T., Schnider, U., Vosard, C., Wuthrich, B., Defago, G., 1991. Secondary metabolites of Pseudomonas fluorescens strain CHAO involved in the suppression of root diseases. In: Hennecke, I.H., Verma, D.P.S. (Eds.), Advances in Molecular Genetics of Plant-Microbe Interactions, Vol. I. Kluwer Academic Publishers, Dordrecht, Boston & London, pp. 450e456. Hayakawa, M., Sadakata, T., Kajiura, T., Nonomura, H., 1991. New methods for the highly selective isolation of Micromonospora and Microbispora from soil. J. Ferment. Bioeng. 72, 320e326. Hendricks, C.W., Doyle, J.D., Hugley, B., 1995. A new solid medium for enumerating cellulose-utilizing bacteria in soil. Appl. Environ. Microbiol. 61, 2016e2019. Hirano, S., Nagao, N., 1988. An improved method for the preparation of colloidal chitin by using methanesulfonic acid. Agric. Biol. Chem. 52, 2111e2112. Hofheinz, W., Grisebach, H., 1965. Die Fettsauren von Streptomyces erythreus and Streptomyces halstedii. Z. Naturforsch. 20B, 43. Hossain, M.N., Rahman, M.M., 2014. Antagonistic activity of antibiotic producing Streptomyces sp. against fish and human pathogenic bacteria. Brazilian Archi 57 (2), 233e237. Indiragandhi, P., Anandham, R., Madhaiyan, M., 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, 327e333. Kekuda, P.T.R., Onkarappa, R., Jayanna, N.D., 2014. Characterization and antibacterial activity of a glycoside antibiotic from Streptomyces variabilis PO-178. Sci. Technol. Arts Res. J. 3, 116e121. Korn-Wendisch, F., Kutzner, H.J., 1992. The family Streptomycetaceae. In: Balows, A., Truper, H.G., Dworkin, M., Harder, W., Schleifer, K.H. (Eds.), The Prokaryotes. Springer, New York, pp. 921e995. Kroppenstedt, R., Stackebrandt, E.M., Goodfellow, M., 1990. Taxonomic revision of the actinomycete genera Actinomudura and Microtetruspora. Syst. Appl. Microbiol. 13, 148e160. Kroppenstedt, R., 1985. Fatty acid and menaquinone analysis of actinomycetes and related organisms. In: Chemical Methods in Bacterial Systematics. SAB Technical Series, Vol. 20. Academic Press, London, United Kingdom, pp. 173e199. Labeda, D., 1987. Actinomycete taxonomy: generic characterization. Dev. Ind. Microbiol. 28, 115e121. Lechevalier, H.A., Lechevalier, M.P., 1965. Classification des actinomycetes aérobies basée sur leur morphologie et leur composition chimique. Ann. Inst. Pasteur. 108, 662e673. Lechevalier, M.P., Lechevalier, H.A., 1980. The chemotaxonomy of actinomycetes, p 225e292. In: Dietz, A., Thayer, D.W. (Eds.), Actinomycetes Taxonomyvol A6. Virginia Society of Industrial Microbiology, Arlington, VA. Lechevalier, M.P., 1977. Lipids in bacterial taxonomy: a taxonomist’s view. CRC Crit. Rev. Microbiol. 5, 109e210. Lima, L.H.C., Marco, J.L., Felix, J.R., 1998. Enzimas hidroliticas envolvidas no controle biologico por miciparasitisma. In: Melo, I.S., Azevedo, J.L. (Eds.), Controle Biologico. 11 Jaguraiuna: EMBRAPA-Meio Ambiente, pp. 263e304. Logan, N.A., 1994. Bacterial Systematics. Blackwell Scientific Publications, Oxford London, Edinburgh Boston, pp. 1e269. Lorck, H., 1948. Production of hydrocyanic acid by bacteria. Plant Physiol. 1, 142e146. Lugtenberg, B.J.J., Dekkers, L.C., 1999. What makes Pseudomonas bacteria rhizosphere competent? Environ. Microbiol. 1, 9e13. Martinez-Hidalgo, P., Olivares, J., Delgado, A., Bedmar, E., Martínez-Molina, E., 2014. Endophytic Micromonospora from Medicago sativa are apparently not able to fix atmospheric nitrogen. Soil Biol. Biochem. 74, 201e203. Masayuki, H., Nideo, N., 1989. A New method for the intensive isolation of actinomycetes from soil. Actinomycetologica 3 (2), 95e104. Misk, A., Franco, C., 2011. Biocontrol of chickpea root rot using endophytic actinobacteria. BioControl 56, 811e822. Nassar, A.H., El-Tarabily, K.A., Sivasithamparam, K., 2003. Growth promotion of bean (Phaseolus vulgaris L.) by a polyamine producing isolate of Streptomyces griseoluteus. Plant Growth Regul. 40, 97e106.

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Nonoh, J.O., Lwande, W., Masiga, D., Herrmann, R., Presnail, J.K., Schepers, E., Okech, M.A., Bagine, R., Mungai, P., Nyende, A.B., Boga, H.I., 2010. Isolation and characterization of Streptomyces spp. with antifungal activity from selected national parks in Kenya. Afr. J. Microbiol. Res. 4, 856e864. Pandey, P., Kang, S.C., Maheswari, D.K., 2005. Isolation of endophytic plant growth promoting Burkholderia spp. MSSP from root nodules of Mimosa pudica. Curr. Sci. 89, 177e180. Patten, C., Glick, B.R., 2002. Role of Pseudomonas putida in indole acetic acid in development of host plant root system. Appl. Environ. Microbiol. 68, 3795e3801. Penrose, D.M., Glick, B.R., 2003. Methods for isolating and characterizing ACC deaminase-containing plant growthpromoting rhizobacteria. Physiol. Plant. 118, 10e15. Porter, J.N., Wilhelm, J.J., Tresner, H.D., 1959. Method for the Preferential Isolation of Actinomycetes from Soils. Biochemical Research Section. Lederle Laboratories, American Cyanamid Company, Pearl River, New York. Pridham, T.G., 1965. Color and Streptomyces. Appl. Microbiol. 13 (1), 43e61. Pridham, T.G., Gottlieb, D., 1948. The utilization of carbon compounds by some Actinomycetales as an aid for species determination. J. Bacteriol. 56 (1), 107e114. Pridham, T.G., Hesseltine, C.W., Benedict, R.G., 1958. A guide for the classification of Streptomycetes according to selected groups; placement of strains in morphological sections. Appl. Microbiol. 6, 52e79. Pridham, T.C., Tresner, H.D., 1974. Family VII Streptomycetaceae, Waksmand and Herici 1943. In: Buchanan, R.E., Gibons, N.E. (Eds.), Bergey’s Manual of Determinative Bacteriology, eighth ed. Williams and Wilkins, Baitlmore, pp. 747e829. Quecine, M.C., Araujo, W.L., Marcon, J., Gai, C.S., Azevedo, J.L., Pizzirani-Kleiner, A.A., 2008. Chitinolytic activity of endophytic Streptomyces and potential for biocontrol. Lett. Appl. Microbiol. 47, 486e491. Richardson, A.E., Barea, J.M., Mcneill, A.M., Combaret, C.P., 2009. Acquisition of phosphorus and nitrogen in the rhizosphere and plant growth-promotion by microorganisms. Plant Soil 321, 305e339. Rothrock, C.S., Gottlieb, D., 1984. Role of antibiosis in antagonism of Streptomyces hygroscopicus var. geldanus to Rhizoctonia solani in soil. Can. J. Microbiol. 30, 1440e1447. Sadeghi, A., Karimi, E., Dahazi, P.A., Javid, M.G., Dalvand, Y., Askari, H., 2012. Plant growth-promoting activity of an auxin and siderophore producing isolate of Streptomyces under saline soil condition. World J. Microbiol. Biotechnol. 28, 1503e1509. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406e425. Sanglier, J.J., Whitehead, D., Saddler, G.S., Ferguson, E.V., Goodfellow, M., 1992. Pyrolysis mass-spectrometry as a method for the classification, identification and selection of actinomycetes. Gene 115, 235e242. Sathya, A., Vijayabharathi, R., Srinivas, V., Gopalakrishnan, S., 2016a. Plant growth-promoting actinobacteria on chickpea seed mineral density: an upcoming complementary tool for sustainable biofortification strategy. 3 Biotech 6 (2), 1e6. Sathya, A., Vijayabharathi, R., Vadlamudi, S., Sharma, H.C., Gopalakrishnan, S., 2016b. Assessment of tryptophan based diketopiperazine, cyclo (L-Trp-L-Phe) from Streptomyces griseoplanus SAI-25 against Helicoverpa armigera (Hübner). J. Appl. Entomol. Zool. 51, 11e20. Sathya, A., Vijayabharathi, R., Gopalakrishnan, S., 2017. Plant growth-promoting actinobacteria: a new strategy for enhancing sustainable production and protection of grain legumes. 3Biotech 7, 102. Schwyn, B., Neilands, J.B., 1987. Universal chemical assay for the detection and determination of siderophore. Annu. Biochem. 160, 47e56. Sharma, M., 2014. Actinomycetes: source, identification and their application. Int. J. Curr. Microb. App. Sci. 3, 801e832. Shaukat, K., Affrasayab, S., Hasnain, S., 2006. Growth responses of Triticum aestivum to plant growth-promoting rhizobacteria used as a biofertilizer. Res. J. Microbiol. 1, 330e338. Shirling, E.B., Gottlieb, D., 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16, 313e340. Singh, S.P., Gaur, R., 2016. Evaluation of antagonistic and plant growth-promoting activities of chitinolytic endophytic actinomycetes associated with medicinal plants against Sclerotium rolfsii in chickpea. J. Appl. Microbiol. 121, 506e518. Singh, S.P., Gaur, R., 2017. Endophytic Streptomyces spp. underscore induction of defense regulatory genes and confers resistance against Sclerotium rolfsii in chickpea. Biol. Contr. 104, 44e56.

