Microbial endophytes: functional biology and applications 9780128196540, 0128196548

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Microbial Endophytes

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Microbial Endophytes Functional Biology and Applications

Edited by

Ajay Kumar Department of Post Harvest, Agriculture Research Organization, Volcani Centre, Rishon Lezion, Israel

Radhakrishnan E.K School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, India

Woodhead Publishing is an imprint of Elsevier The Officers’ Mess Business Centre, Royston Road, Duxford, CB22 4QH, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, 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: 978-0-12-819654-0 For information on all Woodhead publications visit our website at https://www.elsevier.com/books-and-journals

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Contents

Contributorsxi 1

Entry, colonization, and distribution of endophytic microorganisms in plants 1 Ajay Kumar, Samir Droby, Vipin Kumar Singh, Sandeep Kumar Singh, James Francis White 1.1 Introduction 1 1.2 The rhizosphere and its role in endophytic associations 2 1.3 Endophytes and host plant surfaces 3 1.4 Entry and colonization of plants by bacterial endophytes 5 1.5 Plant internalization and extraction of nutrients from microbes in the rhizophagy cycle 7 1.6 Genomic insights into host and endophyte interaction 8 1.7 Transmission of endophytes 10 1.8 Endophytic diversity 11 1.9 Conclusion or future prospective 21 References 22

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Benefits of plant-endophyte interaction for sustainable agriculture Aswani R., Vipina Vinod T.N., Ashitha Jose, Radhakrishnan E.K. 2.1 Introduction 2.2 Endophytic microorganisms 2.3 Endophytic bacteria 2.4 Endophytic bacterial colonization in plants 2.5 Transition from rhizospheric bacteria to endophytic bacteria 2.6 Molecular mechanisms of endophytic bacteria involved in its interactions with plants 2.7 Molecular mechanisms of plants involved in its interaction with endophytic bacteria 2.8 Benefits of plant–endophytic interactions in plant growth promotion 2.9 Benefits of plant–endophytic interactions in biocontrol of plant diseases 2.10 Conclusions References

3  

Plant growth-promoting mechanisms of endophytes Aswathy Jayakumar, Veena P. Kumar, Meritta Joseph, Indu C. Nair, Remakanthan A., Radhakrishnan E.K. 3.1 Introduction

35 35 36 37 37 38 38 40 41 45 48 57 57

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3.2 Bacterial endophytes and their diversity 3.3 Current understanding on the mechanisms of plant growth promotion by bacterial endophytes 3.4 Various mechanisms 3.5 Microbial production of IAA 3.6 ACC deaminase production 3.7 Phosphate solubilization 3.8 Nitrogen fixation 3.9 Siderophore production 3.10 Biocontrol 3.11 Competition 3.12 Antibiotics 3.13 Lytic enzymes 3.14 Induced systemic resistance 3.15 Ethylene 3.16 Quorum quenching 3.17 Plant probiotics 3.18 Conclusions References 4

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Endophytic bacterial strains induced systemic resistance in agriculturally important crop plants Jubi Jacob, Gopika Vijayakumari Krishnan, Drissya Thankappan, Dileep Kumar Bhaskaran Nair Saraswathy Amma 4.1 Introduction 4.2 Endophytes providing disease resistance and mode of action 4.3 Endophytes providing ISR against wilt diseases 4.4 Optimization of bioactive metabolite production by endophytes through statistical approach 4.5 Bioformulation of endophytes 4.6 Challenges related to the development of endophytic formulation 4.7 Future prospective in endophytic research 4.8 Conclusion References

58 59 59 59 63 64 64 65 65 65 66 67 67 68 68 68 69 70 75 75 77 89 91 92 93 95 96 97

Endophytes and seed priming: agricultural applications and future prospects 107 Ajay Kumar, Samir Droby, James Francis White, Vipin Kumar Singh, Sandeep Kumar Singh, V. Yeka Zhimo, Antonio Biasi 5.1 Introduction 107 5.2 Types of seed priming 109 5.3 Factors affecting seed priming processes 112 5.4 Role of endophytes in seed priming 112

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5.5 Future perspectives 5.6 Conclusion References

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117 118 118

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The chemical warfare involved in endophytic microorganisms-plant associations 125 Éder de Vilhena Araújo, João Guilherme de Moraes Pontes, Stephanie Nemesio da Silva, Luciana da Silva Amaral, Taicia Pacheco Fill 6.1 Introduction 125 6.2 Mechanisms used by endophytic fungal to colonize plant tissues 127 6.3 Mechanisms used by endophytic fungi to promote growth of plant 133 6.4 Increase of resistance of plant to biotic and abiotic stresses 138 References 147

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Endophytic microbial influence on plant stress responses Vinaya Chandran, Hitha Shaji, Linu Mathew 7.1 Introduction 7.2 How do endophytes help in stress tolerance? 7.3 Plant growth promotion by the endophytes 7.4 Abiotic stress alleviation by the endophytes 7.5 Biotic stress 7.6 Commercial applications of stress tolerant endophytes 7.7 Conclusion References

161 161 162 163 164 177 180 181 182

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Microbial bioformulation-based plant biostimulants: a plausible approach toward next generation of sustainable agriculture 195 Mohd Aamir, Krishna Kumar Rai, Andleeb Zehra, Manish Kumar Dubey, Sunil Kumar, Vaishali Shukla, Ram S. Upadhyay 8.1 Introduction 195 8.2 Concept of bioformulation, composition, and microbial metabolites 198 8.3 Production and marketing constraints 200 8.4 Bioformulation as biocontrol agents 206 8.5 Formulation and application methods 207 8.6 Microbial bioformulation-based plant biostimulants 209 8.7 Mechanisms implicated in plant biostimulatory effects on crop productivity 213 8.8 Current Scenario/Market Trends 215 8.9 Regulatory framework 216 8.10 Conclusion 217 References 218

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Genomic insights of plant endophyte interaction: prospective and impact on plant fitness Tejas C. Bosamia, Kalyani M. Barbadikar, Arpan Modi 9.1 Introduction 9.2 Microbial diversity

227 227 229

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9.3 Omics–unraveling plant–endophytic interaction 9.4 What makes microbes an endophyte? 9.5 Plant fitness: Plant–endophyte interaction 9.6 Perspectives: a way ahead References

230 234 234 241 241

10 Fungal endophytes-induced gene expression studies in biotic and abiotic stress management 251 Arpan Modi, Poonam Kanani, Ajay Kumar 10.1 Introduction 251 10.2 Endophytic fungi and their importance in agriculture 252 10.3 Gene expression analysis 254 10.4 Gene expression analysis in plant–microbe interactions 259 10.5 Conclusion and future prospects 265 References 266 11 Endophytic fungi from medicinal plants: biodiversity and biotechnological applications Kusam Lata Rana, Divjot Kour, Tanvir Kaur, Rubee Devi, Chandranandani Negi, Ajar Nath Yadav, Neelam Yadav, Karan Singh, Anil Kumar Saxena 11.1 Introduction 11.2 Endophytic fungi from medicinal plants 11.3 Biodiversity and distribution of fungal endophytes 11.4 Biotechnological applications 11.5 Conclusion and future prospects References

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273 274 275 283 293 294

12 Biosynthesis of silver nanoparticles from endophytic fungi and their role in plant disease management 307 B. Shankar Naik 12.1 Introduction 307 12.2 Mechanism of mycosynthesis of silver nanoparticles 309 12.3 Silver nanoparticles from endophytic fungi and their efficacy in biocontrol 311 12.4 Mechanism of antimicrobial action by silver nanoparticles 315 12.5 Factors affecting the mycosynthesis of metal nanoparticles 315 12.6 Nanoparticles in plant disease management 316 12.7 Conclusions 317 References 317 13 Biocommercial aspects of microbial endophytes for sustainable agriculture 323 H.C. Yashavantha Rao, N. Chandra Mohana, Sreedharamurthy Satish 13.1 Introduction 324 13.2 Incidence of microbial endophytes diversity 325

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13.3 Comparison of native and alien endophyte inoculants 13.4 Growth promotional aspects due to symbiosis 13.5 Deciphering disease suppressive mechanisms 13.6 Commercialization of endophyte products for sustainable agriculture 13.7 Bio market 13.8 Recent developments and applications of microbial endophytes 13.9 Conclusion and future perspectives References

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326 327 329 334 336 338 339 339

14 Microbial endophytes and their intellectual property rights 349 Ashutosh Rai, Avinash Chandra Rai 14.1 Introduction 349 14.2 Endophytes and their patents 352 14.3 Microbe’s intellectual properties-related conflicts 374 14.4 Role of WIPO governing intellectual properties of microbial organisms 376 14.5 Implementation of IP protection of microorganisms 378 14.6 The Budapest Treaty 379 14.7 Conclusion and prospects 384 References 385 Index389

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Contributors

Remakanthan A.  Department of Botany, University College, Thiruvananthapuram, Kerala, India Mohd Aamir  Laboratory of Mycopathology and Microbial Technology, Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India Luciana da Silva Amaral  Department of Chemistry, Universidade Federal de São Carlos, São Carlos, Brazil Éder de Vilhena Araújo  Institute of Chemistry, Universidade Estadual de Campinas, Campinas, Brazil Dileep Kumar Bhaskaran Nair Saraswathy Amma  Agro-Processing and Technology Division, CSIR – National Institute for Interdisciplinary Science and Technology (NIIST), Thiruvananthapuram, Kerala; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India Kalyani M. Barbadikar  ICAR-Indian Institute of Rice Research, Hyderabad, Telangana, India Antonio Biasi  Agriculture Research Organization, Volcani Centre, Rishon LeZion, Israel Tejas C. Bosamia  Biotechnology Lab, ICAR-Directorate of Groundnut Research, Junagadh, Gujarat, India Vinaya Chandran  School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, India Rubee Devi  Department of Biotechnology, Eternal University, Baru Sahib, Sirmour, Himachal Pradesh, India Samir Droby  Agriculture Research Organization, Volcani Centre, Rishon LeZion, Israel Manish Kumar Dubey  Laboratory of Mycopathology and Microbial Technology, Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India Radhakrishnan E.K.  School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, India

xii Contributors

Taicia Pacheco Fill  Institute of Chemistry, Universidade Estadual de Campinas, Campinas, Brazil Jubi Jacob  Agro-Processing and Technology Division, CSIR – National Institute for Interdisciplinary Science and Technology (NIIST), Thiruvananthapuram, Kerala; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India Aswathy Jayakumar  School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, India Ashitha Jose  School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, India Meritta Joseph  School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, India Poonam Kanani  Department of Agriculture Biotechnology, Anand Agriculture University, Gujarat, India Tanvir Kaur  Department of Biotechnology, Eternal University, Baru Sahib, Sirmour, Himachal Pradesh, India Divjot Kour  Department of Biotechnology, Eternal University, Baru Sahib, Sirmour, Himachal Pradesh, India Gopika Vijayakumari Krishnan  Agro-Processing and Technology Division, CSIR – National Institute for Interdisciplinary Science and Technology (NIIST), Thiruvananthapuram, Kerala; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India Ajay Kumar  Agriculture Research Organization, Volcani Centre, Rishon LeZion; Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, Rishon LeTsion, Israel Sunil Kumar  Laboratory of Mycopathology and Microbial Technology, Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India Veena P. Kumar  School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, India Linu Mathew  School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, India Arpan Modi  Agricultural Research Organization, Volcani Center; Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, Rishon LeTsion, Israel N. Chandra Mohana  Microbial Drugs Laboratory, Department of Studies in Microbiology, Manasagangotri, University of Mysore, Mysuru, Karnataka, India

Contributors

xiii

Indu C. Nair  Department of Biotechnology, SAS SNDP Yogam College, Konni, Kerala, India B. Shankar Naik  Department of P.G. Studies and Research in Applied Botany, Kuvempu University, Shimoga; Department of Biology, Government Science College, Chikmagalur, Karnataka, India Chandranandani Negi  Department of Biotechnology, Eternal University, Baru Sahib, Sirmour, Himachal Pradesh, India João Guilherme de Moraes Pontes  Institute of Chemistry, Universidade Estadual de Campinas, Campinas, Brazil Aswani R.  School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, India Ashutosh Rai  Division of Biotechnology, Indian Institute of Vegetable Research, Varanasi, Uttar Pradesh, India Avinash Chandra Rai  Institute of Plant Sciences, The Volcani Center, Agriculture Research Organisation, Bet-Dagan, Israel Krishna Kumar Rai  Laboratory of Mycopathology and Microbial Technology, Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India Kusam Lata Rana  Department of Biotechnology, Eternal University, Baru Sahib, Sirmour, Himachal Pradesh, India H.C. Yashavantha Rao  Department of Biochemistry, Indian Institute of Science, Bengaluru, Karnataka, India Sreedharamurthy Satish  Microbial Drugs Laboratory, Department of Studies in Microbiology, Manasagangotri, University of Mysore, Mysuru, Karnataka, India Anil Kumar Saxena  ICAR- National Bureau of Agriculturally Important Microorganisms, Mau, Uttar Pradesh, India Hitha Shaji  School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, India Vaishali Shukla  Laboratory of Mycopathology and Microbial Technology, Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India Stephanie Nemesio da Silva  Institute of Chemistry, Universidade Estadual de Campinas, Campinas, Brazil Karan Singh  Department of Chemistry, Eternal University, Baru Sahib, Sirmour, Himachal Pradesh, India Sandeep Kumar Singh  Centre of Advance Study in Science, Department of Botany, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India

xiv Contributors

Vipin Kumar Singh  Centre of Advance Study in Science, Department of Botany, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India Drissya Thankappan  Agro-Processing and Technology Division, CSIR – National Institute for Interdisciplinary Science and Technology (NIIST), Thiruvananthapuram, Kerala; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India Vipina Vinod T.N.  School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, India Ram S. Upadhyay  Laboratory of Mycopathology and Microbial Technology, Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India James Francis White  Department of Plant Biology, Rutgers University, New Brunswick, NJ, United States Ajar Nath Yadav  Department of Biotechnology, Eternal University, Baru Sahib, Sirmour, Himachal Pradesh, India Neelam Yadav  Gopi Nath P.G. College, Veer Bahadur Singh Purvanchal University, Salamatpur, Uttar Pradesh, India Andleeb Zehra  Laboratory of Mycopathology and Microbial Technology, Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India V. Yeka Zhimo  Agriculture Research Organization, Volcani Centre, Rishon LeZion, Israel

Entry, colonization, and distribution of endophytic microorganisms in plants

1

Ajay Kumar a, Samir Droby a, Vipin Kumar Singhb, Sandeep Kumar Singh b, James Francis Whitec a Agriculture Research Organization, Volcani Centre, Rishon LeZion, Israel; bCentre of Advance Study in Science, Department of Botany, Institute of Science, Banaras Hindu University, Varanasi, India; cDepartment of Plant Biology, Rutgers University, New Brunswick, New Jersey, United States Chapter outline head 1.1 Introduction  1 1.2 The rhizosphere and its role in endophytic associations  2 1.3 Endophytes and host plant surfaces  3 1.4 Entry and colonization of plants by bacterial endophytes  5 1.5 Plant internalization and extraction of nutrients from microbes in the rhizophagy cycle  7 1.6 Genomic insights into host and endophyte interaction  8 1.7 Transmission of endophytes  10 1.8 Endophytic diversity  11 1.9 Conclusion or future prospective  21 References  22

1.1 Introduction Plants interact with large numbers of microbial communities, in which some of them enter and reside in the plant tissue without causing any disease or otherwise negative impact. These intimately associated microbes are called as endophytes. The word “endophyte” is derived from two Greek words “endon” means within, and “phyton” means plant (Chanway, 1996). The term “endophyte” was first introduced by De Bary (1866) for the microorganisms growing inside plant tissues. Later on, definition and types of endophytes were modified as per researcher observations. Hallmann et al. (1997) have defined endophyte as the microbes that can be isolated from the surfacedisinfected tissues of plants, and those microbes that could survive inside their host system without causing any disease. Some researchers have also categorized endophytes on the basis of their types, that is, bacteria or fungi, and their relationship with the plants such as facultative or obligate (Rosenblueth and Martínez-Romero, 2006). However, Hardoim et al. (2015) have characterized endophytes on the basis of Microbial Endophytes. http://dx.doi.org/10.1016/B978-0-12-819654-0.00001-6 Copyright © 2020 Elsevier Inc. All rights reserved.

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Microbial Endophytes

colonization niche instead of their function. Initially, the term “endophyte” was used for the fungi that were documented from the internal cells/tissues of host plants but later on the concept had been changed and the bacterial communities also considered as endophytes (Chanway, 1996; Hardoim et al., 2015). Currently, it has been considered that endophytes are present in all plant species (Strobel and Daisy, 2003; Huang et al., 2007), and have been demonstrated to share a complex relationship with their host plants.