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C H A P T E R

6 Azospirillum Raúl O. Pedraza1, María P. Filippone1, Cecilia Fontana2, Sergio M. Salazar1, 2, Alberto Ramírez-Mata3, Daniel Sierra-Cacho3, Beatriz E. Baca3 Facultad de Agronomía y Zootecnia, Universidad Nacional de Tucumán, San Miguel de Tucumán, Tucumán, Argentina; 2INTA EEA Famaillá, Tucumán, Argentina; 3Centro de Investigaciones en Ciencias Microbiológicas, Benemérita Universidad Autónoma de Puebla, Ciudad Universitaria, Puebla Pue, México 1

Agricultural production has to face to new challenges that go beyond the merely technical ones, which consider social, economic, and especially environmental concerns. Therefore, it is necessary to approach agricultural activities from a new technologic and development focus within a framework of environmental sustainability that allows satisfying the needs of present generations without affecting the natural resources that future generations will need. In this sense, agricultural activities under the principles of agroecology are presented as a viable alternative, as it is more sensitive to the complexity of agricultural systems in diverse social and ecologic contexts and has a multidisciplinary approach that includes concepts of ecologic sustainability, food security and safety, economic viability, conservation of resources, and social equity, as well as an increase in production. Agroecology is considered both a science and a set of practices (Altieri, 2002). Altieri, Gliessman and other researchers agree that “Agroecology, as a science, integrates traditional knowledge and advances in ecology and agronomy and provides tools to design systems that, based on the interactions of biodiversity, work by themselves, and they sponsor their own fertility, pest regulation, health and productivity, without requiring technologic packages” (Altieri, 2002; Gliessman, 2016; Nicholls et al., 2016). Agroecology is based on the application of basic principles of ecology to the design and management of sustainable agroecosystems. The principles of agroecology include different aspects, generally violated or ignored for conventional agriculture (De-Schutter, 2010); these aspects are very diverse, such as the conservation of natural and agricultural resources (capital, energy, water, soil,

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and genetic varieties), the use of renewable resources, the reduction of the use of chemical synthesis products, the management of biodiversity, and the direct connection between farmers; however, the enhancement of biodiversity and the strengthening of biologic regulations are considered the two pillars upholding agroecology. Ecologic principles can be applied through various practices and strategies, and each has different effects on productivity, stability, and resilience within the agricultural system. Agroecological cropping practices are based on different ecologic processes, such as the nutrient cycle, biologic nitrogen fixation, natural regulation of pests, conservation of soil and water, and the carbon sequestration. The practices aimed at improving the quality of the soil, in terms of fertility and health, have a high impact on the productivity and sustainability of agroecological systems. Among these, biofertilization with products containing living cells of different microorganisms has taken on a generalized renewed importance since a few years ago (Itelima et al., 2018). Three major groups of microorganisms are considered biofertilizers: the plant growth-promoting bacteria (PGPB), arbuscular mycorrhizal fungi, and nitrogen-fixing rhizobia (Malusá et al., 2012). These microorganisms have several effects on soil quality and plant production, such as improving nutrients availability and the structure of the soil, decomposing toxic substances, inducing plant growth by the production of hormones, in particular indole-3-acetic acid (IAA), and also increasing plant tolerance to biotic or abiotic stress by direct or indirect mechanisms. In this chapter an updated status of the genus Azospirillum, one of the most widely studied and commercially employed plant growth-promoting bacterium in agriculture, is presented. This includes a revision of its taxonomy, isolation and identification strategies, plant growth-promoting characteristics, biofilm formation, and root colonization as a starting point to obtain benefits in the plantebacteria interaction, and its use as biofertilizer, abiotic stress tolerance induction, bioremediation, biofortification, and the activity as a biocontrol agent. Therefore, this chapter is intended to revise the role of the genus Azospirillum in agroecology.

1. Taxonomy of Azospirillum The genus Azospirillum (Tarrand et al., 1978) constitutes a subcluster within the family Rhodospirillaceae. Almost all known members of this family are found in aquatic environments, suggesting that Azospirillum represents a lineage that might have transitioned to terrestrial environments much later than the Precambrian split of ‘‘hydrobacteria’’ and ‘‘terrabacteria’’ (Wisniewski-Dyé et al., 2011). Azospirillum spp. belong to the a-subclass of Proteobacteria, and this genus was originally described by Krieg and Döbereiner (1984) to include a species formerly named Spirillum lipoferum (Beijerinck, 1925). These bacteria are spiral or slightly curved rod-shaped nonspore-forming cells with polyhydroxybutyrate granules that can form cysts. The Gram staining is negative, and the cells are very motile with a single polar flagellum and several lateral flagella, shorter in length. Azospirillum has a large amount of C18:1 o7c lipids (55.3%) and contains also 16:1o7c, 16:0 as a major component; the major hydroxy fatty acids are 3-OH C14:0 and 3-OH C16:0. When grown aerobically, species of this genus exhibit a quinone system with ubiquinone 10 (Q-10). The polar lipids consist mainly of phosphatidylglycerol,

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phosphatidylcholine, and one unidentified phospholipid. The DNA GþC content varies between 64 and 71 mol % (Baldani et al., 2014b). The occurrence of Azospirillum species is widespread in the environment, including tropical, subtropical, and temperate regions; most of them were described from plant roots and soil samples. However, besides its association with plants, Azospirillum spp. have also been associated with other environments under extreme conditions of temperature or contamination. Some species have significant agricultural importance, specifically as aerobic nitrogen-fixing species, with considerable plant growth-promoting abilities. Until now, a total of 22 species have been described: A. lipoferum, A. brasilense, A. amazonense (now reclassified as Nitrospirillum amazonense; Lin et al., 2014), A. halopraeferens, A. irakense (now reclassified as Niveispirillum irakense; Lin et al., 2014), A. largimobile (a reclassification of Conglomeromonas largomobilis subsp. Largomobilis; Sly and Stackebrandt, 1999), A. doebereinerae, A. oryzae, A. melinis, A. canadense, A. zeae, A. rugosum, Candidatus A. massiliensis (a provisional name for a well-characterized but as-yet uncultured organism), A. picis, A. palatum (not validated), A. thiophilum, A. formosense, A. fermentarium, A. humicireducens, A. himalayense (not validated), A. soli, and A. agricola. However, in terms of physiology, genetics, and agricultural utilization the most studied ones are A. brasilense and A. lipoferum described by Tarrand et al. (1978), associated with forage grasses, maize, wheat, rice, sorghum, sugarcane, and several other plants (Hartmann and Baldani, 2006; Zambrano et al., 2007). The importance and application of the recently described species is not yet known. Table 6.1 shows the alphabetical listing of the different species of Azospirillum, including the type strains, habitat from where they were first isolated, and respective reference. To summarize the information accumulated about the genus Azospirillum up to now, we can assume that Azospirillum spp. are Gram-negative bacteria that belong to the alphaproteobacteria phylum. According to the old and the newly discovered species, they are present in a widespread diversity of environments and plants, including not only those of agronomic importance such as cereals, sugarcane, and forage grasses, but also from other plant species such as coffee, fruit, and floral plants, including orchids. They are aerobic nonfermentative chemoorganotrophs, vibroid, produce several hormones, mainly auxins (not described for all species yet), and most of them are diazotrophic.

2. Isolation of Azospirillum According to Döbereiner and Pedrosa (1987), the introduction of the N-free semisolid medium NFb was the key that allowed for the isolation and identification of Azospirillum and many other nitrogen-fixing bacteria associated with poaceous plants. Generally, the semisolid NFb medium occupies half of the vial volume (10 mL) and permits the inoculation of a single drop of sample (soil suspension, plant tissue, or cell) into the center of the medium. The original NFb semisolid medium established for Azospirillum by Döbereiner and Day (1976) permitted the development of other media just by substituting the carbon source, and changing the pH and osmotic concentration, omitting and adding vitamins, salts, amino acids, root/shoot extracts, etc., to mimic the environment or plant of interest. These modifications allowed the isolation of the different Azospirillum species after A. lipoferum and A. brasilense were isolated and characterized; however, in some cases the NFb semisolid

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6. Azospirillum

Species of Azospirillum, habitat of first isolation, and references.

Species

Type strains

Habitat of first isolation

References

Azospirillum agricola

CC-HIH038; BCRC 80909; JCM 30827

Cultivated soil in Taiwan

Lin et al. (2016)

Azospirillum brasilense

ATCC 29145; DSM 1690; JCM 1224; LMG 13127; NBRC 102289; NRRL B-14647; VKM B-1547; sp7

Tropical grasses in Brazil

Tarrand et al. (1978)

Azospirillum canadense

DS2; LMG 23617; NCCB 100108

Rhizosphere of corn planted in Ontario, Canada

Mehnaz et al. (2007a)

Azospirillum doebereinerae

DSM 13131; KCTC 12904; GSF71

Washed roots and rhizosphere Eckert et al. (2001) soil of Miscanthus sinensis cv. Giganteus and Miscanthus sacchariflorus grown in Germany

Azospirillum fermentarium

CC-LY743; BCRC 80505; JCM 18688; LMG 27264

Fermentative tank in Taiwan

Lin et al. (2013)

Agricultural soil collected in Taiwan

Lin et al. (2012)

Kallar grass (Leptochloa fusca) growing under saline conditions in Pakistan

Reinhold et al. (1987)

Himalayan valley soil, India

Tyagi and Singh (2014)

Azospirillum formosense BCRC 80273; DSM 24137; JCM 17639; CC-Nfb-7; CECT 9202 Azospirillum halopraeferens

ATCC 43709; DSM 3675; LMG 7108; Au 4

Azospirillum himalayense pt-3; CCUG 58760; KCTC 23189 Azospirillum humicireducens

CCTCC AB 2012021; KACC 16605; SgZ-5

Microbial fuel cell in Guangdong, China

Zhou et al. (2013)

Azospirillum largimobile

ACM 2041; UQM 2041

Freshwater sample collected in Australia

Ben Dekhil et al. (1997)

Azospirillum lipoferum

ATCC 29707; CIP 106280; DSM 1691; JCM 1247; LMG 13128; NBRC 102290; NCAIM (B)01801; NRRL B-14654; VKM B-1519; 59b; NCIMB 11861

Tropical grasses in Brazil

Tarrand et al. (1978)

Azospirillum melinis

CCBAU 5106001; CGMCC 1.5340; Forage grass (Melinis LMG 23364; TMCY 0552 minutiflora Beauv) planted in China

Peng et al. (2006)

Azospirillum oryzae

CCTCC AB204051; IAM 15130; JCM 21588; NBRC 102291; COC8

Paddy soil of rice plants in China

Xie and Yokota (2005)

Azospirillum palatum

10; LMG 24444; CCTCC AB 207189

Forest soil in Zhejiang province, China

Zhou et al. (2009)

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TABLE 6.1

Species of Azospirillum, habitat of first isolation, and references.dcont’d

Species

Type strains

Habitat of first isolation

References

Azospirillum picis

CCUG 55431; DSM 19922; IMMIB TAR-3

Discarded road tar in Taiwan

Lin et al. (2009)

Azospirillum rugosum

CCUG 53966; DSM 19657; IMMIB AFH-6

Oil-contaminated soil in Taiwan

Young et al. (2008)

Azospirillum soli

CC-LY788; BCRC 80569;JCM 18820 Agricultural soil in Taiwan

Lin et al. (2015)

Azospirillum thiophilum

BV-S;DSM 21654; VKM B-2513

Sulfur bacterial mat collected from a sulfide spring in Russia

Kwak and Shin (2016) Lavrinenko et al. (2010)

Azospirillum zeae

LMG 23989; NCCB 100147; N

Rhizosphere of corn planted in Ontario, Canada

Mehnaz et al. (2007b)

Azospirillum irakense/ Niveispirillum irakense comb. nov.

ATCC 51182; CCUG 30621; CIP 103311; DSM 11586; LMG 10653; KBC1

Rice roots and rhizosphere soil in Iraq

Lin et al. (2014)

Azospirillum amazonense/ Nitrospirillum amazonense gen. Nov

ATCC 35119 DSM 2787; LMG 6509; NRRL B-23163; Am 14 (Y1); LMG 2223

Forage grasses, Amazonian region of Brazil

Lin et al. (2014)

Water in human environments in France

Pagnier et al. (2008)

Candidatus Azospirillum URAM1 massiliensisa a

A provisional name for well characterized but as-yet uncultured organism.

medium was not used. For example, A. palatum (Zhou et al., 2009) and A. picis (Lin et al., 2009) were described using solid medium. A. humicireducens was isolated from microbial fuel cells (Zhou et al., 2013) using a complete new medium, and A. fermentarium was isolated from a fermentative tank in Taiwan using the standard tenfold dilution plating technique (Lin et al., 2013). In the work “The art of isolating nitrogen-fixing bacteria from nonleguminous plants using N-free semisolid media: a practical guide for microbiologists,” Baldani et al. (2014a) describe in detail the procedures used for bacterial isolation, counting, and identification either from rhizosphere soil or on the surface of or within plant tissues, including colony and cell morphologies. More importantly, they describe the appropriate recipes available for each N-free semisolid culture medium that are used to count and isolate some Azospirillum species. However, a brief description of the strategy used to count and isolate Azospirillum, according to Baldani et al. (2014a), is now presented. The serial dilution technique facilitates the procedure to isolate N2-fixing bacteria and is also used to count the number of cells per unit of sample. The most probable number (MPN) technique using McCrady’s probability tables, together with the serial dilution technique, has been usually used to count and isolate culturable diazotrophic bacteria from different parts and tissues of plants. With the MPN technique, also, it is possible to differentiate the external from the internal bacterial colonization by applying a surface disinfection procedure in advance. In the case of surface-sterilized tissue (roots), to obtain bacteria colonizing internal tissues, the roots are immersed into 1% chloramine-T solution.