1.2  The rhizosphere and its role in endophytic associations The entry or colonization of endophytic bacteria into the host plant is a complex phenomenon and involves a series of events. The process of colonization usually starts from the communication between the specific components of the root exudates and the associated microbial communities (de Weert et al., 2002; Rosenblueth and MartínezRomero, 2006). The rhizosphere can be described as the region of soil adhered to the root and is directly influenced by plants and their associated microbiota, species, growth stages, and the physiology of the host plant. The roots of plants release significant amounts of exudates that influence diverse microbial communities in the rhizosphere (Singh et al., 2017, 2018). Root exudates are rich in organic substrates, such as carbohydrates, lipids, phenolics, amino acids, phytosiderophores, and flavonoids, these serve as chemoattractants and facilitate the communication between roots and microbes that ultimately help in recruiting bacterial endophytes from the rhizosphere and start colonization of host plant tissue (Badri and Vivanco, 2009). There are various reports available showing the evidence of direct involvement of root exudates in initializing the host tissue colonization by microbial entities. Oku et al. (2012) reported the role of amino acids present in root exudates of tomato plants and noticed their role as chemoattractants in the colonization of Pseudomonas fluorescens Pf0-1. The evidence in support was gathered from genomic studies pertaining to three genes namely ctaA, ctaB, and ctaC coding for sensory proteins Pfl01_4431, Pfl01_0124, and Pfl01_0354, respectively. These genes are homologous to Pseudomonas aeruginosa PAO1 pctA gene, exhibiting positive response toward 20, L-amino acids during initial root colonization in tomato (Oku et al., 2012). In another study Kost et al. (2014) reported the role of oxalate in root colonization by a strain of Burkholderia. The plant growth promoting strains were reported to utilize oxalate as a carbon source but pathogenic strains such as B. glumae and B. plantarii did not degrade the oxalate. Interestingly, the mutant strain Burkholderia phytofirmans PsJN lacked the ability to metabolize oxalate could not colonize lupin and maize, indicating oxalotrophy as a prerequisite for colonization by this endophytic species. In similar ways metabolites like malate and benzoates also act as chemoattractants and provide help in effective colonization of the plant (Lopez-de-Victoria and Lovell, 1993). Flavonoids have also been recognized as one of the important components of root exudates secreted by several plant species and could play effective role

Entry, colonization, and distribution of endophytic microorganisms in plants

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in endophytic colonization within the root hairs (Khare et al., 2018). The specificity and effectiveness is considerably determined by the chemical structure of flavonoids as reported by Scervino et al. (2006). There are various reports available confirming the participation of flavonoids as chemotaxis agents during host tissue colonization by endophytic strains of Rhizobium (Dharmatilake and Bauer, 1992; Khandual, 2007; Faure et al., 2009). Furthermore, some of the authors have also illustrated the role of flavonoids in effective colonization of host tissue by the endophytic strain Serratia sp. EDA2 and Azorhizobium caulinodans ORS571 (Webster et al., 1998; Balachandar et al., 2006). In this connection, Steinkellner et al. (2007) also studied the various functional aspects of flavonoids including hyphal growth differentiation and root colonization and concluded the role of flavonoids as effective signaling molecule in the various plant species and their active participation in plant–microbe interaction. In case of legume–rhizobium endophytic association, flavonoids facilitates chemotactic response and start signaling through nod factors, culminating into symbiotic association (Garg and Geetanjali, 2007). Strigolactone (SL), the phytohormones secreted by plant roots has also been demonstrated to act as signaling molecules. In a study, López-Ráez et al. (2017) have discussed the role of SL hormone and concluded that treatment was able to activate the release of oligomers acting as signaling molecules and provided help in tissue colonization. Further, Rozpądek et al. (2018) also reported the role of strigolactone as a signaling molecule during the initial colonization of host tissue by endophytic strain of Mucor sp.

1.3  Endophytes and host plant surfaces The entry or host tissue colonization by microbes is a complex phenomenon and is controlled by signaling molecules, proteins, and/or the secretory products of the plants as well as microbes. Generally, adhesion of a particular microbial strain to the host surface is considered as the first step of colonization. Subsequently, the microbes migrate toward the host surface in response to root exudates via chemotactic movement that precedes attachment (Begonia and Kremer, 1994) (Fig. 1.1). The attachment of bacterial cells to the plant surface is one of the most crucial step during endophyte colonization; in this process various structural components, such as flagella, fimbriae, pili, and the secretory products like exopolysaccharides (EPS), lipopolysaccharide (LPS), or cell surface polysaccharides may directly involve in the attachment (Sauer and Camper, 2001). There are various reports available that confirm the role of microbial appendages in surface attachment. Croes et al. (1993) have reported the role of flagella in the primary attachment of Azospirillum brasilense with the root surface of wheat. However, flagella-deficient mutants did not show attachment with the wheat roots. Dörr et al. (1998) have reported the association of type IV pili in the attachment of endophytic strain Azoarcus sp. BH72, with the root surface of rice. These small appendages flagella and pili have also been described to act as propellers leading to movement of microbes toward the plant surface via chemotactic

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Microbial Endophytes

Figure 1.1  Schematic representation of endophytic bacterial colonization and distribution in the endosphere of a plant root. (A) Invasion of bacteria into a plant using several root zones. White arrows shows translocation of bacteria in to the phloem and xylem and colonization type represented by different colored ovals. (B) Occurrence of endophytes either at the site of entry (indicated in blue) or in the intercellular space of the cortex and xylem vessels (indicated in green). Red and yellow spheres represents rhizospheric bacteria, which are unable to colonize inner plant tissues.

response and developed a weak attractive force to withstand any repulsive barriers that might originate due to electrostatic charges present on the cell envelope (Berne et al., 2015; Zheng et al., 2015). The bacterially synthesized product EPS could also facilitate bacterial attachment to the host plant surface during early stages of colonization as mentioned by Janczarek et al. (2015) for Rhizobium leguminosarum. Similarly, Meneses et al. (2011) have mentioned the role of EPS secreted by endophyte Gluconacetobacter diazotrophicus in the attachment and colonization of rice root endosphere. However, besides attachment EPS have also been reported to offer other advantages including protection of bacterial cell and host plant from oxidative damage and elevated level of free radicals. Similarly, Marczak et al. (2017) reported the role of EPS secreted by Rhizobium in symbiosis and colonization with the legume plants. Balsanelli et al. (2010) have described the role of LPS secreted by endophytic bacterial strain Herbaspirillum seropedicae in attachment and colonization of maize root. In addition, reports are also available in literature emphasizing the role of LPS N-acetyl glucosamine in binding with lectins present in maize root, and concluded their involvement as an essential in bacterial attachment and subsequent colonization in the host roots (Balsanelli et al., 2013).

Entry, colonization, and distribution of endophytic microorganisms in plants

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Plants respond differentially after attachment of microbial strain with the host surface that leads to significant variation in the pattern of gene expression as reported by Sauer and Camper (2001) in case of Pseudomonas putdia. Further, in depth study was conducted by De Mot and Vanderleyden (1991) pertaining the proteomics of outer membrane porin F (OprF) proteins and their role in attachment and host tissue colonization by P. fluorescens. OprF is a multifunctional outer membrane proteins commonly present on the outer surface of Pseudomonas and helps in the attachment with various surfaces and molecules (Bodilis and Barray, 2006). The function of OprF proteins as adhesive had also been observed in various plant species, such as barley, sunflower, maize (De Mot and Vanderleyden, 1991) cucumber, and tomato roots (Crespo and Valverde, 2009). Similarly, arabinogalactan proteins (AGPs), a glycoprotein present on the plant’s cell wall has also been documented to help in initial colonization of microbes at the different growth stages of the plants (Nguema-Ona et al., 2013). In a similar fashion, the important contribution of flagellin, a globular proteins of flagella during the attachment with host surface as reported in case of A. brasilense strain (RodríguezNavarro et al., 2007) is also evidenced. The responsible genes for the glycosylation of flagellin and LPS are the same, and it had been seen that mutation in these genes results in impairment of the attachment of A. brasilense (Rossi et al., 2016).

1.4  Entry and colonization of plants by bacterial endophytes After establishing in the rhizosphere and rhizoplane, bacterial endophytes are known to make their way inside the plant root, with subpopulations ranging from 105 to 107 cfu/g fresh weight (Hallmann, 2001). During colonization, pattern and sites are specific for each of the endophytic strain (Zachow et al., 2010). After attachment to the host surface, endophytes start penetrating in order to enter to the host tissue. Endophytic bacteria, however, may prefer various sites to enter the plant tissue; the most preferred entrance path is via root zone, aerial parts of the plants, including stems, leaves, flowers, and cotyledons (Zinniel et al., 2002). The process of penetration into the host can be mediated by passive or active process. The passive penetration occurs at the site of cracks present in the areas of root emergence, root tips that are created by deleterious organisms (Hardoim et al., 2008), whereas active penetration is achieved through attachment and proliferation of EPS, LPS, structural components, quorum sensing, providing considerable help in the movement, and multiplication of endophytes inside the plant tissues (Böhm et al., 2007; Dörr et al., 1998; Duijff et al., 1997; Suárez-Moreno et al., 2010). There are numerous reports present that have shown different entry modes and colonization patterns of endophytic strains. Apart from this, specialized and frequently studied interaction between nodulating bacteria and legumes is less well-understood. Although not experimentally proven, it has been proposed that endophytic bacteria produce low levels of cell-wall degrading enzymes as compared to phytopathogens that could produce deleteriously high levels of these enzymes and thus endophytes may avoid triggering plant

6

Microbial Endophytes

defence systems (Elbeltagy et al., 2000). Furthermore, another way by which endophytic bacteria escape their detection as a pathogen by host tissue is maintenance of low cell den­ sities (2–6 log cfu/gfw) as compared to pathogenic bacteria. During entry or colonization, microbial strains prefer the site having thin surfaces such as root hairs, or the elongation zone of the apical root meristem serving as one of the preferred site of rhizoplane. At favorable sites, endophytic microbial strain se­ cretes some lytic enzymes, such as lysozymes, cell wall degrading enzymes, cellu­ lases, facilitating the entry of bacterial strain through hydrolyzing external covering or plant cells (Compant et al., 2005; Reinhold-Hurek et al., 2006; Naveed et al., 2014). Reinhold-Hurek et al. (2006) have reported Azoarcus sp. BH72 species at the entry site having endoglucanase, a kind of cellulase and further confirmed the role of endoglucanase in endophyte colonization by mutant analysis of eglA gene. The mutant endophyte lacking eglA genes was unable to colonize plant tissues, whereas wild type strain invaded and colonized the host surface. Suzuki et al. (2005) have reported a nonspecific wax-degrading enzyme helping in colonization of Streptomyces galbus on the Rhododendron. Taking together, all these previous investigations have shown the ability of bacteria to utilize certain plant metabolites as an essential mechanism for successful establishment as endophyte. Successful colonization of endophyte involves compatible plant–microbe interactions. As the endophyte invades the host surface, it is recognized by the plant and crosstalk of signaling molecules is initiated (Rosenblueth and Martínez-Romero, 2006; Compant et al., 2010; Brader et al., 2014). The colonization of endophytic microbes depends upon various factors including microbial strains, host genotype, biotic and abiotic factors, nutrients limitation, UV light, etc. and most importantly, the strains better adapted to these factors are comparatively more efficient in getting entry into the plant tissues via various routes like natural opening such as hydathodes, stomata, etc., followed by colonization of host tissue (Hallmann, 2001; Hardoim et al., 2015). To date, numerous reports have presented the details regarding the colonization routes of endophytic microbial strains. In a study, Álvarez et al. (2010) reported the colonization pattern of Ralstonia solanacearum strain and concluded that strains firstly attached to surface followed by invasion of the extension of roots such as root hairs, root tips, lateral roots; however, they may also prefer to enter through mechanical binding during initial colonization. After entering the host tissues, strain may spread themselves upwardly in the plants via xylem vessels. In another study, Compant et al. (2005, 2008) studied the colonization route of strain Paraburkholderia phytofirmans PsJN and reported that endophytic strain entered through the exodermis layer of roots following cortical cells and crossed the barrier of endodermal layer leading to its access to the central zone. From this zone, the endophyte spread toward the upper part of plants through xylem vessels. At the site of xylem colonization, very few bacterial strains are able to cross the endodermal layer. Generally, the endophytic strains prefer unsuberized endodermal cells of the apical root zone to get entry inside host tissues (James et al., 2002; Roncato-Maccari et al., 2003; Compant et al., 2005; Gasser et al., 2011). Studies have demonstrated low concentrations of nutrients in the xylem tissues or plant sap that could be sufficient for the growth of endophytic bacteria (Madore and Webb, 1981; Sattelmacher, 2001; Bacon and Hinton, 2007). At the site of

Entry, colonization, and distribution of endophytic microorganisms in plants

7

cortex colonization, once the bacterial strains have crossed the exodermal barrier, they may remain localized at the site of entry (Timmusk et al., 2005) or move deeper into the host system such as cortex of the plant (Roncato-Maccari et al., 2003; Compant et al., 2005; Gasser et al., 2011). In phyllosphere colonization, bacterial strains are firstly attached to the surface of leaf and randomly distributed throughout. Some of them may enter into the leaf tissue via natural openings such as stomata, hydathodes, and influence their local environment. At this site, bacterial strains multiply and form a thin layer of biofilm, however, some of them may enter into the leaf tissue and start surviving as endophytes (Yaron and Römling, 2014). In a study James et al. (2001) have reported stomata as an entry site during colonization of Gluconobacter diazotrophicus strain in the sugarcane. Currently, various reports have confirmed the utilization of plant nutrient as source of energy by the endophytic microbes (Rasche et al., 2009) and carbon has been reported as the most preferred source for growth and survival of endophytes (Krause et al., 2006; Malfanova et al., 2013). However, Iwai et al. (2003) have reported endophytic pseudomonads isolated from cucumber plants with the ability to utilize l-arabinose as one of the most abundant sugars available in the xylem fluid utilized by endophyte as nutrient source. In another study, Krause et al. (2006) reported alcohol dehydrogenases as an essential component in the colonization of Azoarcus sp. BH72 in waterlogged rice. They also concluded from their study that, in waterlogged rice, alcohol was present abundantly and may be utilized as carbon source by the colonizing bacterial strain Azoarcus sp. BH72. Some reports have described the local colonization of endophytic strains and further there was no transmission to other parts of the host after successful colonization as documented in the case P. fluorescens strain invading olive plants (Prieto et al., 2011). Similar studies by Moulin et al. (2015) have also reported the colonization of Rhizobium strain only in the symbiotic zone of root nodule of legume. After colonization or entry of endophytic strains into the plant tissue, they may colonize locally or spread systemically (Afzal et al., 2019) to the upper parts of the host tissues. It has been mentioned that 103–104 cfu/gfw population density is established in the ground tissue of root and stem (Compant et al., 2010). The above ground migration of endophytes depends upon their functional and physiological requirements and the strain could move upwardly as above ground tissues are well-adapted for the particular environment and endophytic niche (Hallmann, 2001). The movement of the endophytic strain within the host tissue is however, largely executed by lateral appendages such as flagella, pilli, or the transpiration stream of the plants similar to transport of plant nutrients (Compant et al., 2005; James et al., 2002).

1.5  Plant internalization and extraction of nutrients from microbes in the rhizophagy cycle Recent studies have shown that plants internalize soil microbes (bacteria and fungi) into plant roots and oxidatively extract nutrients from them in a process that has been termed “rhizophagy” (Paungfoo-Lonhienne et al., 2010, 2013) or “rhizophagy cycle”

8

Microbial Endophytes

(White et al., 2018). In the rhizophagy cycle, plants attract soil microbes to the root tip meristem with root exudates, and then internalize microbes into root meristem cells, which have soft cell walls. The precise mechanism by which microbes are internalized into root meristem cells remains unknown but may involve previously discussed processes. After internalization microbes become situated in the periplasmic space (between cell wall and plasma membrane) of root cells. The root cell plasma membrane secretes superoxide (produced on membrane bound NADPH oxidases) onto microbes and this strips the cell walls from microbes, resulting in formation of microbe protoplasts (White et al., 2018). Superoxide causes microbe protoplasts to become porous and leak nutrients that are absorbed by root cells. Through the action of cyclosis (cytoplasm rotation or streaming) in root cells, microbe protoplasts are circulated around the periphery of root cells and broken into many smaller protoplasts, rapidly replicating the intracellular microbes. Intracellular microbes in root cells accumulate in the tips of root hairs and trigger root hair elongation by an as yet unknown mechanism; without microbes root hair elongation does not occur (Verma et al., 2017). Microbe protoplasts are ejected into the soil through pores that form in the elastic wall at tips of elongating root hairs after a wave of vacuolar expansion propagates from the base of the root hair to the tip. It is unknown what triggers the periodic ejection of microbes from root hairs. Once ejected from root hairs, microbes reform cell walls and move out into the rhizosphere soil to acquire additional nutrients. The rhizophagy cycle appears to occur in all plants that form root hairs, and may be an important mechanism for acquisition of nitrogen and soil micronutrients like iron, zinc, and magnesium (White et al., 2015, 2018). It seems evident that the rhizophagy cycle is a mechanism whereby plants use soil microbes as carriers of difficult to acquire nutrients (Fig. 1.2). Work is still being done to evaluate details of the rhizophagy cycle mechanism and determine its importance to plant growth (Domka et al., 2019).