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6. Azospirillum

The MPN method using N-free semisolid media relies upon the appearance of a typical diazotrophic bacterial pellicle in the subsurface of the medium after incubation for 2e10 days at 30 C. Counting can normally be performed after 5e7 days of growth. The typical bacterial pellicles in vials with the highest dilution are transferred to a fresh N-free semisolid medium, and once the bacterial growth is confirmed, a loopful of the new pellicle is streaked onto the corresponding solid semispecific medium containing a trace amount of yeast extract (about 40 mg/L) to isolate the target bacterium according to the phenotypic characteristics of the colonies. A single purified colony is another time tested in the same N-free semisolid medium, and the vials that originally contained the characteristic pellicle are used for isolation of the bacteria and subsequent purification on potato agar medium. The basic NFb medium contains (g/L): malic acid, 5.0; K2HPO4, 0.5; MgSO4.7H2O, 0.2; NaCl, 0.1; CaCl2. 2H2O, 0.02; micronutrient solution (CuSO4.5H2O, 0.04; ZnSO4.7H2O, 0.12; H3BO3, 1.40; Na2MoO4.2H2O, 1.0; MnSO4.H2O, 1.175. Complete volume to 1000 mL with distilled water), 2 mL; Bromothymol blue (5 g/L in 0.2 N KOH), 2 mL; FeEDTA (solution 16.4 g/L), 4 mL; vitamin solution (biotin, 10 mg; pyridoxal-HCl, 20 mg. Dissolve in hot water bath. Complete to 100 mL by adding distilled water), 1 mL; KOH, 4.5 g. Distilled water to bring the final volume to 1000 mL and adjust pH to 6.5. A quantity of 1.6e1.80 g agar/L should be added to prepare the semisolid medium and 15 g agar/L for the solid medium. It is important to add the ingredients in the given sequence to avoid precipitation of iron or of other salts due to the high pH (Baldani et al., 2014a). The following is a brief example of the steps generally used to isolate A. brasilense: This species requires inoculation of the sample into semisolid NFb medium. After incubation for 2e7 days at 30 C, the typical white pellicle appears and should be reinoculated approximately five times in new semisolid NFb and incubated at 30 C until the white pellicle is observed again. Then, the cells can be streaked onto BMS (Batata-Malato-Sacarose) agar medium, also known as potato medium. In BMS the colonies grown are firstly yellowishwhite color, becoming pinkish as they become larger. Alternatively, colonies grown on NFb medium with Bromothymol blue replaced with Congo red indicator are scarlet color (Rodriguez-Cáceres, 1982). One colony from one of these solid media inoculated and grown in semisolid NFb medium constitutes a pure isolate to further continue with its characterization (Fig. 6.1).

3. Biochemical and genetic methods for the identification of Azospirillum Bacteria of the genus Azospirillum can use several carbon sources, such as sugar, amino acids, and sugar alcohols (Table 6.2), and the pattern of carbon utilization has been used for discriminatory purpose between species of the genus. The ability of the isolates to use different carbon sources for growth, among other analysis, such as those involving biomolecular techniques, is one analysis necessary to describe new species. Different commercial kits can be used, such as API (bioMérieux), Biolog, or medium prepared in the laboratory by changing the carbon source. In this case, the main carbon source is omitted and the carbon sources of interest are evaluated. The genus Azospirillum is widely known to contain N2-fixing PGPB, and the versatile carbon and N-metabolism

I. Bacteria

3. Biochemical and genetic methods for the identification of Azospirillum

FIGURE 6.1

79

Diagram of the steps generally used to isolate A. brasilense.

within the genus makes it well adapted to numerous soil conditions and competent to colonize the rhizosphere and, in some cases, the inner plant tissues. Members of the genus Azospirillum are recognized to use tricarboxylic acid as the sole carbon source, but there is variability among the species in the use of sugars and sugar alcohols (Table 6.2). L-arabinose and D-fructose are used for most of the species included in Table 6.2, while lactose is used only by A. irakense and A. melinis, and A. canadense is the only species that does not use any of the carbon sources presented in Table 6.2. This latter species uses malic acid, potassium gluconate, acetic acid, and pyruvic acid, among others, as a sole carbon source. Genetic characterization of putative Azospirillum isolates can be performed by using phylogenetic markers such as the 16S rDNA/RNA gene. For example, an almost full-length polymerase chain reaction (PCR) from the genomic DNA of the 16S rDNA gene can be amplified using universal bacterial primers such as 1F (50 -GAG TTT GAT CAT GGC TCA GA-30 ) and 9R (50 -AAG GAGGTG ATC CAA CCG CA-30 ) and then sequenced. A neighbor-joining phylogenetic analysis conducted in MEGA6 (Tamura et al., 2013) based on 16S rRNA gene sequences for 21 Azospirillum species obtained from the Ribosomal Database Project (https://rdp.cme.msu.edu/index.jsp) is shown in Fig. 6.2. The most recently species described for this genus, such as A. soli, A. fermentarium, A. formosense, A. himalayense, and A. thiophilum (Kwak and Shin, 2016; Lin et al., 2012, 2013, 2015; Tyagi and Singh, 2014), constituted a solid lineage, placed nearest to A. brasilense, A. lipoferum, and A. halopraeferens in the evolutionary tree. Lin et al. (2014) reclassified Azospirillum irakense (Khammas et al., 1989) and Azospirillum amazonense (Magalhães et al., 1983; Falk et al., 1985) as Niveispirillum irakense comb. nov. and Nitrospirillum amazonense gen. nov, respectively. In fact, these species were placed in an independent clade with Candidatus Azospirillum massiliensis more distant from the rest of Azospirillum spp. Even though the taxonomic name for A. himalayense was effectively published by Tyagi and Singh (2014), until now, it was not validated under the rules of the International Code of Nomenclature of Bacteria (Bacteriological Code). A more detailed study on close species and strains has been shown to not be robust enough, principally because 16S rDNA is present in several copies and in different replicons

I. Bacteria

80 TABLE 6.2

6. Azospirillum

Pattern of carbon sources used by different species of Azospirillum.

Species

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

A. agricola

nd

nd nd nd nd nd nd nd 

þ

nd nd nd 

þ

nd nd 

nd nd

A. amazonense

v

þ

þ

þ

þ

þ

þ



þ



þ

v

þ



þ

v

þ



þ

þ

A. brasilense



v



þ



v



þ

v

þ





















A. canadense















nd 















nd 





þ

nd v



v





þ





19

20

A. doebereinerae



nd 

v

þ



þ





A. fermentarium

þ



nd 

nd nd nd nd 



nd nd nd nd nd þ

nd þ

þ

nd

A. formosense

nd

þ

nd þ

nd þ

nd nd nd þ

nd 

nd 

nd nd

A. halopraeferens

nd

v

nd þ

nd 

nd nd 

nd nd nd þ

þ

nd nd nd nd nd nd nd nd þ

A. humicireducens þ

þ



nd þ



þ

nd 



18

þ

nd þ





nd

þ

þ

nd

þ

nd

þ

A. himalayense

nd

þ

þ

nd nd nd nd nd 

A. irakense

þ

þ

þ

v

þ

þ

þ



A. largimobile

þ

þ



þ



þ

þ

A. lipoferum

þ

þ



þ

v

þ

A. melinis

þ

þ

þ

þ

þ

þ

A. orizae

nd

þ



þ

nd þ

A. palatum

v

v

þ

þ

nd nd nd nd þ

A. picis

þ

þ



nd 

A. rugosum





nd þ

A. soli



þ

nd nd nd nd nd nd 



nd nd nd nd þ

nd nd nd

nd nd

A. thiophilum

nd

þ

nd þ

nd þ

nd nd þ

þ

nd 

nd þ





nd þ





A. zeae

v

þ



þ



þ









nd þ





þ

þ

nd nd 

nd nd nd þ

nd nd

nd 

þ





þ

þ



þ

þ

v



þ



þ

þ







þ





þ

þ

nd 



v

þ

þ

v





þ

v



þ

þ





þ

þ

þ

þ

þ

þ

þ

þ

þ

þ

nd þ

þ

þ

nd 







nd þ

þ





nd

nd nd þ

þ

nd nd nd 

þ

þ





nd nd þ

þ

þ

nd nd nd 

þ



nd nd nd þ

þ

nd nd 



nd nd nd nd

þ





nd v





þ



þ

þ

þ

nd nd

Carbon sources: 1. N-acetylglucosamine, 2. L-arabinose, 3. D-cellobiose, 4. D-fructose, 5. L-fucose, 6. D-galactose, 7. Gentiobiose, 8. D-gluconate, 9. D-glucose, 10. Glycerol, 11. Myo-inositol, 12. Lactose, 13. Maltose, 14. D-mannitol, 15. D-mannose, 16. L-rhamnose, 17. D-ribose, 18. D-sorbitol, 19. Sucrose, 20. D-trehalose. Symbols: þ, positive; , negative; v, variable or inconsistent; nd, not determined. Data from Eckert et al. (2001), Sly and Stackebrandt (1999), Ben Dekhil et al. (1997), Khammas et al. (1989), Reinhold et al. (1987), Xie and Yokota (2005), Peng et al. (2006), Mehnaz et al. (2007a,b), Lavrinenko et al. (2010), Lin et al. (2012, 2013, 2015, 2016), Zhou et al. (2013), and Tyagi and Singh (2014).

(chromosome and plasmids), which exhibited variations in their sequences and were divergent within the same species (Maroniche et al., 2017). To be more confident, Lin et al. (2011) designed the genus specific primers Azo494-F/Azo756-R to amplify the 16S rDNA that were sufficient to differentiate Azospirillum from other closely related genera. Moreover, the sequence diversity of the internal transcribed spacer (ITS) regions between 16S and 23S rDNA is useful for differentiating among Azospirillum species and even among strains (Jijón-Moreno et al., 2015; Vezyri et al., 2013). Furthermore, it was observed that

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3. Biochemical and genetic methods for the identification of Azospirillum

81

FIGURE 6.2

Evolutionary relationships of taxa within the genus Azospirillum based on the 16S rRNA gene sequences of 21 Azospirillum species obtained from the Ribosomal Database Project. The evolutionary history was inferred using the UPGMA method. The optimal tree with the sum of branch length ¼ 0.27983176 is shown. The tree is drawn to scale, with branch lengths (next to the branches) in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated. There were a total of 1254 positions in the final dataset.

the ITS-RFLP analysis enables intraspecies differentiation, particularly of the close species (Jijón-Moreno et al., 2015). In addition, PCR from the genomic DNA profile of nif genes, such as the nifD and nifH genes, as well as the rpoD gene has revealed intergeneric and interspecific diversity among the strains and identified the probable nitrogen fixation activity from Azospirillum species (Maroniche et al., 2017; Pedraza et al., 2007). To identify genes involved in the biosynthesis of indole-3-acetic acid (IAA), such as those participating in the principal pathway of IAA production, the intermediate indole-3-pyruvic acid (IPyA), the hisC1 and hisC2 genes, encoding for the aromatic amino acid aminotransferases, and the ipdC gene, encoding for the phenyl pyruvate decarboxylase have been analyzed by PCR (Carreño-López et al., 2000; Jijón-Moreno et al., 2015). The primers employed for PCR clearly discriminated between strains that biosynthesize IAA, which was confirmed in the supernatant from cultures producing IAA. Additionally, the identification of such genes allows their use as alternative phylogenetic markers for Azospirillum strains producing IAA (Jijón-Moreno et al., 2015). To search for new alternative genetic markers from a list of housekeeping genes, the correlation between pairwise gene and whole-genome similarities

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6. Azospirillum

was examined by Maroniche et al. (2017). They found that PCR amplification and sequencing of the gene rpoD, which encodes the sigma 70 factor of RNA polymerase, is a suitable alternative method for a confident molecular identification in Azospirillum spp. The most valuable information regarding to the evolution of Azospirillum species was obtained from the recent analysis of the complete genome sequences through the use of bioinformatics tools. The genomes of A. brasilense, A. lipoferum, A. thiophilum, Nitrospirillum amazonense, A. halopraeferens, A. oryzae, A. humicireducens, and Niveispirillum irakense were sequenced and published. Genome assembly and annotation information are available at the NCBI data base (https://www.ncbi.nlm.nih.gov/genome/?term¼azospirillum). The first studies on the genomic organization of Azospirillum species show that the genome size ranges from a minimum of 4.8 Mbp for A. irakense to 9.7 Mbp for A. lipoferum strain Sp59b, being the genetic information distributed on several mega-replicons (Martin-Didonet et al., 2000). These largest replicons in Azospirillum species were described as chromosomes, chromids (intermediates between chromosomes and plasmid), and plasmids ranging in size from 6 kb to over 300 kb (Givaudan and Bally, 1991; Martin-Didonet et al., 2000; Vanbleu et al., 2004; Wood et al., 1982). Different reports show that plasmids harbored by Azospirillum species confer to them some advantages and play an important role in bacteriumeplant interactions (García-de los Santos et al., 1996; Michiels et al., 1989; Vieille and Elmerich, 1990; Wood et al., 1982). The best characterized with their complete annotation are those present in A. brasilense strain Sp245 and in A. brasilense Sp7 (Wisniewski-Dyé et al., 2011) carrying several loci involved in the production of different surface polysaccharides, in motility, and flagellar synthesis (Vanbleu et al., 2004). As a consequence of large-scale genomic rearrangements, a very little synteny between replicons of Azospirillum strains was observed (Petrova et al., 2010; Vial et al., 2006; Wisniewski-Dyé et al., 2011, 2012). Additionally, ancestral and horizontally transferred genes were identified in Azospirillum, revealing that members from this genus transitioned from aquatic to terrestrial environments (Wisniewski-Dyé et al., 2011). Genes that differentiated Azospirillum species from one another and from their closest relatives, encoding functions that are critical for adaptation to the rhizosphere and interaction with host plants, were horizontally acquired (Wisniewski-Dyé et al., 2011). Throughout a comparative genomics analysis, Wisniewski-Dyé et al. (2012) reported the presence in Azospirillum of a core genome dominated by ancestral genes that contained a small percent of horizontal transferred genes, so genes involved in direct plant growth promotion do not belong to the Azospirillum core genome and seem to have been gained specifically after speciation events or by individual strains. A common set of gene coding functions related with genetic information processing, amino acid metabolism, metabolism of cofactors and vitamins, nucleotide metabolism, human diseases, and metabolism of terpenoids and polyketides was identified by Hongsheng et al. (2018) to be present in the core genome of 151 plant-associated bacteria. Since a large amount of experimental data has been collected over several years about the ability of A. brasilense strains to increase plant productivity in several important crops, the mechanism of the plantebacterial interaction is not yet entirely understood. During the last 10 years, the number of complete genomes from A. brasilense strains sequenced has increased notably, enabling in silico studies to better understand their beneficial effects on plant growth. Table 6.3 summarizes the general features of the A. brasilense genomes sequenced to date.