1.6  Genomic insights into host and endophyte interaction Comparative genomics studies of close mutualistic or pathogenic endophytic strains have shown very similar genetic contents (Lòpez-Fernàndez et al., 2015; SheibaniTezerji et al., 2015) and this similarity may be used in differentiating strains as a pathogen or beneficial microbe for the host on the basis of genetic analyses. LòpezFernàndez et al. (2015) when comparing the virulence genes in endophytes and other symbiotic bacteria lead to the conclusion that there are only minor differences between endophytes and pathogens and that the similarities between these two groups are set above the species level. In plant–microbe interactions secretion of protein is a determinant factor and is required for beneficial interaction. The transport of specific proteins for particular functions such as biocontrol is of considerable importance as the immunity of host plant is enhanced multiple orders after transport of effector proteins from microbes to the host, and thus helping in marking a particular microbe as a endophyte or parasite (Jones and

Entry, colonization, and distribution of endophytic microorganisms in plants

9

Figure 1.2  Diagrammatic representation of nutrient mining by rhizophagy microbes. Plant roots secrete organic acids (citric, malic, and acetic acids) into the soil. Organic acids complex with metals in the soil (Fe3+, Mg2+, Co2+, Mn2+, Co2+, Cu2+, Ni2, etc.). Rhizophagy cycle microbes possess transporters that bind to these organic acid-metal complexes and absorb them into the microbe cells. Microbes then return to the plant root and enter into root cells where nutrients are extracted from microbes oxidatively.

Dangl, 2006). These effector proteins are recognized by the plant immune system and are demonstrated to participate in activation of effector-triggered immune responses particularly T3SSs and T6SSs genes in the plant (Jones and Dangl, 2006). Interestingly, in the case of mutualistic endophytes, genes for T3SSs are missing (Hardoim et al., 2015; Mitter et al., 2017; Reinhold-Hurek and Hurek, 2011). Reinhold-Hurek and Hurek (2011) have proposed the view that missing T3SSs showed characteristics of an endophytic lifestyle. Iniguez et al. (2005) have also reported a similar observation; mutants of T3SSs of Typhimurium showed increased endophytic colonization in Medicago truncatula. However, some reports are also available that have shown endophytic establishment of Pseudomonas strain in the root even in the presence of T3SS gene (Preston et al., 2001). Endophytic microbes generally contain genes for T6SSs, conferring them with the potential for plant–microbe interaction (Mitter et al., 2013; Reinhold-Hurek and Hurek, 2011). There are various reports in the literature showing the contribution of T6SSs genes in the control of phytopathogens and disease management (Mattinen et al., 2008; Schell et al., 2007). In addition, nod genes are also responsible factors for nodulation and symbiotic association between host and

10

Microbial Endophytes

bacterial strain. Various authors have reported nod genes in the genome sequence of nodule-forming bacteria such as Burkholderia phymatum strain STM815A (Amadou et al., 2008), Bradyrhizobium japonicum USDA110 (Kaneko et al., 2002), and Frankia spp. strain CcI3 (Normand et al., 2007).

1.7  Transmission of endophytes Plant-associated microbes interact with plants through various ways, and during colonization they may vector horizontally (plant or soil to plant), vertically (parent plant to seed), or in a mixed way (Bright and Bulgheresi, 2010). The transmission mode may also depend upon the ecological and evolutionary relationship between host and microbe. Microbes displaying symbiotic relationship with the host plant generally follow vertical transmission (Moran, 2006) and during transmission, parents (seeds and pollens) fulfill nutrient requirements. In many vertically transmitted symbiosis, the symbiont is obligate and spends its entire life inside the host plant (Bright and Bulgheresi, 2010; Herre et al., 1999). Some of the fungal species are known to prefer vertical mode of transmission via the seeds and is well-documented by various authors (Schardl, 2001; Wilkinson and Sherratt, 2001; Foster and Wenseleers, 2006). Generally, bacterial endophytes employ a horizontal route of transmission and it has been also seen that bacterial count in the soil or in other environment is higher than the seed or the seed grown under artificial conditions (Hardoim et al., 2012). Some of the naturally existing bacteria, after entry to the host tissue, may act as endophytes and thus may be transmitted to the next generation in a similar way as pathogens. Many of the bacterial species are known to infect different plant species through the similar horizontal mode (Ma et al., 2011; Compant et al., 2005; Khan et al., 2012). The horizontal transmission mode of beneficial bacteria appears optimal for the host system, because endophytic strains provide resistance against various biotic and abiotic stresses that may directly influence the plants (Carroll, 1988; Schlaeppi and Bulgarelli, 2015; Bulgarelli et al., 2012; Lundberg et al., 2012; Peiffer et al., 2013; Schlaeppi et al., 2014; Edwards et al., 2015; Verma et al., 2017). Some of the endophytic bacterial strains, however, may employ a mixed mode of transmission and this may depend upon the surrounding environmental conditions. There are various reports available that have confirmed the existence of bacterial inhabitants as endophyte inside the seed or the vertical mode of transmission. In the last few decades, the microbiome of seeds is gaining high importance and attracting researchers to explore their hidden potentials (Verma and White, 2019). The endophytic microbial isolates from different plant seeds have been reported by various authors from hosts such as alfalfa (Charkowski et al., 2001), rice (Hardoim et al., 2012; Cottyn et al., 2001; Bacilio-Jiménez et al., 2001; Kaga et al., 2009; Okunishi et al., 2005; Verma et al., 2017), maize (Liu et al., 2013; Johnston-Monje and Raizada, 2011), tobacco (Mastretta et al., 2009), coffee (Vega et al., 2005), quinoa (Pitzschke, 2016), common bean (López-López et al., 2010), grapevine (Pitzschke, 2016), barley (Zawoznik et al., 2014), and pumpkin (Fürnkranz et al., 2012). Different parts of seeds such as seed coat, endosperm,

Entry, colonization, and distribution of endophytic microorganisms in plants

11

and embryonic tissue have been reported to be occupied by various types of bacterial communities (Mitter et al., 2017; Compant et al., 2011; Glassner et al., 2018). Rhizomes of plants may also act as seed and harbour various groups of bacteria as endophyte (Kumar et al., 2016). There are numerous bacterial genera such as Bacillus, Pseudomonas, Klebisella, Burkholderia, Penibacillus, Staphylococcus, Pantoea, Acinetobacter that have been the most commonly reported seed endophytes. Inside seeds, these endophytic bacterial strains mediate various beneficial interactions such as nutrient acquisition, synthesis of growth regulators, along with biotic and abiotic stress management. However, it is not necessary that all the inhabiting seed bacteria colonize the seedlings or are transferred from parent to offspring plants. The best evidence in support of vertical transfer of endophytes via seed comes from the studies demonstrating overlap in endophyte taxa between seed and seedling (Ferreira et al., 2008; Gagne-Bourgue et al., 2013; Ringelberg et al., 2012; Verma and White, 2019). Other studies have also reported the continued transfer of particular endophytic strains across generations in rice and maize (Mukhopadhyay et al., 1996; Liu et al., 2012), thus supporting vertical transfer. And at least in maize, there is some evidence of long-term conservation in the seed endophytic community; noteworthy, seeds of some genetically related maize hybrids have been found to host similar bacterial taxa (Liu et al., 2012). In an experimental investigation based on terminal restriction fragment length polymorphism (RFLP) of 16S rDNA, the presence of the same genera across several genotypes of maize, including its ancestor teosinte was documented (Johnston-Monje and Raizada, 2011). Further, different bacterial species can colonize the seeds horizontally from the external environment via flowers, fruits, and during seed dispersal.

1.8  Endophytic diversity In the last few years, exploration and isolation of endophytic microbes have been carried out using new technologies and “omics.” Every plant species, which is growing in the natural environment, has endophytic microbial communities, and it is a peculiar exception, if any plant does not have an endophytic community of microbes (Partida-Martinez and Heil, 2011; Afzal et al., 2019). Currently, more than 16 phyla or 200 genera of bacteria have been reported as endophytes in various plant species. These bacterial genera include both cultivable and uncultivable strains (Malfanova et al., 2013); Proteobacteria followed by Actinobacteria, Firmicutes, and Bacteroidetes (Edwards et al., 2015) are the most dominant phyla, and contain numerous groups of bacteria such as Pseudomonas (Kumar et al., 2016), Bacillus (Deng et al., 2011) Burkholderia (Weilharter et al., 2011), Enterobacter (Taghavi et al., 2010), and Serratia (Taghavi et al., 2009). There are various reports that show similar types of observations inside roots. Marques et al. (2015) reported Gamma-Proteobacteria (including Enterobacter, Pseudomonas, and Stenotrophomonas genera) was the dominant group in the endosphere of sweet potato. Sun et al. (2008) studied endophytic bacterial diversity of rice roots

12

Microbial Endophytes

and revealed Beta-Proteobacteria (27.08% of the total clones) was the most dominant phylum among bacteria communities, whereas Stenotrophomonas was the dominant genus among all the endophytes. Similar observations in rice were reported by Ferrando and Fernández Scavino (2015) and Ren et al. (2015). Mendes et al. (2007) studied the endosphere of sugarcane, and found Burkholderia, Pantoea, Pseudomonas, and Microbacterium were the common genera whereas Burkholderia genus was the most dominant in the endosphere. Similarly Han et al. (2009) studied the interior root tissues of moso bamboo, and reported, 22 bacterial genera in which majority of root endophytic bacteria belong to phyla of Proteobacteria (67.5%). Burkholderia was the most common genus inside the roots, comprising 35.0% of the total isolates from root domain. However, in the leaf microbiome the endophytic bacterial genera were also dominated by Proteobacteria, Actinobacteria, and Firmicutes as reported by Costa et al. (2012) in the common bean plants. The overlapping of endophytic bacterial communities in the root and leaf confirm upward movement of bacterial group with the translocation through xylem. It is likely that the concentration of available nutrients in xylem is decreasing along the plant axis. This can explain the facts that the diversity and population density of endophytic bacteria decreases with the distance from the root and only a small number of bacteria reaches the upper parts of shoots, the leaf apoplast, and reproductive organs such as flowers, fruits, and seeds (Compant et al., 2010; Fürnkranz et al. 2011). Endophytic bacteria are generally present in plant parts including roots, stems, leaves, seeds, fruits, tubers, and ovules (Benhizia et al., 2004; Hallmann et al., 1997) (Table 1.1). Since 1940, there have been numerous reports of indigenous endophytic bacteria in various plant tissues including seeds and ovules (Mundt and Hinkle, 1976), tubers (Trevet, 1948), roots (Philipson and Blair, 1957), stems, leaves (Henning and Villforth, 1940), and fruits (Samish et al., 1961; Sharrock et al., 1991). The microbiomes of the root endosphere is significantly less diverse than the rhizosphere and bulk soil (Liu et al., 2017) and it has been estimated, inside the root microbial population varies in between 104 and 106 per gram of root tissues which is very much less than the bulk or rhizospheric soil (106–109) bacterial cells (Bulgarelli et al., 2013). The diver­ sity of endophytic microbes varies with their height, altitude, and organs. The diversity of endophytic communities varies with the differences in host plant species, genotype, location, growth stages of the host plant, and the local environment. (Hallmann and Berg, 2006; Shi et al., 2014; Ding and Melcher, 2016). Besides these factors, the omics approaches used to enumerate endophytic populations are also major limiting factors. During isolation of endophyte strains surface sterilization of the host tissues is the first or important phenomenon that influences the diversity and composition of endophytic communities, which in nature, concentration, and treatment time of the sterilizing agent also influences the diversity and population of endophytic microbial communities (Hallmann and Berg, 2006; Hallmann et al., 1997). It has been found that different plant species growing in the same soil have different patterns of microbial communities. Granér et al. (2003) reported diversity in the endophytic bacterial communities in different cultivars of Brassica napus, which were growing in the same type of soil. In another study, Rashid et al. (2012) observed different endophytic bacterial communities in the same tomato species which were growing in different types of

Beneficial features/PGP characters

Endophytic bacteria

Plant

Bacillus cereus and Bacillus subtilis

Teucrium polium L.

Bacillus sp. BETS11; B. subtilis, and Arthrobacter sp. BECS1 Bacillus sp. and Stenotrophomonas sp.

Seed Lycopersicon esculentum; Capsicum annuum Rawalakot, Azad Jammu Root Triticum aestivum IAA production, Phosphate solubilization, Plant biomass and Kashmir (AJK), L. and N accumulation Pakistan Synthesis of phytohormone Banaras Hindu University Root Cassia tora L. indole-3- acetic acid, campus, India ammonia, siderophores, HCN, and by solubilization of phosphate

Bacillus subtilis, Agrobacterium tumefaciens, Bacillus sp., Pseudomonas putida, and Pseudomonas sp. Klebsiella sp. PnB 10 and Enterobacter sp. PnB 11

Pseudomonas fluorescens G10 and Microbacterium sp. G16

Piper nigrum

Brassica napus

Collected from

Produced indole-3- acetic acid Wadi al-Zwatin, Saint (IAA), ammonia, phosphate Katherine Protectorate, solubilization Sinai Peninsula, Egypt Siderophore production, Andaman and Nicobar P-solubilization, IAA Islands, India production

References

Leaves

Hassan (2017)

Amaresan et al. (2012)

Majeed et al. (2015)

Kumar et al. (2015)

Stem

Jasim et al. (2013)

Root

Sheng et al. (2008)

13

Phosphate solubilization, Avajyothisree ACC deaminase production, Karunakara Guru siderophore production Research Centre for Ayurveda and Siddha, Uzhavoor, Kottayam, India Produced indole-3- acetic Nanjing, China acid, siderophores and 1-aminocyclopropane-1carboxylate deaminase, Increases in biomass production

Plant part

Entry, colonization, and distribution of endophytic microorganisms in plants

Table 1.1  Beneficial endophytic bacteria associated with different plants and their plant growth promoting properties.

(Continued)

Beneficial features/PGP characters Collected from Phosphate solubilization, and Brazil potential for plant growth promotion

Plant Cymbidium eburneum

Pseudomonas sp. ZoB2

Zingiber officinale IAA, ACC deaminase and siderophore

Paenibacillus sp. RM

Tridax procumbens

Plant part Meristem tissues

References Faria et al. (2013)

Ipomoea batatas (L.) Lam.

Produced indole-3- acetic acid, Seattle, Washington fix nitrogen, exhibit stress tolerance

Stems

Khan and Doty, (2009)

Glycine max L.

Increase nodule number per plant, increased soybean weight

Seed

Bai et al. (2002)

Rhizome

Jasim et al. (2014)

Root

Govarthanan et al. (2016)

Produce secondary metabolites, indole acetic acid, siderophores, ACC deaminase, biosurfactant and solubilize phosphate

A.E. Lods Agronomy Research Centre, Macdonald Campus, McGill University, Sainte-Anne-deBellevue Navajyothisree Karunakara Guru Research Centre for Ayurveda and Siddha, Uzhavoor, Kottayam, India Namakkal, India

Microbial Endophytes

Endophytic bacteria Paenibacillus lentimorbus and Paenibacillus macerans Enterobacter sp., Rahnella sp., Rhodanobacter sp., Pseudomonas sp., Stenotrophomonas sp., Xanthomonas sp. and Phyllobacterium sp. Bacillus subtilis and Bacillus thuringiensis

14

Table 1.1  Beneficial endophytic bacteria associated with different plants and their plant growth promoting properties. (Cont.)

Bacillus sp., Sphingopyxis sp.

Fragaria ananassa

Klebsiella sp. PnB 10 and Enterobacter sp. PnB 11

Piper nigrum

Bacillus subtilis OS-11

Ocimum sanctum

Bacillus sp. and Enterobacter sp.