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3. Biochemical and genetic methods for the identification of Azospirillum

TABLE 6.3

General features of Azospirillum brasilense complete genomes. Isolation source

Collection year and place

Genome size (Mb) GC% Level

Azospirillum brasilense Sp245

Wheat root

1986 Brazil

7.682.39

68.44

Complete 7043

2011

WisniewskiDyé et al. (2011)

Azospirillum brasilense FP2

Spontaneous mutant from A. brazilense Sp7

1984 Brazil

6.885.11

68.08

Contig (413)

6728

2013

Unpublished

Azospirillum brasilense Az39

Wheat rhizosphere

1982 Argentina

7.391.28

68.53

Complete 6713

2014

Rivera et al. (2014)

Azospirillum brasilense Sp 7

Root

1978 Brazil

7.082.77

68.33

Complete 5951

2015

Unpublished

Azospirillum brasilense V6

Plant rhizosphere

2014 Israel

7.092.52

68.39

Contig (595)

6381

2017

Unpublished

Azospirillum brasilense 2A1

Root

2013 Argentina

7.257.81

68.46

Scaffold (137)

6656

2017

Unpublished

Azospirillum brasilense REC3

Root

2006 Argentina

7.228.95

68.71

Scaffold (137)

6574

2018

Fontana et al. (2018)

Azospirillum brasilense Ab-V5

Maize field

1998 Brazil

6.934.49

68.41

Contig (63)

6314

2018

Hungria et al. (2018)

Azospirillum brasilense Ab-V6

Soil

1998 Brazil

7.197.20

68.29

Contig (63)

6604

2018

Hungria et al. (2018)

Azospirillum brasilense HAMBI_3172 (LGM4376)

Oryza sativa, root

1977 Bogor Indonesia

7.139.72

68.98

Scaffold (181)

6386

2018

Unpublished

Azospirillum brasilense SR80

Seedlings

1988 Russia

7.146.59

68.26

Scaffold (228)

6635

2018

Unpublished

Strain

Release Genes date References

The genome availability improved the possibility of reconstructing the metabolic pathway and predicting metabolic models, integrating data, and biologic interpretation on niche adaptation of A. brasilense. Hongsheng et al. (2018), comparing 151 plantassociated bacteria genomes, showed that specific biosynthesis pathways are possibly involved in the plantebacteria interactions. Probably due to their high genome plasticity, Azospirillum members are heterogeneous and possess multiple mechanisms involved in plant growth promotion and bacterial adaptation. The availability of complete genome sequences of PGPB collections will allow the identification of appropriate genomic markers and/or novel PGP-associated genes, providing a faster screening and the opportunity for the discovery of new strains with plant promotion or beneficial properties.

I. Bacteria

84

6. Azospirillum

At present, researchers possess an assortment of primers to amplify different genes to identify new Azospirillum isolates for phylogenetic, biotechnologic, and molecular studies. Till whole-genome sequencing is broadly accepted as a standard method in bacterial identification, single-gene genotyping, such as that presented here, stands as the relatively confident and low-cost technique that permits quick identification of new Azospirillum isolates.

4. Beneficial role of the genus Azospirillum in agroecology The genus Azospirillum is one of the best studied PGPB and the most used in agriculture. Although more than 20 species have been described within the genus at present (DSMZ, 2018; Bashan and de-Bashan, 2010; Fukami et al., 2016), A. brasilense and A. lipoferum are the best characterized both biochemically and genetically (Fibach-Paldi et al., 2012) and the most widely used in commercial inoculants. This microorganism colonizes more than 100 plant species, and abundant information exists about the improvement in plant growth, development, and production in field conditions (Bashan and de-Bashan, 2010; Cassan and Diaz-Zorita, 2016). Bashan and Levanony (1990) proposed the “additive hypothesis” that considers multiple mechanisms rather than one mechanism participating in the association of Azospirillum with plants. Although early studies regarding the effects of Azospirillum to induce plant growth were mainly associated with the capacity to fix N2, today it is known that this bacterium has different additional mechanisms that contribute to greater growth and crop yield. Among them, the phosphate solubilization, hormone and/or siderophore production (Puente et al., 2009), phytopathogens control (Bashan and de-Bashan, 2010), and protection against abiotic stress like drought, salinity, or toxic compounds have been characterized.

4.1 Plant growth-promoting properties Azospirillum can promote growth indirectly, that is, by mechanisms that occur outside the plant, or by direct mechanisms that occur within the plant and directly affect its metabolism (Glick, 1995; Vessey, 2003). Within the latter, the most outstanding feature of the genus, and to which it owes its reclassification as Azospirillum, has been the ability to fix atmospheric N2 (Okon et al., 1983) through the process known as biologic nitrogen fixation. In this process occurs the conversion of atmospheric dinitrogen (N2) to ammonia (NH3), catalyzed by the enzyme nitrogenase. Then, plants can readily assimilate NH3 to produce different nitrogenous biomolecules such as chlorophyll, amino acids, ATP, and nucleic acids. The level of nitrogen fixation is established by several factors, for example, soil temperature (Azospirillum species thrive in more temperate and/or tropical environments), the capacity of the host plant to provide a rhizosphere environment low in oxygen pressure, the availability of host photosynthates for the bacteria, the competitiveness of the bacteria, and the efficiency of nitrogenase. Numerous evidences have contributed to support this mechanism, such as the increase of the total nitrogen content in the vegetal tissues, the improvement of the N balance of plants and the reduction in the doses of nitrogenous fertilizations required in numerous plant species, after the inoculation with Azospirillum (Dalla-Santa et al., 2004).

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4. Beneficial role of the genus Azospirillum in agroecology

85

However, Bashan and Levanony (1990) determined that of the total N increase in the plant, less than 20% corresponded to nitrogen fixation by Azospirillum. After the fixation of nitrogen, the production of hormones is the best characterized of Azospirillum features. Azospirillum can synthesize hormones and other compounds, as polyamines and amino acids in culture media. Among the hormones, auxins, mainly indole-3-acetic acid and gibberellins, seem to play a protagonist role (Spaepen et al., 2014). Also, it was reported to be able to synthesize in vitro other hormones such as abscisic acid, salicylic acid, and ethylene (Bashan and de-Bashan, 2010). Hormones increase the development and improve the architecture of the roots; therefore, a better absorption of minerals and water is obtained, which in the end has repercussions for the benefit of the whole plant (Bashan and de-Bashan, 2010). It was reported that IAA is produced during all stages of culture growth and, even more, it was reported that Azospirillum produces auxin-type molecules, such as indole acetamide, indole lactic acid, indole butyric acid, indole acetaldehyde, indole ethanol and indole methanol, and phenyl acetic acid (Bashan and de-Bashan, 2010; Somers et al., 2005; Spaepen et al., 2014). Some authors postulate that the production of hormones would have a higher incidence than the N2-fixation; so, for example, de-Bashan et al. (2008b) did not observe an increase in root growth of inoculated plants with a mutant of A. brasilense with deficient production of IAA and high N2-fixation activity. However, similar studies also showed that IAA alone cannot explain the total growth-promoting effect observed by Azospirillum inoculation (Spaepen et al., 2014). The wild-type A. brasilense Sp245 can produce nitric oxide (NO) in vitro under anoxic and oxic (or aerobic) conditions (Creus et al., 2005). The latter can be achieved by possible different pathways, such as aerobic denitrification and heterotrophic nitrification. NO is produced during the middle and late logarithmic phases of growth (Molina-Favero et al., 2008). It was observed that an NO-dependent promoting activity in A. brasilense Sp245 induces morphologic changes in tomato roots, regardless of the full bacterial capacity for IAA synthesis. An IAA-attenuated mutant of this strain, producing up to 10% of the IAA level, compared with the wild-type strain, had the same physiologic characteristics and slightly less effect on root development. When the NO was removed, using a chemical NO scavenger, both types of root formation were inhibited. This demonstrates that NO-mediated Azospirillum induced branching of roots. These results provide further evidence of an NO-dependent promoting activity of tomato root branching, regardless of the bacterium’s capacity for synthesizing IAA (Molina-Favero et al., 2008). Although the IAA and other metabolites produced by the bacteria promote a beneficial effect for the plant, this is as collateral effect of the real objective by which the bacteria produce them, which is for their own survival. Van Puyvelde et al. (2011) proposed that IAA allows a bacterium to adapt itself to the plant rhizosphere, by changing its arsenal of transport proteins and cell surface proteins. Azospirillum is among the PGPB that produce the enzyme 1-aminociclopropane-1carboxylate (ACC) deaminase, which decreases ethylene levels by cleavage of the plant ethylene precursor, ACC, into ammonia and ketobutyrate. Some authors consider that the production of ACC-deaminase is one of the key characteristics of PGPB in the promotion of plant growth since ethylene is usually induced by different stresses and, when present in high concentrations, can lead to plant growth inhibition or even death (Glick, 2012). Plant growth promotion results from a direct relationship between IAA, ethylene, and