Phoenix dactylifera L.

Rhizomes

Kumar et al. (2016, 2017)

Roots containing rhizospheric soil Meristematic tissues

Forchetti et al. (2007)

Dias et al. (2009)

Jasim et al. (2013)

Tiwari et al. (2010)

Yaish et al. (2015)

15

Indole acetic acid production, Tissue culture solubilizing inorganic laboratories Pouso phosphate Alegre (Minas Gerais, Brazil) Phosphate solubilization, Navajyothisree Stem ACC deaminase production, Karunakara Guru siderophore production Research Centre for Ayurveda and Siddha, Uzhavoor, Kottayam Increase the content of Central Institute of Leaves essential oil, enhancement Medicinal and of growth Aromatic Plants Lucknow, India ACC deaminase, IAA Sultan Qaboos University Seed production, chelate (Muscat), Al-sharqia, ferric iron and solubilize Al-Dakhilia and Alpotassium, phosphorus and Batinah regions, Oman zinc, and produce ammonia

Entry, colonization, and distribution of endophytic microorganisms in plants

Botanical garden of Bacillus cereus (ECL1), Curcuma longa L. Produced IAA, solubilized phosphate and siderophore Banaras Hindu Bacillus thuringiensis University, Varanasi, (ECL2), Bacillus India sp. (ECL3), Bacillus pumilis (ECL4), Pseudomonas putida (ECL5), and Clavibacter michiganensis (ECL6) Showed positive catalase and Córdoba, Argentina Achromobacter Helianthus oxidase activities, phosphate xiloxidans;Alcaligenes annuus L. solubilization sp., Bacillus pumilus

(Continued)

Endophytic bacteria

Plant

Psuedomonas resinovorans, Paenibacillus polymaxa, and Acenitobacter calcoaceticus Arthrobacter humicola YC6002

Gynura procumbens (Lour.) Merr.

Beneficial features/PGP characters

Seed germination and stem growth by producing phytotoxic compound Pseudomonas aeruginosa Triticum aestivum Phosphate solubilization PW09

Pseudomonas sp. Ph6gfp Bacillus subtilis EPC8 Acinetobacter sp. ALEB16

Jinju, Korea

Agricultural Farm of Banaras Hindu University, Varanasi, India Nanjing Agricultural Trifolium pratense Degrades phenanthrene, a toxic metabolite that enters University, China L. plant Plant length increases and Tamil Nadu agriculture Cocos nucifera fruit yield University, India Activating accumulation of China Atractylodes plant volatile oils, induced lancea abscisic acid and salicylic acid production ACC deaminase activity, Abandoned mine in Pinus sylvestris Indole acetic acid, South Korea siderophore production, and P solubilization

Plant part

References

Leaves

Bhore et al. (2010)

Root

Chung et al. (2010)

Stem

Pandey et al. (2012)

Root

Sun et al. (2014)

Root Leaves

Rajendran et al. (2010) Wang et al. (2015)

Root

Babu et al. (2013)

Microbial Endophytes

Bacillus thuringiensis GDB-1

Zoysia japonica

Collected from

Produce cytokinin compounds Forest research institute of Malaysia

16

Table 1.1  Beneficial endophytic bacteria associated with different plants and their plant growth promoting properties. (Cont.)

Achromobacter xylosoxidans strain AUM54

Indole acetic acid production; Xiaotangshan Seed phosphate solubilization; Geothermal Special siderophores production; Vegetable Base, nitrogen fixation and ACC Beijing deaminase activity ACC deaminase, increased Parangipettai region of Root Catharanthus plant ethylene levels and Cuddalore, Tamil Nadu roseus increased antioxidative enzyme content Valleyfield, QC, Canada Leaf Panicum virgatum Produced cellulases and capable of solubilizing L. inorganic phosphorus Lycopersicum esculentum Mill.

Microbacterium testaceum, Curtobacterium flaccumfaciens, Bacillus subtilis, Bacillus pumilus, Pseudomonas fluorescens, Sphingomonas parapaucimobilis, Serratia sp. and Pantoea ananatis Bacillus megaterium, Plectranthus Bacillus pumilus, tenuiflorus Bacillus licheniformis, Micrococcus luteus, Paenibacillus sp., Pseudomonas sp., and Acinetobacter calcoaceticus

Exhibited extracellular enzymatic activity

Al hada and Al shafa, Taif province, Northwest of Saudi Arabia

Xu et al. (2014)

Karthikeyan et al. (2012)

Gagne-Bourgue et al. (2013)

Root, stem, and El-Deeb et al. (2013) leaves

Entry, colonization, and distribution of endophytic microorganisms in plants

Bacillus subtilis HYT12-1

(Continued) 17

Plant Scutellaria baicalensis

Serratia nematodiphila LRE07, Enterobacter aerogenes LRE17, Enterobacter sp. LSE04 and Acinetobacter sp. LSE06 Acinetobacter sp, Agrobacterium sp, Bacillus sp, Brevibacillus sp, Burkholderia sp, Curtobacterium sp, Erwinia sp, Lactococcus sp, Pantoea sp, and Pseudomonas sp. Alcaligenes faecalis subsp. faecalis str. S8

Solanum nigrum L.

Beneficial features/PGP characters Collected from Produce secondary metabolites Chinese medicine with broad-spectrum plantation in antibacterial and antifungal Anhui Science and activities Technology University ACC deaminase, indole Sewage discharge canal acetic acid, siderophore bank of Zhuzhou and phosphate solubilizing Smeltery, Hunan activity Province, China

Plant part Root

References Sun et al. (2006)

Roots, stems, and leaves

Chen et al. (2010)

Eucalyptus sp.

Control of diseases and plant Piracicaba, site P growth promotion, as well (Instituto de Pesquisa e as for the production of new Estudos Florestais, SP, metabolites and enzymes Brazil)

Stem

Araújo et al. (2009)

Withania somnifera

Production of indole-3acetic acid and phosphate solubilization

Stems and fruits

Singh et al. (2019)

Teboulba, Tunisia

Microbial Endophytes

Endophytic bacteria Bacillus amyloliquefaciens ES-2

18

Table 1.1  Beneficial endophytic bacteria associated with different plants and their plant growth promoting properties. (Cont.)

Papaver somniferous

Bacillus subtilis LK14

Moringa peregrina

Rahnella aquatilis, Pseudomonas sp. Paenibacillus validus, Lysinibacillus fusiformis, Bacillus licheniformis, Pseudomonas putida, Microbacterium oleivorans, and Serratia plymutica

Picea abies

Pantoeaa ananatis, Pseudomonas putida, Brevibacillus agri, Bacillus subtilis and Bacillus megaterium

Oryza sativa

Citrus sinensis

Benzylisoquinoline alkaloid National Gene Bank (BIA) biosynthesis, IAA for Medicinal and production, ACC deaminase Aromatic Plants at CSIR-CIMAP, Lucknow Phosphate solubilization, Mountains of Jabal AlACC deaminase and acid Akhdar, Sultanate of phosphatase activity Oman Biological control Pokljuka, Slovenia

Roots, leaves, Pandey et al. (2016) capsules, and seeds

Phosphate solubilization, Fort Pierce, Florida siderophore production, nitrogen fixation, IAA synthesis, production of antibiotic and lytic enzymes (chitinase), induction of systemic resistance [salicylic acid production], and production of quorum sensing [N-acyl homoserine lactones] signals Phosphate solubilization Lakhimpur district of Assam, India

Bark

Latif Khan et al. (2016)

Seed

Cankar et al. (2005)

Root

Trivedi et al. (2011)

Leaves, stems, and roots

Matos et al. (2017)

Entry, colonization, and distribution of endophytic microorganisms in plants

Arthrobacter sp. SMR3, B. subtilis SMR15

(Continued)

19

20

Table 1.1  Beneficial endophytic bacteria associated with different plants and their plant growth promoting properties. (Cont.) Endophytic bacteria

Plant

Bacillus sp., Bauhinia Pseudomonas sp. purpurea Azotobacter sp., Vigna radiata Azotobacter vinelandii, Azotobacter chroococcum Acinetobacter sp. Phyllanthus ACMS25 and Bacillus amarus sp. PVMX4

Collected from Chennai district, Tamil Nadu Mandalay, Myanmar

Phosphate solubilization, IAA Foot hills of Western production, siderophore Ghats of Kalakad production, and ACC region, Tamil Nadu, deaminase activity and India hydrolytic ezymes such as cellulose, protease, and pectinase Intertidal zone of Vellar Avicennia marina Produce siderophore, phosphate solubilization, estuary, Tamil Nadu IAA production

Plant part References Stems, leaves Sunkar et al. (2018) and flowers Root and leaves Aung et al. (2011) tissues

Root

Joe et al. (2016)

Pneumatophores

Sona Janarthine et al. (2011) Microbial Endophytes

Bacillus sp., Enterobacter sp. and Sporosarcina aquimarina

Beneficial features/PGP characters Appreciable antioxidant and antimicrobial activity IAA production, phosphate solubilizing activities

Entry, colonization, and distribution of endophytic microorganisms in plants

21

soil; similar observations were reported in the in roots of canola plants grown at three different places (Germida et al., 1998). The surrounding of the host plant, including biotic and abiotic stress factors, also govern the diversity of endophytic microbial communities. In a study Siciliano et al. (2001) reported that plants growing in a petroleum contaminated site had endophytic bacterial strains that contained genes for degrading the contaminants.

1.9  Conclusion or future prospective Endophytic microbes are fascinating life forms surviving in a range of host plants. Their entry inside the intricate system of diverse plants holds a promising research area in the field of microbiology and agricultural sciences. Involvement of different plant as well as microbe-derived molecules has been proposed to play an important role in development of symbiotic relationship with different plants. Their entry inside the plants has been documented to offer different advantages to host systems including tolerance to numerous biotic and abiotic stresses and enhancement in crop nutrients and productivity. In spite of well-developed plant immune system, endophytes have evolved the strategy for entry into the host system. It has also been proposed that plants have evolved to internalize microbes for purposes of acquiring nutrients and defence. So far limited numbers of genes have been identified and proposed to contribute in the invasion of hosts in order to enter tissues of the host plant. Many endophytes employ natural openings for entering into the host system and their survival and transfer to other parts of the host system is considerably determined by the nutrient materials available in the xylem sap. In the rhizophagy cycle microbial endophytes are internalized into root cells prior to hardening of the plant root cell walls in the root tip meristem; and they are ejected back into the soil from tips of root hairs through pores that form in the wall of the expanding root hair tip. Identifying the genes facilitating the entry of endophytes inside the host tissue could be utilized to inoculate desirable microbial endophytes into plants. For instance, the identification of genes favoring the colonization of nitrogen-fixing microorganism and exploring the existing biochemical mechanisms to overcome the plant immune response could be a viable option to enhance the agricultural productivity without, or minimizing, the application of fertilizers. Further, detailed investigations of a huge diversity of endophytic microorganisms could help in identifying unexplored genes having possible application as drugs or medicines. The integrated involvement of scientists from different disciplines, including microbiology, agriculture, biochemistry, genetics, and molecular biology, could be helpful in developing a better understanding of how plant endophytes function, and in identifying applications.

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Aswani R., Vipina Vinod T.N., Ashitha Jose, Radhakrishnan E.K. School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, India Chapter outline head 2.1 Introduction  35 2.2 Endophytic microorganisms  36 2.3 Endophytic bacteria  37 2.4 Endophytic bacterial colonization in plants  37 2.5 Transition from rhizospheric bacteria to endophytic bacteria  38 2.6 Molecular mechanisms of endophytic bacteria involved in its interactions with plants  38 2.6.1 Chemotaxis  39 2.6.2 Adherence to the plant root surface  39 2.6.3 Penetration and colonization of bacteria in the internal parts of the plant  39

2.7 Molecular mechanisms of plants involved in its interaction with endophytic bacteria  40 2.7.1 Plant receptors  40 2.7.2 Plant hormone-signaling pathways  41

2.8 Benefits of plant–endophytic interactions in plant growth promotion  41 2.8.1 Mobilization of nutrients  42 2.8.2 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity  44 2.8.3 Production of phytohormones  44

2.9 Benefits of plant–endophytic interactions in biocontrol of plant diseases  45 2.9.1 Production of antimicrobial compounds  46 2.9.2 Cell wall-degrading enzymes  47 2.9.3 Induced systemic resistance  47

2.10 Conclusions  48 References  48

2.1 Introduction The agricultural sector is facing various challenges to produce sufficient amount of quality food in a sustainable manner with the increasing global population and decreasing food resources. The productivity is mainly affected by a plethora of biotic stresses caused by pathogens and herbivores (Iriti and Faoro, 2009; Gust et al., 2010; Thakur Microbial Endophytes. http://dx.doi.org/10.1016/B978-0-12-819654-0.00002-8 Copyright © 2020 Elsevier Inc. All rights reserved.

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and Sohal, 2013). However, the use of agrochemicals, which have been recommended to manage this problem and their inappropriate usage cause environmental toxicity and thereby negative impact on humans and animals (Aktar et al., 2009; Bhandari, 2014; Ferreira et al., 2012). In this context, agricultural bioproducts using plant beneficial microorganisms provide a promising alternative to the existing agrochemicals. This also provides effective and environment friendly solution to potentially ensure the production of good quality agricultural products to meet the agricultural requirement. Microorganisms and plants coexist in nature since time of evolution and their close association is required to promote plant growth and productivity. The establishment of plant–microbe interaction generally involves diverse chemical communication and crosstalk (Fernanda Chagas et al., 2018). Diverse range of microorganisms have been associated with plants including these at the aerial surfaces (phyllosphere), internal tissues of plants (endosphere), and root-associated soils (rhizosphere). However, the diversity, dynamic nature, and the composition of plant microbiota is based on a complex of multilateral interactions and several environmental factors including climate change, type of species, developmental stage of host plant, and the region compartmentalization. These plant-associated microorganisms have already been welldemonstrated for its plant beneficial features such as acquisition and mobilization of nutrients, production of phytohormones, and production of antimicrobial compounds for the growth and disease resistance of the host plant (Jimtha et al., 2014; Rohini et al., 2018). Although several metabolites from plants and microbes have been fully characterized, their roles in the chemical interplay between these partners are not well studied. Hence, the current chapter describes the interaction, colonization, and mechanisms of plant growth promotion by endophytic organisms which can be utilized for the sustainable agricultural practices.

2.2  Endophytic microorganisms Microbial endophytes are microsymbionts, which includes culturable and nonculturable microorganisms that inhabit the interior of the plant tissue and spend their life cycle without causing any detrimental effect to the host plant. The ability to enter and thrive in the plant tissues makes the endophytes unique and it exhibits multidimensional interactions within the host plant. The term “Endophyte” was first introduced by De Barry (1866). The most popular definition of endophyte is proposed by Hallmann et al. (1997) who stated that endophytes can be defined as the microorganisms that can be isolated from surface-disinfected plant tissue or extracted from within the plant. Hardoim et al. (2015) defined endophytes as microbes including bacteria, archaea, fungi, and protists that colonize the plant interior regardless of the outcome of the association. Usually, these endophytic microorganisms can be isolated by traditional culture-based method such as surface sterilization of the plant tissue followed by the inoculation onto the culture media (Jasim et al., 2013). Recently, many cultureindependent approaches are introduced such as genome sequencing of the 16S rRNA gene, the internal transcribed spacer regions, or through the whole genome sequencing of plant material to identify the plant-associated microorganisms at the genus and

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the species level by comparing with the available databases (Sessitsch et al., 2012; Taghavi et al., 2009; Ikeda et al., 2010; Turner et al., 2013).

2.3  Endophytic bacteria The endophytes can be from bacterial, fungal, or actinomycetes groups. Bacterial endophytes are well characterized from many plants and their intimate association with plant cell can implement a direct benefit to the host plant (Rosenblueth and Martinez-Romero, 2006). Generally, these endophytic microbial communities are considered as the subset of rhizophere population. Because most of the genera identified as endophytes are common rhizospheric organisms such as Bacillus, Pseudomonas, Burkholderia, Micrococcus, Microbacterium, Stenotrophomonas, and Pantoea (Sun et al., 2008; Romero et al., 2014; Hallmann et al., 1997; Sturz et al., 2000; Rosenblueth and Martinez-Romero, 2006; Marquez-Santacruz et al., 2010; Shi et al., 2014; Moreno-hagelsieb et al., 2015). Normally, endophytes originate from the rhizosphere and phyllosphere. Some of them are transmitted via seeds. Endophytes contain many genes, which code for plant beneficial traits and provide modulatory effects in growth and development of the host plant (Khare et al., 2018). They play a major role in the plant growth promotion mainly through the production of various phytohormones, acquisition of nutrients, biodegradation, and tolerance to biotic and abiotic stress. Various mechanisms of biocontrol properties have been demonstrated for these endophytic bacteria. They act against diverse phytopathogens through various strategies involving competition for ecological niche, production of antimicrobial substances, production of siderophores, and induced systemic resistance (ISR).

2.4  Endophytic bacterial colonization in plants Plant–endophytic interaction is a biological mutualistic relationship in which both the partners are benefited. Here the bacterial partner helps to increase nutrient absorption, plant growth promotion, gain of biomass, and enhanced disease resistance in plants mediated by the metabolites produced by them and in turn the plants provide shelter for the growth and survival of these microbes. The endophytes adopt various mechanisms to colonize within the host according to their habitat as a result of vertical or horizontal transmission. Based on the different mechanisms established by endophytes to colonize the plant tissues, they can be classified as “obligate,” “facultative,” or “passive” endophytes. Obligate endophytes are transmitted by seed and not through rhizosphere and they spread inside the host via vertical colonization. In the case of facultative endophytes, they are free living in soil and colonize internally under favorable conditions by horizontal transmission. Whereas passive endophytes colonize via open wounds along with root hairs (Khare et al., 2018). The roots and root hair cells contain a high number of endophytes

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and microbial entry here is directed by exudates which are rich in biomolecules, phenolic compounds, enzymes, nutrients, and other gene activation factors (Khare et al., 2018). Other routes of entry of microorganisms include tissue damage, natural openings such as lenticels (present in the periderm of stem), and stomata mainly present on leaves and young stems (Moreno-hagelsieb et al., 2015). Endophytic colonization within the host plant tissues occurs through entry followed by growth and multiplication of microorganisms (Kandel et al., 2017). Influence of biotic and abiotic stress, organic matter, nitrogen availability, and other physiological parameters also determine the survival of these microorganisms in plants and its colonization processes (Langner et al., 2018).

2.5  Transition from rhizospheric bacteria to endophytic bacteria During the invasion of microorganisms from the host rhizosphere to endosphere, the colonizing bacteria must be able to adapt the new environment and it should have the ability to overcome the plant defense responses such as production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). ROS- and RNS-scavenging enzymes such as glutathione peroxidase, glutathione S-transferase, catalase, and nitric oxide reductase participate in alleviating the harmful effects of stress (Hardoim et al., 2015; Santoyo et al., 2016). Therefore, the endophytic bacteria should possess the ability to detoxify ROS and RNS for its growth and survival within the plant tissues. The importance of ROS-detoxification in the early stages of root colonization in Gluconacetobacter diazotrophicus PAL5 has already been described and suggested that superoxide dismutase and glutathione reductase mutants were not able to colonize in the plants (Alqueres et al., 2013). Production of various phytoalexins by plants has also known to prevent the growth and survival of rhizospheric and endophytic bacteria. In such cases, different microorganisms have developed mechanisms to deal with these compounds through the activity of efflux pumps. One of them is the flavonoid–responsive RND family of efflux pumps, which includes several members such as MexAB-OprM from Pseudomonas syringae, AcrAB from Erwinia amylovora, XagID2689 from Xanthomonas axonopodis, and BjG30 from Bradyrhizobium japonicum (Stoitsova et al., 2008; Pletzer and Weingart, 2014; Chatnaparat et al., 2016; Takeshima et al., 2013). Some of these efflux pumps are responsible for plant colonization whereas some others are involved in the symbiotic association.

2.6  Molecular mechanisms of endophytic bacteria involved in its interactions with plants The successful colonization of endophytic bacteria in plants relay on various processes and can be divided into three stages (1) chemotaxis, (2) adherence to the plant root surface, and (3) penetration and colonization in the internal parts of a plant (Hardoim et al., 2015; Kandel et al., 2017). Each of these stages is mediated by

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various biomolecules, which drives dynamic changes in the expression of bacterial genes and in the colonized plants. The changes in the gene expression in plants and microorganisms can be determined by the holistic approaches of “omics” technology such as genomics, transcriptomics, proteomics, and metabolomics analysis and this could provide the detailed understanding on the upregulation and downregulation of genes encoding various metabolic profile (Kaul et al., 2016).

2.6.1 Chemotaxis Chemotaxis is the phenomenon behind bacterial motility to plant. Here, the bacteria is attracted by the exudates produced by root and several rhizodeposits facilitate the interaction of beneficial microbes with plants (Compant et al., 2010; Langner et al., 2018; Moreno-hagelsieb et al., 2015). The endophytic bacteria Serratia sp. EDA2 and Azorhizobium caulinodans ORS571 have been reported to colonize wheat through various mechanisms (Khare et al., 2018). The chemotaxis of free-living bacteria toward the roots primarily starts with the attachment to the rhizoplane through methyl–accepting chemotaxis proteins (MCPs). MCPs are transmembrane sensors that direct the bacteria toward attractants or away from the repellents (Scharf et al., 2016). However, previous reports on the inactivation of MCPs encoding gene Hsero-3720 has resulted in the twofold reduction in the efficiency of Herbaspirillum seropedicae SmR1 colonization and the inactivation of another MCP chemotaxis-like protein encoding tlp1 gene has displayed impaired colonization of Azospirillum brasilense sp7. in the plant roots (Greer-Phillips et al., 2004).

2.6.2  Adherence to the plant root surface Although the release of exudates can attract a wide variety of bacteria to the plant, only those which have the capability to adhere to the root surface colonize the internal tissues of plants. The attachment of bacteria to the plant roots is expected to be due to the formation of biofilm. Biofilms are bacterial communities in which cells are embedded in a matrix of extracellular polymeric compounds, which protect bacteria from deleterious conditions (Davey and O’Toole, 2000). Bacterial surface components and extracellular compounds like pili, flagella, lipopolysaccharides (LPSs), and exopolysaccharides (EPSs) in combination with quorum-sensing signals play important role in the adherence and biofilm formation. Previous report on the reduced colonization of rice root by G. diazotrophicus, PAL5 has revealed the deficiency of biofilm formation due to the inactivation of gumD gene coding the EPS. This implies the crucial role of biofilm formation in the colonization process of plant microbe interaction (Meneses et al., 2011).

2.6.3  Penetration and colonization of bacteria in the internal parts of the plant Rhizophagy is defined as the phenomenon in which the attached endophyes can enter into the plant tissues through the secretion of cell wall degrading enzymes (Singh et al., 2017; Paungfoo-lonhienne et al., 2013). Some of the endophytic bacterial

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population have been previously reported to possess the ability to produce the plant cell wall–degrading enzymes, which act against cellulose, xylulose, and pectins. This inturn facilitates the disruption of plant cell wall and makes the possible entry of bacteria. This it also facilitates its colonization through out the plant. This has been well studied in Azoarcus sp. BH72 where in mutants devoid of endoglucanase activity had a decreased ability to colonize the rice roots (Reinhold-Hurek et al., 2006). The importance of cell wall–degrading enzymes in the entry of bacteria is further confirmed by the upregulation of these enzymes in presence of root exudates. When exposed to root exudates, Bacillus mycoides EC18 have been demonstrated to exhibit the upregulation of genes encoding for hydrolytic enzymes and increased the penetration ability of this bacterium in plants (Yi et al., 2017). Root exudates primarily consist of sugars, polysaccharides, amino acids, aromatic acids, aliphatic acids, sterols, phenolics, plant growth regulators, secondary metabolites, proteins, and enzymes (Badri et al., 2009). In order to utilize these root exudates, the bacteria possess adequate transporters and enzymes. For example, the root exudates of maize and lupin contain the compound oxalate, which is utilized by bacteria through the activity of oxalate decarboxylase. This was further supported by the study on Burkholderia phytofirmans PsJN with the reduced colonization on maize and lupin upon the inactivation of the oxalate decarboxylase gene (Kost et al., 2014). In addition to these, the bacteria produce polyhydroxybutyrate (PHB) for its survival under adverse environmental conditions and a higher level expression of PHA biosynthesis genes has been observed in A. brasilense FP2 during the wheat-endophyte interactions (Camilios-Neto et al., 2014).

2.7  Molecular mechanisms of plants involved in its interaction with endophytic bacteria Plants mediate endophytic interactions through various receptors and phytohormone signaling pathways, which are involved in either symbiosis or defense mechanisms. Some of them are described as follows:

2.7.1  Plant receptors The ability of plants to recognize bacterial signals is primarily mediated by the family of receptor-like kinases (RLK), which include a leucine-rich repeat receptor-like kinases (LRR-RLKs), wall-associated kinases (WAK), lectin receptor-like kinases (LecRLKs), and Lys-motif receptors (LysM). The RLK can further be functioned as pattern recognition receptors (PRR), a key component of the plant innate immune system (Medzhitov and Janeway, 1997). The pattern-recognition receptors perceive the conserved molecular signatures present in the pathogenic microorganisms called pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) and act as the defense systems to counteract attack from phytopathogens (Jones and Dangl, 2006). PAMP recognition and subsequent initiation of defense signaling are referred to as PAMP-triggered immunity (PTI) and is generally considered as the first line of plant

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defense. But during the coevolution with plants, pathogens developed the strategy of injecting effectors to suppress PAMP-triggered immunity. In response, plants developed a second line of defense called effector-triggered immunity (ETI) also known as vertical resistance accompanied with a hypersensitive response (HR) (Chisholm et al., 2006; Jones and Dangl, 2006). In ETI, the interaction occurs through the plantderived resistance (R) genes and pathogen-derived avirulence (Avr) genes. Here the six distinct secretion systems (Type I–VIII) are responsible for the bacterial effectors. Among which, the type III system is used to secrete the effector into the plants which interfere with PTI to ensure the viability of the pathogen within the cell. When this occurs, the ETI is activated, leading to the release of the antimicrobial compounds and hydrolytic enzymes which finally leads to the encasement of pathogen as a callus at the infection site (Rajamuthiah and Mylonakis, 2014). The endophytic bacteria produce their own MAMPs distinct from the phytopathogens and thereby avoid the elimination by the plant's immune system (Vandenkoornhuyse et al., 2015). For example, the RLK receptor flagellin sensing 2 (FLS2) involved in recognizing the MAMP and binds to flg22 a 22-amino-acid peptide present in the N-terminal part of a flagellin. Recognition of flg22 leads to the rapid extracellular alkalization, ROS production, activation of a mitogen-activated protein kinase (MAPK) cascade, and the upregulation of pathogenesis-related genes in A. thaliana. At the same time, flagellin-derived epitopes of endophytic B. phytofirmans in grapevine differentially recognized from those of a bacterial pathogen such as Pseudomonas aeruginosa or Xanthomonas campestris and suggested that the flagellin of endophytic bacteria may have evolved to circumvent from the recognition by the plant's immune system (Trda et al., 2014).

2.7.2  Plant hormone-signaling pathways Phytohormones and their related signaling pathways play critical role in plant defense. The major influence of plant defense signaling pathways on its microbiome could be attributed to the following reasons. Firstly, these pathways can be activated by external stimuli and potentially alter the microbiome structure in the plant. Secondly, it helps to illustrate the role of plant-associated microbiomes in plant nutrition and disease resistance against biotic stress (Liu et al., 2017). Several studies have also reported the role of ethylene (ET), salicyclic acid (SA), and jasmonic acid (JA) in regulating the colonization of bacteria and their diversity distribution (Pinski et al., 2019).

2.8  Benefits of plant–endophytic interactions in plant growth promotion Recently, more ecofriendly and lesser toxic biological methods are in practice to replace the toxic agrochemicals to control infections in the agricultural sector. The aim of integrated management of plant infection is to incorporate biological, cultural and chemical methods to reduce the crop loss. The asymptomatic internal survival

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of the endosymbiotic microorganisms has a holistic signature in the disease management and plant growth executed through various direct and indirect mechanisms (Table 2.1). The direct mechanisms of plant growth promotion include mobilization of nutrients (e.g., phosphorous, nitrogen, and iron), lowering of ethylene-related stress in plants, and the production of plant hormones (Santoyo et al., 2016). Whereas, the indirect mechanisms involve the antagonistic effects toward phytopathogens and protection of the plant from pathogen attack (Compant et al., 2010). The mechanisms underlying this include the production of cell-wall degrading enzymes (chitinase and β-1,3-glucanase), antimicrobial compounds, and induction of ISR (Muthukumar et al., 2017). Through ISR, the endophytes can trigger the defense response in plants without forming any visible symptoms, whereas the systemic acquired resistance (SAR) is activated upon a primary infection by the pathogen followed by hypersensitive reaction and ISR.

2.8.1  Mobilization of nutrients Nitrogen is one of the important nutrients essential for the growth of all living organisms including plants and bacteria. The observation on nitrogen deficiency in the soil has recommended the use of large amounts of nitrogenous fertilizers to achieve the maximum crop yield (Zhang et al., 2015). Although nitrogen is abundant in the earth's atmosphere, about 78% of this gaseous nitrogen is not readily accessible by the bacteria. However, it can be assimilated efficiently by plants once it is converted into ammonia (Baas et al., 2014). Broad range of bacteria known as nitrogen-fixing bacteria have been known to convert this atmospheric nitrogen and fix nitrogen symbiotically in plants (Babalola, 2010; Jasim et al., 2014; Pérez-Montaño et al., 2014; Turan et al., 2016). Phosphorous, is the second most essential macronutrient after nitrogen for biological growth and development of plants. Eventhough soil is a large reservoir of inorganic phosphate, the concentration of soluble phosphorus in soil is very low. The phosphate solubilizing microorganisms are reported to have the ability to transform insoluble phosphorous to soluble form and make it available to the plants. The core mechanism behind the mineral phosphate solubilization is the production of organic acids and acid phosphatases (Illmer and Schinner, 1995). Among various organic acids produced, the production of gluconic acid play a major role in inorganic phosphate solubilization (Rodríguez et al., 2006). Previous reports suggested that the acids are produced in the periplasm of Gram-negative bacteria by a direct oxidation pathway of glucose (DOPG; non-phosphorylating oxidation) (Anthony, 2004). In the DOPG, the enzyme glucose dehydrogenase (GCD/GDH) and gluconate dehydrogenase (GAD) orient to the outer face of the cytoplasmic membrane and are able to oxidize the substrate in the periplasmic space (Chhabra et al., 2013). As a result, the organic acids diffuse freely outside the cells releasing high quantities of soluble phosphate from mineral phosphates by the release of both protons and metal complexing organic acid (Rodríguez and Fraga, 1999). Gluconic acid biosynthesis is commonly carried out by the enzyme glucose dehydrogenase (GCD) in the presence of the cofactor pyrroloquinoline quinone (PQQ) (Sharma et al., 2013). PQQ is a small, redox active molecule that serves

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Table 2.1  Impact of endophytic microorganisms on plant growth promotion. Endophytic bacteria

Plant growth promotion

References

Bacillus cereus and B. subtilis Penicillium chrysogenum and P. crustosum. P. phytofirmans PsJN

Plant growth promoting properties

Hassan (2017)

Biofilm formation, activation of plant defense-signaling pathways (salicylic acid, jasmonate, and ethylene) Nitrogen fixation, indoleacetic acid (IAA) production

Esmaeel et al. (2016)

Burkholderia sp., Herbaspirillum seropedicae

Siderophores production; IAA synthesis, ACC-deaminase, antifungal activity, nitrogen fixation Gluconacetobacter diazotrophicus, Biological nitrogen fixation, production of siderophores, Azospirillum amazonense, IAA synthesis and phosphate Burkholderia tropica, solubilization Herbaspirillum seropedicae, H. rubrisubalbicans, G. diazotrophicus Nitrogen fixation; IAA A. brasilense, Burkholderia synthesis, growth promotion, cepacia, Bacillus subtilis, B. antagonistic activity lentimorbus, Streptomyces sp. Azospirillum lipoferum Plant growth promotion Enterobacter sp., Pseudomonas sp., Stenotrophomonas sp. Shoot growth, cellular redox Bacillus spp. and Pseudomonas balance, and protein expression spp IAA production, siderophore Paenibacillus sp. Pantoea sp., production, phosphate Bacillus sp. solubilization, antifungal activity Phytoremediation, antifungal Enterobacter sp. strain PDN3, activity Siderophores, phosphorus Pseudomonas, Pantoea, and solubilization Bacillus Plant growth promoting Pseudomonas stutzeri A15 properties Antifungal activity, plant growth Bacillus, Paenibacillus, promotion Enterobacteriaceae, Staphylococcus, Microbacterium, Stenotrophomonas sp. Promotes early nodulation and Serratia grimesi BXF1 plant growth promotion Production of lipopeptide and Pseudomonas poae RE1-1-14 antifungal activity Bacillus amyloliquefaciens, B. japonicum, Azospirillum brasilense

Bao et al. (2013); Guimaraes et al. (2015) Sharma et al. (2013) Hungria et al. (2013)

Gírio et al. (2015) Pereira et al. (2013) Beneduzi et al. (2013)

Ferreira et al. (2013) Wang et al. (2010) Shiomi et al. (2008) Hungria (2011) Mastretta et al. (2009) Tamošiûnë et al. (2018) Herrera et al. (2016)

Pham et al. (2017) Scott et al. (2018) Tavares et al. (2018) Khalaf and Raizada (2018), Mastretta et al. (2009) Tavares et al. (2018) Zachow et al. (2015)

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as a cofactor for several bacterial dehydrogenases and the production of the PQQ molecule is encoded in the pqq operon which consists of six core genes pqq A, B, C, D, E, and F, of which PqqA, PqqC, PqqD, and PqqE are essential (Shen et al., 2012). Although iron is essential for all living organisms in many cellular functions, its bioavailability in the soil is limited. In these circumstances, the microorganisms associated with plants developed specific mechanisms for the assimilation of iron by the production of low molecular weight iron-chelating compounds called siderophores. These compounds chelate iron in the soil and generate soluble complexes that can be absorbed by plants (Ahmed and Holmström, 2014). Siderophore forms a complex with free iron and transport it into the cell by membrane receptor molecules through an energy-dependent process. These membrane receptor molecules are encoded by five genes, which are turned off when sufficient iron has been taken into the cell (Lewin, 1984). Siderophores are part of multicomponent system that transport the iron into the cell. Under iron deficiency, bacteria synthesize siderophore to increase number of receptor molecules, once the siderophore is excreted outside of cell thorough membrane receptor, it binds with the iron complex and transport the iron in to the cell (Davidson and Nikaido, 1991; Boos and Eppler, 2001). Later siderophore iron complex release into the cytoplasm with the help of membrane protein. In cytoplasm, the iron released from the complex may involve hydrolytic destruction of the siderophore molecule or the reduction of Fe3+ by a NAD (P) H-linked siderophore reductase. The resulting Fe2+ does not have a high affinity for siderophore and therefore dissociate easily from the complex. Therefore, this mechanism helps the plants to thrive in low iron soils. A key role for siderophore production has also been reported from endophytic Streptomyces sp. GMKU 3100 (Rungin et al., 2012) and its beneficial properties in rice plants have been established via studying siderophore-deficient mutants.