I. Bacteria

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ACC-deaminase. The bacterium produces IAA that stimulates the multiplication and/or elongation of the plant cells but also induces the synthesis of the enzyme ACC-synthase and the formation of ACC, the precursor of ethylene. Bacteria that have the ACC-deaminase can regulate the high ethylene levels, avoiding the deleterious effects of this. As a result of this interaction between IAA and ACC-deaminase, the ethylene levels of the plant decrease, and in this way, the ACC-deaminase facilitates the stimulation of plant growth by IAA (Glick, 2012). For this reason, among the PGPB that produce IAA, those that have the ACC-deaminase are more efficient in stimulating plant growth than the PGPB that do not have it (Glick, 2012). The lipopolysaccharides (LPS) of the bacteria would be other molecules involved in the effect of promoting plant growth. For example, Vallejo-Ochoa et al. (2018) showed that LPS of A. brasilense Sp245 produced early plant growth and biochemical responses in wheat (Triticum aestivum L.) seedlings. They suggest that the ionically bound cell wall peroxidase enzymes could be a molecular target of A. brasilense LPS and that the recognition or association of LPS by plant cells could play a critical role during plant growth regulation by A. brasilense LPS. Other molecules related to the ability of plant growth induction by Azospirillum are the polyamines. These are low molecular weight aliphatic polycations present in all eukaryotes and most prokaryotes, and they play multiple roles in cell growth, survival, and proliferation and also in stress resistance. Several authors have reported that Azospirillum produces different polyamines like spermidine, spermine, putrescine, and cadaverine; however, the latter is the one that has been most related with root development and adventitious root formation (Niemi et al., 2001). Cassán et al. (2009) indicate that the strain A. brasilense Az39 promoted root growth and helped mitigate osmotic stress in rice seedlings due, in part, to cadaverine production, and Bashan et al. (2004) proposed cadaverine as the main polyamine contributing to the whole plant response to Azospirillum inoculation. Phosphorus is considered the most limiting macronutrient for plants after nitrogen, mainly because it is found in an unavailable form, usually precipitated by reacting with Ca, Fe, or Al, depending on the soil pH. Some microorganisms, such as Azospirillum sp., solubilize phosphate by pH modification through the secretion of organic acids from sugar metabolisms present in plant exudates (Goswami et al., 2014). However, not all Azospirillum strains have the same capacity to solubilize phosphates or do so in different ways. For example, some strains of Azospirillum have been described that can solubilize phosphate without the presence of root exudates (Puente et al., 2009). In soils with high pH and calcium content, typically of arid areas, a lowering pH of the rhizosphere increases the solubility of phosphorus and also iron, improving their availably for the plants. It was proposed that lowering of the pH effect is induced by Azospirillum, which produce a signal cascade (still of unknown nature) that alters the plant membrane permeability, affecting proton and organic acid extrusion (proton pump) (Bashan and de-Bashan, 2010; Bashan and Levanony, 1990). An evidence of this was determined by the exposure of wheat roots to A. brasilense Cd that enhanced the proton efflux of the root after inoculation (Bashan and Holguín, 1997). Amooaghaie et al. (2002) determined that this effect would be dependent on both bacterial strain and plant genotypes combination. Azospirillum halopraeferens does not produce acid in the presence of glucose but can solubilize insoluble inorganic phosphate in vitro by unknown mechanisms (Seshadri et al., 2000). Results of

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Mohan and Radhakrishnan (2012) about P solubilization efficiency of diazotrophic bacteria determined that different isolates of Azospirillum analyzed did not show the highest solubilization potential compared to other PGPB species such as Bacillus or Pseudomonas. However, Collavino et al. (2012) have determined in a collection of phosphatesolubilizing bacteria isolated from acid soils of the northeast region in Argentina that the capacity of phosphate solubilization in vitro was not necessarily associated with the promotion of plant growth. The ability to produce siderophores is another mechanism by Azospirillum that can contribute to the promotion of plant growth. Siderophores are ferric iron-specific ligands of low molecular weight that the bacteria produce when the concentration of iron in the environment is limiting. Siderophores chelate the ferric ion (Fe3þ) with a high specific activity, and thus, serve as vehicles for the transport of Fe (III) into a microbial cell, but also make it available to the host organisms in favor of the plant nutrition since, in general, iron is a commonly limiting element (Fernández-Scavino and Pedraza, 2013). In addition to the classically recognized mechanisms that directly affect plant growth, the literature also mentions other mechanisms possessed by Azospirillum and other PGPB that can indirectly improve the growth of plants, as they can improve the performance of the plants against different biotic or abiotic factors. For example, volatile organic compounds (VOCs), characterized by low molecular weight and high vapor pressure, are produced by all organisms, including PGPB, as part of normal metabolism, and play important roles in communication within and between organisms. Santoro et al. (2011) examined the effects of VOCs released by Pseudomonas fluorescens, Bacillus subtilis, and A. brasilense on growth parameters in the aromatic plant Mentha piperita (peppermint). Growth parameters of plants exposed to VOCs of P. fluorescens or B. subtilis were significantly higher than those of controls or A. brasilense-treated plants. However, Amavizca et al. (2017) observed that remote effects (occurring without physical contact) of A. brasilense Cd and Bacillus pumilus ES4 on growth of the green microalga Chlorella sorokiniana UTEX 2714 enhanced the growth of the microalga, up to sixfold, and its cell volume by about threefold. Additionally, both bacteria remotely induced increases in the amounts of total lipids, total carbohydrates, and chlorophyll a in the cells of the microalga, indicating an alteration of the microalga’s physiology. A. brasilense Cd and Bacillus pumilus ES4 produced large amounts of VOCs, including CO2, and the known plant growth-promoting volatile 2,3-butanediol and acetoin. This constitutes a new model on how PGPB promote growth of microalgae that may serve to improve performance of Chlorella spp. for biotechnologic applications (Amavizca et al., 2017).

4.2 Biofilm production and colonization of plant roots The attachment of Azospirillum to the roots is considered the first requisite step for colonization of host plants (Steenhoudt and Vanderleyden, 2000). It is known that Azospirillum mainly colonizes the root surface; nonetheless, some strains are able to colonize plant internal tissues as endophytic bacteria that occupy tissues of living plants without showing external signs of infection or negative effects on the host (Schulz and Boyle, 2006; Steenhoudt and Vanderleyden, 2000). In this case, normally the bacteria enter the inner part of the plant using opportunities such as disrupted cortical tissues at the lateral root junction, lysed root hairs, or natural cracks on the plant tissues (Steenhoudt and Vanderleyden, 2000).