2.8.2  1-aminocyclopropane-1-carboxylate (ACC) deaminase activity Ethylene-related stress at higher concentrations may lead to the reduction in plant growth and development (Glick, 2014). Some microbes including bacterial endophytes use 1-aminocyclopropane-1-carboxylate (ACC) the immediate precursor of ethylene biosynthesis. As, the presence of increased level of ethylene negatively affects the plant growth (Ali et al., 2014; Glick, 2014), the enzyme ACC deaminase of microbes cleaves ACC through fragmentation of cyclopropane ring to yield ammonia and α-ketobutyrate. This facilitates the plant growth by lowering the ethylene levels Inoculation with bacterial ACC deaminase producers may decrease the endogenous ACC level in plant roots and therefore increases plant tolerance to stresses (Glick, 2014).

2.8.3  Production of phytohormones Phytohormones can act as a flexible signaling molecules which influences the gene expression, metabolism, and many other physiological processes of plant growth and

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development even at the low concentration (Mehmood et al., 2018). Endophytic bacteria have already been reported to have the presence of genes encoding for biosynthesis of indole 3-acetic acid (IAA), and cytokinins (CKs) (Zúñiga-Mayo et al., 2014) to boost plant growth and increase plant stress tolerance. Previous report on endophytic bacterium Sphingomonas sp. LK11 have demonstrated the enhanced growth of tomato mediated through the production of IAA (Khan et al., 2014). This highlights the role of phytohormone produced by endophytic bacteria for the increased agricultural productivity. IAA is considered as the biologically active form of auxin, one of the major phytohormone, which take part in the root initiation, cell division, cell enlargement, extension, and differentiation. They also impart effect on photosynthesis, regulating vegetative growth, lateral and adventitious root initiation, enhance the rate of xylem functioning, pigmentation, and various metabolic processes. There are mainly five metabolic pathways involved in the microbial biosynthesis of IAA and are indole-3-acetamide (IAM) pathway, indole-3-acetonitrile (IAN) pathway, tryptamine pathway, indole-3-acetaldoxime pathway and indole-3-pyruvate (IPyA) pathway. Many reports have demonstrated the ability of endophytic bacteria to produce IAA and therby influence the enhanced plant growth (Aswathy et al., 2013; Jasim et al., 2014; Jimtha et al., 2014; Sabu et al., 2019). Cytokinins are produced in the root tips and transported through the xylem to the shoot by translocation and also control cell differentiation in plant meristematic tissues (De Rybel et al., 2016). Several plant-associated bacteria have been found to express the cytokinin gene. Their addition to the growing plants can largely alter the plant's phytohormone composition. The increased cytokinin content and plant growth upon inoculation of lettuce with Bacillus subtilis further supported the association of cytokinin producing bacteria in plant growth (Arkhipova et al., 2005).

2.9  Benefits of plant–endophytic interactions in biocontrol of plant diseases The intimate association and similar pattern of colonization as that of the phytopathogen make the endophytic bacteria to have its potential application in agriculture as the biocontrol agent (Santoyo et al., 2016). The biocontrol efficiency of endophytic bacteria can be achieved either by direct inhibition of pathogens or through the indirect strengthening of the plant immune system (Fig. 2.1). Direct inhibition of pathogens is mainly mediated through the synthesis of antimicrobial compounds (Compant et al., 2010). Quenching quorum signals by degrading autoinducer signals of pathogens is also among the direct modes of biocontrol activity of endophytic bacteria (Miller and Bassler, 2001). Indirect biocontrol mechanisms of endophytic bacteria include the induction of plant systemic resistance that inhibits a broad spectrum of phytopathogens (Niu et al., 2011; Conrath et al., 2015). This indicates the agricultural promises of endophytic bacteria for various field applications (Fig. 2.2).

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Figure 2.1  Plant disease protective property of endophytic bacteria.

Figure 2.2  Preparation of bacterial bioformulations for agricultural applications.

2.9.1  Production of antimicrobial compounds The antimicrobial compounds produced by endophytic bacteria can act against various phytopathogens and protect plant from fungal diseases (Brader et al., 2014). The exogenous treatment with antifungal compound, iturin has been reported

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toinduce MAMP-triggered immunity defense in cotton plants. This triggered ROS burst, disruption of cell-wall integrity, and affected the fungal signaling pathways (Han et al., 2015). Likewise, the bacteria and fungi produce a wide range of volatile organic compounds (VOCs). VOC are small bioactive molecules with low molecular weight that can vaporize at normal atmospheric temperature and pressure (Hung et al., 2015; Kanchiswamy et al., 2015). They naturally come under alkenes, alcohols, benzenoids, aldehydes, ketones, or terpenes (Venturi and Keel, 2016). VOCs play an important role in interactions between the microbes and plants (Bitas et al., 2013) and regulate the symbiotic association and the entry and distribution of pathogenic microorganisms (Hung et al., 2015). VOCs-mediated microbe–microbe interactions include antimicrobial activity, interference with quorum sensing systems, coordinating gene expression, biofilm formation, virulence, and stress tolerance (Audrain et al., 2015). The impact of microbial VOCs on plant health (Bitas et al., 2013) include inhibition of plant-pathogenic bacteria and fungi (Bennett et al., 2012). In addition to these, plant growth-promoting volatiles like acetoin can have the ability to promote plant growth by increasing the photosynthetic capacity, stimulating synthesis of plant hormone-like compounds, and by inducing plant systemic resistance (Bennett et al., 2012).

2.9.2  Cell wall-degrading enzymes Plants are frequently infected with many bacterial and fungal pathogens. Among which, fungal diseases are the major threat to the survival, growth, and development of the plant. The ability of plant-associated microorganisms in synthesizing several lytic enzymes has been reported to cause disruption of cellular material of pathogenic microbes and thereby prevent its multiplication (Zarei et al., 2011; Hong et al., 2017). Hence, the fungal cell wall-degrading enzymes like chitinase, which degrades chitin gained much more attention for the biocontrol of fungal pathogens (Husson et al., 2017). Similarly, protease, which can degrade cell wall proteins; and lipase, which degrade cell wall-associated lipid have also been reported to have the ability to degrade the fungal cells individually upto certain extent (Friedrich et al., 2012). Chitinases, peroxidases and β-1,3-glucanases are part of the PR proteins and their activation can actually induce ISR in plants (Yedidia et al., 1999).

2.9.3  Induced systemic resistance Induced systemic resistance is a state of enhanced defensive capacity developed by a plant. Systemic acquired resistance and ISR are the two forms of induced resistance. Certain bacteria can also trigger a phenomenon known as ISR and is effective against different types of pathogens but differs from SAR in that the inducing resistance does not cause any visible symptoms of disease on the host plant (Van Loon et al., 1998). Plant growth promoting bacteria elicited ISR was first observed in Dianthus caryophillus with reduced susceptibility to Fusarium wilt diseases (Van Peer et al., 1991) and on cucumber (Cucumis sativus) with reduced susceptibility to foliar disease caused by Colletotrichum orbiculare (Wei et al., 1991).

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2.10 Conclusions The immense agricultural promises of endophytic bacteria indicate its promises as potential alternative to the currently used agrochemicals. A detailed mechanistic basis of its interaction with plants especially with respect to plant beneficial features will be useful for understanding the chemical basis of its interaction. This could helps to design these microbial cultures into agribioproducts. Hence, a detailed insight into plant endophytic interactions and its plant growth promoting properties can have promising applications in sustainable agricultural practices.

Acknowledgment The authors are thankful to Kerala State Council for Science, Technology, and Environment— Kerala Biotechnology Commission—Young Investigators Programme in Biotechnology (673/2017/KSCSTE dated 13.10.2017).

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Stoitsova, S.O., Braun, Y., Ullrich, M.S., Weingart, H., 2008. Characterization of the RND-type multidrug efflux pump MexAB-OprM of the plant pathogen Pseudomonas syringae. Appl. Environ. Microbiol. 74 (11), 3387–3393. Sturz, A.V., Christie, B.R., Nowak, J., 2000. Bacterial endophytes: potential role in developing sustainable systems of crop production. Crit. Rev. Plant Sci. 19 (1), 1–30. doi: 10.1080/07352680091139169. Sun, J., Guo, L., Zang, W., Ping, W., Chim, D., 2008. Diversity and ecological distribution of endophytic fungi associated with medicinal plants. Sci. China C. Life. Sci. 51, 751–759. doi: 10.1007/s11427-008-0091-z. Taghavi, S., Garafola, C., Monchy, S., Newman, L., Hoffman, A., Weyens, N., van der Lelie, D., 2009. Genome survey and characterization of endophytic bacteria exhibiting a beneficial effect on growth and development of poplar trees. Appl. Environ. Microbiol. 75 (3), 748–757. Takeshima, K., Hidaka, T., Wei, M., Yokoyama, T., Minamisawa, K., Mitsui, H., Itakura, M., Kaneko, T., Tabata, S., Saeki, K., Oomori, H., Tajima, S., Uchiumi, T., Abe, M., Tokuji, Y., Ohwada, T., 2013. Involvement of a novel genistein-inducible multidrug efflux pump of Bradyrhizobium japonicum early in the interaction with Glycine max (L.). Merr. Microbes Environ. 28 (4), 414–421. Tamošiûnë, I., Stanienë, G., Haimi, P., Stanys, V., Rugienius, R., Baniulis, D., 2018. Endophytic Bacillus and Pseudomonas spp. modulate apple shoot growth, cellular redox balance, and protein expression under in vitro conditions. Front. Plant Sci. 9, 889. doi: 10.3389/ fpls.2018.00889. Tavares, M., Nascimento, F., Glick, B., Rossi, M., 2018. The expression of an exogenous ACC deaminase by the endophyte Serratia grimesii BXF1 promotes the early nodulation and growth of common bean. Lett. Appl. Microbiol. 66, 252–259. doi: 10.1111/lam.12847. Thakur, M., Sohal, B.S., 2013. Role of elicitors in inducing resistance in plants against pathogen infection: a review. ISRN Biochem. 2013, 762412. doi: 10.1155/2013/762412. Trda, L., Fernandez, O., Boutrot, F., Heloir, M.C., Kelloniemi, J., Daire, X., Adrian, M., Clement, C., Zipfel, C., Dorey, S., 2014. The grapevine flagellin receptor VvFLS2 di erentially recognizes flagellin-derived epitopes from the endophytic growth-promoting bacterium Burkholderia phytofirmans and plant pathogenic bacteria. New·Phytol. 201, 1371–1384. Turan, M., Kıtır, N., Alkaya, Ü., Günes, A., Tüfenkçi, Ş., Yıldırım, E., Nikerel, E., 2016. Making soil more accessible to plants: the case of plant growth promoting rhizobacteria. Plant Growth. InTech, Rijeka. Turner, T.R., Ramakrishnan, K., Walshaw, J., Heavens, D., Alston, M., Swarbreck, D., Poole, P.S., 2013. Comparative metatranscriptomics reveals kingdom level changes in the rhizosphere microbiome of plants. ISME J. 7 (12), 2248–2258. doi: 10.1038/ismej.2013.119. Van Loon, L.C., Bakker, P.A., Pieterse, C.M., 1998. Systemic resistance induced by rhizosphere bacteria. Annu. Rev. Phytopathol. 36, 453–483. Van Peer, R., Niemann, G.J., Schippers, B., 1991. Induced resistance and phytoalexin accumulation in biological control of Fusarium wilt of carnation by Pseudomonas sp. strain WCS417r. Phytopathology 81, 728–734. Vandenkoornhuyse, P., Quaiser, A., Duhamel, M., Le Van, A., Dufresne, A., 2015. The importance of the microbiome of the plant holobiont. New Phytol. 206, 1196–1206. doi: 10.1111/nph.13312. Venturi, V., Keel, C., 2016. Signaling in the rhizosphere. Trends Plant Sci. 21, 187–198. doi: 10.1016/j.tplants.2016.01.005. Wang, Y., Li, H., Zhao, W., He, X., Chen, J., Geng, X., Xiao, M., 2010. Induction of toluene degradation and growth promotion in corn and wheat by horizontal gene transfer within endophytic bacteria. Soil Biol. Biochem. 42 (7), 1051–1057.

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Wei, L., Kloepper, J.W., Tuzun, S., 1991. Induction of systemic resistance of cucumber to Colletotrichum orbiculare by select strains of plant growth-promoting rhizobacteria. Phytopathol. 81, 1508–1512. Yedidia, I., Benhamou, N., Chet, I., 1999. Induction of defense responses in cucumber plants (Cucumis sativus L.) by the biocontrol agent Trichoderma harzianum. Appl. Environ. Microbiol. 65, 1061–1070. Yi, Y., de Jong, A., Frenzel, E., Kuipers, O.P., 2017. Comparative transcriptomics of Bacillus mycoides strains in response to potato-root exudates reveals different genetic adaptation of endophytic and soil isolates. Front. Microbiol. 8, 1487. Zachow, C., Jahanshah, G., de Bruijn, I., Song, C., Ianni, F., Pataj, Z., et al., 2015. The novel lipopeptide poaeamide of the endophyte Pseudomonas poae RE*1-1-14 is involved in pathogen suppression and root colonization. Mol. Plant Microbe Interact. 28, 800–810. doi: 10.1094/mpmi-12-14-0406-r. Zarei, M., Aminzadeh, S., Zolgharnein, H., Safahieh, A., Daliri, M., Noghabi, K.A., Ghoroghi, A., Motallebi, A., 2011. Characterization of a chitinase with antifungal activity from a native Serratia marcescens B4A. Braz. J. Microbiol. 42 (3), 1017–1029. Zhang, X., Davidson, E.A., Mauzerall, D.L., Searchinger, T.D., Dumas, P., Shen, Y., 2015. Managing nitrogen for sustainable development. Nat. 528 (7580), 51. Zúñiga-Mayo, V.M., Reyes-Olalde, J.I., Marsch-Martinez, N., De Folter, S., 2014. Cytokinin treatments affect the apical-basal patterning of the Arabidopsis gynoecium and resemble the effects of polar auxin transport inhibition. Front. Plant Sci. 5, 191.

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Aswathy Jayakumara, Veena P. Kumara, Meritta Josepha, Indu C. Nairb, Remakanthan A.c, Radhakrishnan E.K.a a School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, India; b Department of Biotechnology, SAS SNDP Yogam College, Konni, India; cDepartment of Botany, University College, Thiruvananthapuram, India Chapter outline head 3.1 Introduction  57 3.2 Bacterial endophytes and their diversity  58 3.3 Current understanding on the mechanisms of plant growth promotion by bacterial endophytes  59 3.4 Various mechanisms  59 3.5 Microbial production of IAA  59 3.6 ACC deaminase production  63 3.7 Phosphate solubilization  64 3.8 Nitrogen fixation  64 3.9 Siderophore production  65 3.10 Biocontrol  65 3.11 Competition  65 3.12 Antibiotics  66 3.13 Lytic enzymes  67 3.14 Induced systemic resistance  67 3.15 Ethylene  68 3.16 Quorum quenching  68 3.17 Plant probiotics  68 3.18 Conclusions  69 References  70

3.1 Introduction The global changes in climate and increasing population have unfortunate effects in food production and will become insufficient to feed the world. The green revolution could alleviate poor crop production by using high yielding varieties and use of chemical fertilizers and agrochemicals. But excessive use of chemical fertilizers and agrochemicals has resulted in the deterioration of soil fertility. Hence, agronomic practices are moving toward sustainable and environment friendly approach. Now a days several approaches like organic farming, environment friendly fertilizers and pesticides, Microbial Endophytes. http://dx.doi.org/10.1016/B978-0-12-819654-0.00003-X Copyright © 2020 Elsevier Inc. All rights reserved.

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Figure 3.1  Mode of entry of endophytic bacteria.

plant growth-promoting rhizobacteria and endophytic bacteria, highly efficient transgenic plants are in the mainstream of agriculture. Plant microbe interactions have been studied for many decades. Among them, the interaction between endophytic bacteria and its host have of great physiological and ecological significance. Endophytic bacteria are those organisms that reside in plants without causing any harmful effect to the host plant. They are capable of colonizing in any part of a plant like root, stem, leaf, flowers, and nodes. They gain entry into the plant by multiple ways except for the endophytes present in the seeds (Santoyo et al., 2016). The nutrient rich rhizosphere soil harbors wide number of microorganisms, from which the selected ones colonize within the plant. The metabolites secreted or leaked out from the plant determine the characteristics and species of organism get attracted and recruited. The primary entry into plant tissues is through the cracks in roots due to the emergence of lateral roots and different wounds, which allow the leakage of metabolites and attracts the bacteria toward the plant. The other site of entry in aerial portion is via stomata, particularly of leaves and young stems (Roos and Hattingh, 1983). The bacteria also enter through the root hair cells (Huang, 1986) (Fig. 3.1). Rhizobia spp. also colonizes the internal plant tissues and form nodules, where the nitrogen fixation process is carried out. The endophytic bacteria in to the entering host moves to different plant parts for colonization, and continue to reside with in the plant by providing necessary support for its survival.

3.2  Bacterial endophytes and their diversity Bacterial endophytes are found in almost all species of plants that have been analyzed. Endophyte free plant is likely to be absent, because such plant will be more susceptible to both biotic and abiotic stress. Several endophytes are associated with different plant

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organs and are diverse. Within the roots the number of bacterial cells could be found in the range of 104–108 per gram of root tissue, which is less than that in the rizosphere soil. This observation indicates that the roots are effective in selecting and limiting the bacteria in the root endobiome (Bulgarelli et al., 2013). Root endophytes are dominated by Proteobacteria followed by Actinobacteria, Firmiculates, and Bacteroidetes. Studies suggest that the leaf or shoot microbiome are mainly recruited from the soil and translocated to respective tissues via apoplast pathways. There are evidences suggesting the overlapping of these shoot and root microbiome at both taxonomic and functional levels (Bai et al., 2015). The bacterial components present in the interior tissues of plant are harmless to their host. Mostly changes occur in composition and diversity, which could be determined by the ecological factors of plant and soil.

3.3  Current understanding on the mechanisms of plant growth promotion by bacterial endophytes After successful colonization in host plant, endophytes promote the growth of the plant by several mechanisms (Table 3.1). They are sheltered from the majority of biotic and abiotic stress factors by the host plant, which confirms the mutual relationship. The direct mechanisms of growth promotion involve the production of plant beneficial compounds like phytohormones, ACC deaminase, sequestration of iron, and the solubilization of phosphate (Glick, 2012). The deleterious phytopathogens and pests are prevented from attacking the plants by certain indirect methods and involve the production of antibiotic, chelation of iron, and the synthesis of extracellular enzymes for the lysis of fungal cell wall (van Loon, 2007) (Fig. 3.2).

3.4  Various mechanisms Bacterial endophytes have advantages over bacteria inhabiting the rhizosphere. By virtue of being within the tissue, they are having direct contact with the plant and hence easy communication between cells can occur. Therefore, they could exert a direct beneficial effect on host. In this process, compound produced by the bacteria directly influence the physiological activities of the host plant and may result in enhanced biomass production. Bacteriogenic substances include hormones, siderophore, ACC deaminase, etc. resulting in plant beneficial processes such as phosphate solubilization, fixation of atmospheric nitrogen, and chelation of metal ions in absorbable form.

3.5  Microbial production of IAA Indole acetic acid is a phytohormone, which is known to be functionalized in cell division, elongation, and differentiation of plants. Also it aids in the germination of seeds, tubers, and initiates adventitious and lateral root formation. IAA production by

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Table 3.1  Plant growth-promoting properties of bacterial endophytes. Plant growthpromoting properties

References

Saccharum officinarum, Camellia sinensis, Oryza sativa Zea mays

Nitrogen fixation, auxin synthesis

Bertalan et al. (2009)

Nitrogen fixation

Teucrium polium

PGP properties

Guttman et al. (2008) Hassan (2017)

Malus domestica

Shoot growth, cellular redox balance, and protein expression under in- vitro conditions Endophyte-assisted phytoremediation of Trichloroethylene PGP properties

Endophytic bacteria

Source of isolation

Gluconacetobacter diazotrophicus PaI5

Klebsiella pneumoniae 342 Bacillus cereus and B. subtilis Bacillus spp. and Pseudomonas spp

Enterobacter spp. strain Populus deltoides PDN3 Pseudomonas stutzeri A15 Serratia grimesi BXF1

Enterobacter spp.

Oryza sativa Pine pinaster, Solanum lycopersicum and Cucumis sativus Eleusine coracana

Pseudomonas poae RE*1-1-14

Beta vulgaris

Azoarcus spp. BH72

Oryza sativa

Azospirillum lipoferum 4B

Oryza sativa, Zea mays, Triticum

Bacillus mojavensis

B. monnieri

Azospirillum spp. B510

Oryza sativa

Tamošiu¯nė et al. (2018)

Doty et al. (2017)

Pham et al. (2017) promotes early Tavares et al. nodulation and growth (2018) of common bean Mousa et al. Suppressing Fusarium (2015) graminearum in plant tissues and reduction of deoxynivalenol mycotoxin Production of novel Zachow et al. lipopeptide Poaeamide (2015) suppressing Phytophthora capsici and P. infestans zoospores Nitrogen fixation Krause et al. (2006) Nitrogen fixation, Richardson phytohormone et al. (2011) secretion Biocontrol mechanisms Jasim et al. (2016c) Nitrogen fixation, Kaneko et al. phytohormone (2010) secretion

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Table 3.1  Plant growth-promoting properties of bacterial endophytes. (Cont.) Endophytic bacteria Burkholderia phytofirmans PsJN

Burkholderia spp. KJ006 Klebsiella sp.

Enterobacter spp. 638

Burkholderia phytofirmans Burkholderia phytofirmans Burkholderia phytofirmans Ralstonia sp. and Bacillus sp

Plant growthSource of isolation promoting properties Solanum tuberosum, IAA synthesis, ACC deaminase Zea mays,Solanum lycopersicum, Hordeum vulgare,Allium cepa, ACC deaminase, Oryza sativa nif gene cluster, antifungal action Phosphate solubilization, P. nigrum ACC deaminase, Siderophore Siderophore, IAA, Populus acetoin and 2,3-butanediol synthesis, antifungal action Growth enhancement Allium cepa Allium cepa

Growth enhancement

Allium cepa

Growth enhancement, increased chlorophyll content Growth enhancement effect

Rhizobium spp.

Musa accuminata AAA cv. Grand Nain Solanum lycopersicum Zea mays

Bacillus sp.

C. annuum

Biocontrol

Rhizobium spp.

Zea mays, Sorghum bicolor, Oryza sativa Zingiber officinale

Growth enhancement

Rhizobium spp.

Pseudomonas sp. Ralstonia spp.

Pseudomonas spp.

Zea mays, Sorghum bicolor, Oryza sativa Solanum lycopersicum

Growth enhancement Growth enhancement

IAA, ACC deaminase and siderophore Growth enhancement

Growth enhancement

References Weilharter et al. (2011)

Kwak et al. (2012) Jasim et al. (2013b) Taghavi et al. (2008)

Compant et al. (2005b) Kim et al. (2012) Zúñiga et al. (2013) Jimtha et al. (2014) Tian et al. (2017) Patel and Archana (2017) Jasim et al. (2016b) Riggs et al. (2001) Jasim et al. (2013c) Patel and Archana (2017) Tian et al. (2017) (Continued)

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Table 3.1  Plant growth-promoting properties of bacterial endophytes. (Cont.) Endophytic bacteria Burkholderia vietnamiensis, Rhanella spp., Acinetobacter spp., Herbaspirillum spp., Pseudomonas putida, Sphingomonas spp. Burkholderia vietnamiensis, Rhanella spp., Enterobacter spp., Pseudomonas graminis, Acinetobacter spp., Herbaspirillum spp., Sphingomonas yanoikuyae Paenibacillus sp.

Source of isolation Populus deltoides

Plant growthpromoting properties Growth enhancement, increased CO2 assimilation

References Knoth et al. (2013)

Populus deltoides

Growth enhancement

Knoth et al. (2014)

Curcuma longa

IAA production

Herbaspirillum seropedicae Pseudomonas aeruginosa Herbaspirillum seropedicae

Zea mays

Growth enhancement

Zingiber officinale

Biocontrol

Zea mays

Bacillus sp.

Elettaria cardamomum Zea mays

Increased rooting, change in gene expression Plant growth enhancement Nitrogen fixation

Aswathy et al. (2012) Riggs et al. (2001) Jasim et al. (2013a) do Amaral et al. (2014)

Bacillus amyloliquefaciens Pseudomonas fluorescens

Bacopa monnieri

Biocontrol

Miscanthus sinensis

Pseudomonas fluorescens

Brassica napus

Burkholderia spp.

Capsicum frutescens

Growth enhancement in phosphate limited conditions Growth enhancement, increased Pb uptake, root elongation Plant probiotic function

Pseudomonas fluorescens

Solanum nigrum

Growth enhancement

Herbaspirillum seropedicae

Jasim et al. (2015) RoncatoMaccari et al. (2003) Jasim et al. (2016a) Oteino et al. (2015)) Oteino et al. (2015) Sabu et al. (2018) Ausubel et al. (2008)

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Table 3.1  Plant growth-promoting properties of bacterial endophytes. (Cont.) Endophytic bacteria Herbaspirillum spp., Methylobacterium spp., and Brevundimonas spp. Bacillus sp.

Source of isolation Camellia sinensis

Curcuma longa

Bacillus cereus and Zea mays L. Enterobacter cloacae

Plant growthpromoting properties Plant probiotic function

References Yan et al. (2018)

IAA production, ACC Jayakumar et al. deaminase production, (2018) Nitrogen fixation IAA production Abedinzadeh et al. (2019)

Figure 3.2  Plant growth-promoting mechanisms of endophytic bacteria.

endophytic bacteria occurs through tryptophan dependent and tryptophan independent mechanisms. Generally microbial production of IAA occurs via indole-3-acetonitrile (IAN) pathway, indole-3-acetamide (IAM) pathway, and the indole-3-pyruvate (IPyA) pathway (Li et al., 2018). Among these IAM pathway is mainly attributed to the phytopathology, and the IPA pathway is related to epiphytic and rhizosphere fitness. Endophytic IAA can contribute to the increase in shoot length, root length, root number, and also can prevent the plant from pathogenic invasion (Jayakumar et al., 2018). Several other studies also supported the activity of endophytic IAA in plant growth promotion (Bhutani et al., 2018; Lata et al., 2006; Liu et al., 2017).

3.6  ACC deaminase production Ethylene is an important plant hormone produced by higher plants in association with fruit ripening, senescence, and stress response. The presence of increased level of

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ethylene causes stress on plants and thereby inhibits the growth of vegetative tissues. ACC deaminase is an enzyme produced by many plant growth-promoting bacteria (PGPB), which have the ability to uptake ACC and converts to α-ketobutyrate and ammonia. This lowers the ACC levels and thereby decreasing the level of ethylene, which inturn minimize the plant stress level. Several studies reported that the inoculation of ACC deaminase producing bacteria can protect the plant against flooding, salinity, drought, heavy metal toxicity, and the presence of phytopathogens (Glick, 2014; Santoyo et al., 2016; Zhang et al., 2011).

3.7  Phosphate solubilization Phosphorus is an essential macro nutrient, which aids in the growth and development of plants and is present at 400–1200 mg concentration per kg of soil. The soluble phosphate concentration present in soil is very low and is about 1 ppm. Plant absorbable forms of phosphate include monobasic and the dibasic ions. Most of the elemental phosphorous is found to be immobilized in various living organisms and or locked up in sediments. Microbes play an important role in the release and cycling of immobilized phosphorous. These microbes solibilize phosphate by acidification, secretion of organic acids, and through the chelation-based mechanisms. Many bacterial genera are reported as phosphate solubilizers such as Azotobacter, Bacillus, Beijerinckia, Burkholderia, Enterobacter, Erwinia, Azospirillum, Serratia, Flavobacterium, Pseudomonas, Microbacterium, and Rhizobium (de Abreu et al., 2017; Huang et al., 2010; Oteino et al., 2015; Zaidi et al., 2009). Several studies have reported that the inoculation of phosphate solubilizing endophytic bacteria can contribute to the enhancement of growth in plants (Emami et al., 2019; Oteino et al., 2015).

3.8  Nitrogen fixation Nitrogen is one of the macronutrients for the growth and development of plants. About 78% of the nitrogen is in its gaseous form, which is not readily available to plants. And also the nitrogen deficiency in soil has necessitated the use of various nitrogenous fertilizers. Although many endophytes are present in nature, only certain bacteria have the capacity to fix nitrogen. This is because of the inability of these bacteria to produce nitrogenase enzyme. Endophytic diazotropic bacteria have been reported to be present in agriculturally important plants such as Brassica napus, Leptochloa fusca, Oryza sativa, Pennisetum glaucum, Musa acuminata, Saccharum officinarum, and Zea mays (Anand and Chanway, 2013; Andrade et al., 2014; Araújo, 2013; Gupta et al., 2013). Several nitrogen fixers with plant growth enhancement effect have been reported and include Azospirillum spp., Herbaspirillum spp., Burkholderia spp., Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, Pantoea sp., and Bacillus spp. (Govindarajan et al., 2006; Islam et al., 2009; Loiret et al., 2004).

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3.9  Siderophore production Iron (Fe) is the most abundant element on earth, however obtaining sufficient amount of iron is more problematic in the rhizosphere where plants and microorganisms competes for iron. Under such iron limiting conditions, many bacteria produce low molecular weight (∼400–1500 Da) iron-specific ligands, termed as siderophores. Because of its iron chelating activity, siderophores are known as the vehicle for the transport of Fe3+ into microbial cells. The uptake of Fe3+ by microoorganisms is successfully carried through the Fe-siderophore receptors because of the high affinity of these molecules for Fe3+. There are over 500 types of siderophore, out of which the chemical structure of 270 is studied well. The known siderophores are belongs to three main groups like the catecholates, hydroxamate, and carboxylates.

3.10 Biocontrol Biocontrol or biological control can be defined as the reduction or complete inhibition of phytopathogens by the endophytic bacteria. The most studied and commonly reported mechanism is antagonism. It includes most specific mechanisms like competition, antibiosis, hydrogen cyanide production, and siderophore production.Various other mechanisms are also reported like “induced systemic resistance” (ISR) and “systemic acquired resistance” (SAR). ISR is elicited by certain non pathogenic microorganisms, whereas SAR is elicited via pathogens or chemical compounds.