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Ultrastructural studies of Azospirillum localization on root surface have shown that it can be found all along the inoculated root system, but they are concentrated on the elongation zone, root tips, the base of root hairs, and in few cases on root hair tips (Bashan et al., 2004). It is well established that microorganisms in the natural environment live in a complex community bounded by a polymeric matrix comprising diverse structures and molecules such as exopolysaccharides (EPS), lipids, adhesins, extracellular DNA, and pilus, which enable to bacteria to form biofilms attached to inert or biotic surfaces. The formation of biofilms is a very important trait that allows bacteria to increase their capacity to adhere, colonize, and tolerate environmental stresses, as the biofilms provide them a protective niche. The second messenger cyclic di-guanylate monophosphate (c-di-GMP) is a key regulatory player in the transition between bacterial planktonic and sessile (biofilm) lifestyles. This molecule is biosynthesized by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs) enzymes, and numerous genes encoding these proteins occur in the Azospirillum spp. genomes (Ramírez-Mata et al., 2018a). To investigate the participation of DGCs in biofilm production in A. brasilense strains under abiotic conditions and during interaction with plants, Ramírez-Mata et al. (2018b) have used strains tagged with genes encoding autofluorescent enhanced-green fluorescent protein (EGFP) and MCherry fluorescent protein, or stained the EPSs with calcofluor white colorant (CWC) and further examination using confocal scanning laser microscopy (CSLM). The biofilm production was evaluated in A. brasilense Sp245-EGFP wild-type strain and its isogenic mutant, cdgC::smReEGFP, grown in static conditions in a microtiter plate and subsequently stained with CWC. The biofilm of the Sp245 strain was a uniform, a compact-structure with a thickness of 25 mm and abundant EPS production (Fig. 6.3, panels A, B, and C). However, the biofilm of the cdgC mutant was nonuniform in structure with variable thickness, scarce EPS production, and differences in local adhering cells (Fig. 6.3, panels D, E, and F). This clearly indicates that mutation in cdgC gene encoding the putative diguanylate cyclase

(A)

(B)

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Sp245-EGFP

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FIGURE 6.3 Confocal scanning laser microscopy (CLSM) images of A. brasilense EGFP tagged strains showing the biofilm formation and exopolysaccharides production stained with calcofluor white colorant (CWC). (A) A. brasilense Sp245-EGFP. (B) cells stained with CWC. (C) panels A and B merged. (D) A. brasilense cdgC:smR-EGFP. (E) cells stained with CWC. (F) panels D and E merged. Images were obtained from a Nikon CLSM C2þ, with a Plan Apo lambda 20X objective. All images were edited with NIS Elements confocal and ImageJ software. Black arrows indicate Sp245 wild-type strain and its isogenic mutant cdgC:smR strain.

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C affects the production of EPSs, which are important components of the biofilm matrix, leading to a severe reduction in biofilm production. In addition, to test the contribution of the cdgA gene encoding diguanylate cyclase A, which has been shown to be involved in biofilm production under abiotic conditions (Ramírez-Mata et al., 2016), seeds of wheat (Triticum aestivum), variety “Nana” were inoculated with the wild-type A. brasilense Sp7 strain and its isogenic mutant cdgA::kmR, and the strains were tagged with MCherry and EGFP fluorescent proteins, respectively (Fig. 6.4A). The inoculated plantlets were grown under hydroponic conditions and maintained in a growth chamber at 28 C (14 h) and 18 C (10 h). Seven days after bacterial inoculation the wheat roots were analyzed by CSLM, and aliquots were quantified for colonization by determining the CFU/mL (Fig. 6.4B). It was observed that the cdgA::kmREGFP-GmR strain showed a lower root colonization than the wild-type A. brasilense Sp7-MCherry-GmR strain, confirmed by the values of the CFU/mL (Ramírez-Mata et al., 2018b). Moreover, in the competition study a moderate reduction of the colonization of wheat root plants by both strains was observed under the conditions tested, indicating that the cdgA gene plays a role in biofilm development and colonization during the bacteriaeplant interaction. According to the work of Ramírez-Mata et al. (2018b), calcofluor staining of a polysaccharide-binding dye provides rapid and direct information on the involvement of genes in biofilm formation. The findings illustrated in this chapter encourage further studies to test other genes that may be involved in the bacteriaeplant interaction using the methods briefly described herein.

4.3 Use as biofertilizer There are different considerations as to what is defined as a biofertilizer. In a broad sense, this includes all organic resources, from plant, microbial, or animals, which applied to plants, to improve their growth by increasing the availability of primary nutrients; but in a stricter sense, a biofertilizer is restricted to living microorganisms (Vessey, 2003). Although the use of biofertilizers is an old practice in agriculture, since the green revolution, its use has been practically restricted to the family farming of underdeveloped countries. Nowadays, where the negative consequences of practices not compatible with the sustainability of the agroecosystem is already evident and irrefutable, biofertilization has been considered again as a fundamental practice to recover mainly the functionality of soils. The production of biofertilizers is dynamic and abundant, and this is possible thanks to the continuous contributions to the knowledge originated from the research on the properties of the microbiome present in the soil. Although much progress has been made, Arjjumend et al. (2017) argues that “soil biology is still largely a mystery, and scientists have only identified somewhere between 5% and 10% of the microbial species living in soil.” In addition to microorganisms, which include bacteria and fungi, arthropods, nematodes, and earthworms found in the soil, and each one has a specific function, the roots of the plants are also considered within the living organisms of the soil since they are a highly complex living system that is fundamental for the functioning of the soil as a whole (Hooper et al., 2005). The principles on plant nutrition, on which the green revolution was based, are focused on external inputs of chemical fertilizers, which “illusively” did not seem to need the full functionality of a living soil, have now been banished, and it has been understood

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2.5e+10

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2.0e+10 1.5e+10 1.0e+10 5.0e+9 0.0

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FIGURE 6.4 Confocal scanning laser microscopy (CLSM) images and CFU/mL of wheat (Triticum aestivum var. Nana) roots inoculated with 107 CFU/mL of A. brasilense and derivative tagged strains. (A) Panels 1, 2, and 3 show the roots inoculated with the A. brasilense Sp7 (pMP2449-5)-mCherry-GmR strain. Panel 3 shows the panels 1 and 2 merged. Panels 4, 5, and 6 show roots inoculated with the cdgA::kmR (pMP2444)-EGFP strain. Panel 6 shows the panels 4 and 5 merged. Panels 7, 8, and 9 show the wheat roots inoculated with both strains. Panel 7, the Sp7-mCherry strain; panel 8, the cdgA::kmR-EGFP strain; panel 9 shows the panels 7 and 8 merged. The panels 1, 4, and 7 show the visualization of the auto fluorescence of the roots. White arrows indicate A. brasilense cells; dark arrows indicate wheat plant roots. Images were analyzed by CLSM obtained with a Plan Apo lambda 20X objective. All images were edited with NIS Elements confocal and ImageJ software. Scale bar ¼ 10 mm. (B) Wheat plants inoculated with Sp7-mCherry-GmR strain (1), with cdgA::kmREGFP strain (2), and with both strains (3) for competition assays. The plantlets were maintained in a growth chamber for 7 days. Subsequently, plantlets were washed with sterile water, and mashed bacteria were counted using serial dilutions plated on Congo redeagar plates containing gentamycin (Gm) or kanamycin as needed. After 48 h of growth at 30 C, colonies were counted to determine CFU/mL. Data are the result of three independent experiments with each of the seven plants. *P-value of