3.11 Competition The greater incidence of disease can be limited by the competition between pathogenic and nonpathogenic strains of bacteria for colonization. The root surface and rhizosphere soil contain carbon sinks with 40% of photosynthate allocation. Rapid colonization of abundant nonpathogenic bacteria in this nutrient rich area prevents the growth of pathogenic strains. Overall environment of soil is dependent on nutrient rich niches that attract wide variety of microorganisms, forming relationships such as associative, symbiotic, neutralistic, or parasitic. The various parameters that determine the effectiveness of PGPB mediated processes include the strain competence and persistence, root colonizing capacity, ability to synthesize and release various metabolites, plant species, and genotype specificity of the bacterial strain. The crucial and complex process of root colonization is the prerequisite for its effective application like biofertilization, phytostimulation, bicontrol, and phytoremediation. The chemotactic and motile microorganisms are efficient root colonizers, whereas the nonmotile ones are less efficient. The organic acids, amino acids, and specific sugars present in the root exudates attract these organisms to the root. They reach the site of entry by active mobility in response to the chemotactic substance (de Weert et al., 2002). PGPB may be uniquely equipped to sense chemoattractants, for example, rice exudates induce

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stronger chemotactic responses of endophytic bacteria than from nonPGPB present in the rice rhizosphere (Bacilio-Jiménez et al., 2003). Pseudomonas and Bacillus spp. are the most common colonizers in agricultural crops. The exudates may contain antimicrobial compounds of great ecological importance, which inhibit the pathogens. The quantity and composition of these nutrient niches vary with the species of plants, possess a challenge to the colonizing bacteria. Here the colonization completely depends on the bacterial competence to the compounds by taking it as an advantage or by getting adapted to the specific changes in the environment (Bais et al., 2004). Bacterial lipopolysaccharides (LPS), the O-antigen chain, can also contribute to root colonization (Dekkers et al., 1998). The importance of LPS in colonization are strain dependent as the O-antigenic side chain of Pseudomonas fluorescens WCS374 does not contribute to potato root adhesion, whereas the O-antigen chain of P. fluorescens PCL1205 is involved in tomato root colonization. Furthermore, LPS, O antigen does not contribute to rhizoplane colonization of tomato by the plant beneficial endophytic bacterium P. fluorescens WCS417r, but they were involved in endophytic colonization of roots (Compant et al., 2005a).

3.12 Antibiotics The natural products produced by the endophytes can help in protection of the host against pathogen invasion. These chemicals are also of great significance in pharmaceutical, agrochemical, and biotechnological industries too (Harrison et al., 1991). Researches on antibiotics and other microbial natural products are pivotal for global fight against the growing problem of antibiotic resistance. It is necessary to find new antibiotics to tackle this problem, and currently endophytic bacteria are one of the potential sources of novel antibiotics (Christina et al., 2013). Many natural products produced by endophytes have proven to be antibacterial, antifungal, antidiabetic, antioxidants, and immunosuppressives. Thus, endophytes are viewed as great novel sources of bioactive natural products. They are one of the untapped potential sources with majority of them producing different kinds of antibiotics, which has unusual amino acids in it. A wide variety of antibiotics are being produced by plant growth-promoting bacteria (PGPB). Most of them not only inhibit phytopathgens like bacteria, fungus, virus, but also helps in growth enhancement of the plant. They include Bacillus spp., Pseudomonas spp., Azospirillum spp., Rhizobium spp., and Serratia spp. (Haas and Keel, 2003). Antibiotics produced by PGPB include 2,4 diacetylphloroglucinol, phenazine-1-carboxyclic acid, phenazine-1-carboxamide, pyoluteorin, pyrrolnitrin, oomycin A, viscosinamide, butyrolactones, kanosamine, zwittermycin-A, aerugine, rhamnolipids, cepaciamide A, ecomycins, pseudomonic acid, azomycin, antitumor antibiotics FR901463, cepafungins, and antiviral antibiotic karalicin. These antibiotics are known to possess antiviral, antimicrobial, insect and mammalian antifeedant, antihelminthic, phytotoxic, antioxidant, cytotoxic, antitumor, and plant growth-promoting activities (Fernando et al., 2006). The mechanism behind the biocontrol by these compounds include cell distruption and suppression of pathogens, hence it is commercialized and greatly significant to pharmaceutical field (Compant et al., 2005a). The

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free living and endophytic bacteria release allelochemicals, which act antagonistically with the pathogens likewise all other microorganisms (Saraf et al., 2014). Phenazines and pyrrolnitrin are antifungal products that are produced by Pseudomonads. Each antibiotic exhibits a different antifungal mechanism, pyrrolnitrin had shown to have antagonism against Botrytis cinerea, Rhizoctonia solani, and Sclerotinia sclerotiorum. The phenazines have proven to be effective against Gaeumannomyces graminis var. tritici. The antibiotics from Bacillus spp. include lipopeptides, polymyxin, circulin, and colistin are active against Gram-positive and Gram-negative bacteria and pathogenic fungi (Maksimov et al., 2011). The UW85 strain of B. cereus suppressed oomycete pathogens through the production of the antibiotics zwittermicin A (aminopolyol) and kanosamine (aminoglycoside), which contributed to the biocontrol of alfalfa damping off (Beneduzi et al., 2012; Silo-Suh et al., 1994). The PGPB antibiotics were mostly used for the crop plants, where a single infection could badly affect the yield. Gradually the excessive use of these antagonisis lead to development of the resistant strains. To tackle the menace of resistance bicontrol agents that produce cyanides, alcohols, and ketones as secondary metabolites were used. Cyanide is a secondary metabolite produced by Gram-negative P. fluorescens, P. aeruginosa, and C. violaceum. The aerobic, root colonizing biocontrol bacterium CHA0 protects several plants from root diseases caused by soil borne fungi through the production of diverse metabolites (Fernando et al., 2006; Voisard et al., 1994). Antifungal volatiles of P. chlororaphis (PA23) isolated from soybean roots include aldehydes, alcohols, ketones, and sulfides, which were inhibitory to all the stages of S. sclerotiorum root pathogen (Fernando et al., 2006).

3.13  Lytic enzymes Apart from antibiotics, many endophytes produce certain cell wall degrading enzymes to control phytopathogens. These degradative enzymes have the capacity to alter the structural integrity of the fungal cell wall and thereby inhibit or kill the pathogens. These include β-1,3-glucanase, chitinase, cellulase, and protease which inhibit the growth of pathogens in alone and in combination with other biocontrol strategies. Chitinase mediates the degradation of chitin, which is the major cell wall component of fungus. Along with other enzymes, it has been found to be active against various phytopathogens like Botrytis cinerea, Sclerotium rolfsii, Fusarium oxysporum, Phytophthora spp., Rhizoctonia solani, and Pythium ultimum (Aktuganov et al., 2003; Quecine et al., 2008; Zhang et al., 2015).

3.14  Induced systemic resistance ISR caused by plant beneficial bacteria by inducing resistance mechanism in the plant. The plant develops an enhanced defensive state against the pathogen, when stimulated appropriately. It is not pathogen specific, and is effective in controlling disease caused by several phytopathogens by ethylene and jasmonate signaling pathways.

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Pseudomonas and Bacillus spp. are the most studied microbes that trigger ISR and develop resistance against several plant pathogens, including fungal, bacterial and viral pathogens, nematodes, and insects. Several elicitors are reported other than jasmonate and salicylate and include the O-antigenic side chain of outer membrabe of bacteria, chitin, cyclic lipopeptides, flagellar proteins, β-glucans, and pyoverdine.

3.15 Ethylene The smallest simple structured gaseous phytohormones, which allows plant–plant communications. It is a multifaceted hormone, which has various roles in the regulation of leaf development, senescence, fruit ripening, stimulation of germination, etc. It is mainly produced in response to multiple environmental stresses, both abiotic and biotic and acts as a bridge between a changing environment and developmental adaptation. Ethylene synthesis triggering abotic stresses include submergence, heat, shade, exposure to heavy metals and high salt, low nutrient availability, and water deficiency (Dubois et al., 2018). ACC deaminase is the rate determining enzyme that regulates ethylene in plants. The plant growth promotion is directly linked to the levels of ethylene in plants, which is highly produced during stress conditions. The ACC deaminase positive bacterial endophytes are excellent growth promoters because they ameliorate plant stress by blocking the production of ethylene. The ACC deaminase activity and its role in plant growth induction was well demonstrated using endophytic Burkholderia phytofirmans.

3.16  Quorum quenching Quorum sensing mechanism is required for the survival of most of the microorganisms. It is thought to regulate the physiological activities such as cell to cell communication, reproduction, adaptation, biofilm formation, and competence. Endophytic bacteria have been reported to be involved in the quorum sensing quenching mechanism as a strategy to control certain phytopathogens. For example, endophytic bacteria from Cannabis sativa L. have been found to disrupt the cell to cell communication of Chromobacterium violaceum (Kusari et al. 2014).

3.17  Plant probiotics The ecofriendly approach for sustainable agricultural practices has of great significance in day-to-day life. For this, formulations based on endophytic bacteria have of great interest. Bioprimed plant always shows enhanced plant growth and is free from many of the environmental stresses (Mahmood and Kataoka, 2018) (Fig. 3.3). The big challenge associated with the development of such formulations are the isolation, characterization, and studying the field potential of promising bacteria. Formulations with increased shelf life, broad spectrum action, and with good performance under

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Figure 3.3  Effect of endophytic bacterial priming on plants .

field conditions can be commercialized. The commercialization and application of such potential candidate depend mainly on the selection of compatible carriers. Currently organic and inorganic carriers are available with the potential to protect the bacteria from stress conditions. Many inorganic and organic carriers such as talc, alginate, peat, vermiculate, sawdust, zeolite, pyrophyllite, and montmorrilonite are used (Malusá et al., 2012). The shelf life of formulations varies based on the bacteria and the carrier type. Most suitable carrier material, which can extent the bacterial viability can be selected to transform the bacterial formulations into plant probiotics for agricultural field application.

3.18 Conclusions Endophytic bacteria are microorganisms that colonize the interior part of plant without causing any harmful effects. Being inside, they may promote the growth of plants by several direct and indirect mechanisms. Several bacteria have been reported to enhance the growth of plants, and many of them are uder investigation. Currently several endophytic bacteria-based formulations have been developed, and some of them are under commercialization. Hence these endophytic bacteria can replace majority of the chemical fertilizers and pesticides for better agronomic practices and sustainable agricultural productivity.

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Jubi Jacoba,b, Gopika Vijayakumari Krishnana,b, Drissya Thankappana,b, Dileep Kumar Bhaskaran Nair Saraswathy Ammaa,b,* a Agro-Processing and Technology Division, CSIR – National Institute for Interdisciplinary Science and Technology (NIIST), Thiruvananthapuram, Kerala, India; b Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India Chapter Outline head 4.1 Introduction  75 4.2 Endophytes providing disease resistance and mode of action  77 4.2.1 Siderophores  79 4.2.2 Antibiotic production  81 4.2.3 Production of lytic enzymes  84 4.2.4 Induced systemic resistance  85

4.3 Endophytes providing ISR against wilt diseases  89 4.4 Optimization of bioactive metabolite production by endophytes through statistical approach  91 4.5 Bioformulation of endophytes  92 4.6 Challenges related to the development of endophytic formulation  93 4.7 Future prospective in endophytic research  95 4.8 Conclusion  96 References  97

4.1 Introduction Food and Agriculture Organization estimated that the world population would expect to be nearly 8.5 billion by 2025 and this tremendous increase will inevitably require an additional agricultural production of nearly 2.4 billion ton/year, which is 70% of the total food production (Timmusk et al., 2017). In developing countries, agriculture faces several unexpected challenges, as it represents a major economy in those nations. Therefore the production of an adequate amount of balanced and secure foods to meet the crisis is inevitable. The extensive use of synthetic compounds as pesticides and fertilizers to meet the renewed agricultural production may contaminate the food crops *

Corresponding author: [email protected]

Microbial Endophytes. http://dx.doi.org/10.1016/B978-0-12-819654-0.00004-1 Copyright © 2020 Elsevier Inc. All rights reserved.

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and the ecosystem. Moreover, it is detrimental to the soil beneficial bacteria and creates several threats to the consumer’s health and the environment (Kannojia et al., 2019). Hence the use of endophytic plant growth-promoting rhizobacteria (PGPR) and their promising metabolites will be an ideal, viable, and eco-friendly alternative. Crop losses are one of the major concerns to attain food security, especially in developing countries (Savary et al., 2019). In India, 30% crop loss is recording every year due to pests and diseases, where fungi and other microorganisms played an influential role in some food crisis scenarios. Hence the management of crop diseases such as wilt, stem and root rot, smut, rust, blight, blast, cankers, and other diseases pose a major threat in modern agriculture. For example, the sheath blight disease caused by Rhizoctonia solani has become a significant constraint in rice production, leading to an annual yield loss of up to 50% (Kagale et al., 2011). Fusarium species also responsible for a significant reduction in agriculture outputs, predominantly on staple cereal crops, including wheat, maize, barley, and other pulses like peas and beans (Eljounaidi et al., 2016). For instance, in ginger, the crop may get severely affected with rhizome or soft rot caused by F. oxysporum f.sp zingiberi and occasionally by R. solani during cultivation (Das et al., 2019). However, very limited number of biocontrol strategies have been exploited successfully to control these pathogens/diseases (Nguvo and Gao, 2019). Use of beneficial microorganisms such as endophytes were taken much attention due to its eco-friendly and cost-effective approach. It has been reported that at least one or more than one endophyte reside in every three lakh plant species that exist on the earth (Strobel and Daisy, 2003), but only 6%–7% of the endophytes existence has been known (Gupta et al., 2019). Endophytic bacteria are commonly present in every plant parts, including roots (Chen et al., 2019), root nodules (MartínezHidalgo et al., 2015), seeds (Gond et al., 2015), stems (Chung et al., 2015), leaves (Tan et al., 2015), and rhizomes (Jasim et al., 2014). Generally, microbes enter into plant tissues through natural openings like stomata, lenticels, wounds, germinating radicles, etc. The major sites of colonization are the intercellular spaces of the epidermal and cortical regions and lysed plant cells (Gupta et al., 2019). The leakage of plant exudates from the wounded area allowed favorable conditions for bacterial entry and colonization of approaching microbes. But endophytes do not require wounds and lateral roots for their entry as studies shows they can penetrate the plant cells actively. These beneficial microbes can be used as bio-elicitors for the induction of systemic resistance in host plants (Mao et al., 2019), and their application is sustainable from an ecological perspective. The term endophyte means “in the plant” and is derived from two Greek words endon (within) and phyton (plant). It is a general term used for bacteria or fungi that can be detected at any moment within the healthy plants and do not cause any disease (Schulz and Boyle, 2006). General procedures for the isolation of endophytic bacteria consist of plant selection, surface sterilization of plant materials, the choice of culture medium and growth conditions, strain isolation, purification, morphological, and molecular identification (Potshangbam et al., 2017). Isolation from surface disinfected plant tissues is not enough to prove the endophytic nature of the isolate/s. Plant surface sterilization protocols must be sufficiently performed to eliminate the external microbiota without killing bacteria within the tissue. However, the “endophyte” must be

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shown to be a true endophyte by examining its ability to reinfect disinfected seedlings and by microscopic confirmation (Mercado-Blanco, 2015). Recently, the comparative genomics and bioinformatics provided insights into the endophytism exhibited by the microbes. The analysis of biosynthetic gene clusters involved in the biosynthesis of secondary metabolites provided unprecedented opportunities to investigate the diversity and classification of natural products originated from those gene clusters (Tawfike et al., 2019). Also, the modification of the nutritional environment of biocontrol bacteria may result in more production of bioactive compounds (Kilani-Feki et al., 2016). Therefore the commercial development of a bioformulation is preconditioned by the optimization of production conditions, which provide its maximum antifungal activity. Compared to conventional optimization strategies, experimental factorial design and response surface methodology (RSM) is inexpensive, fast for a large number of variables. However, the efficiency of endophytic microorganisms as bioformulation depends on many factors such as host specificity, colonization patterns, population dynamics, the ability to move within host tissue, ability to induce systemic resistance, the physical structure of the soil, environmental conditions, and the growth phase and physiological state of the plant (Bolívar-Anillo et al., 2019). It is necessary for remaining endophytic microorganisms and their novel bioactive metabolites to be explored for their potential exploitation in sustainable agriculture. In this situation, the use of microbial inoculants, which induce systemic resistance in host plants and provide more resistance against pathogenic infection, has recently gained much attention and for efficient management of various crop diseases. Despite the biocontrol mechanisms and their role in stress tolerance, the management of plant diseases through the induction of systemic resistance caused by endophytic bacteria is poorly summarized (Malfanova et al., 2013; Sharma et al., 2018). This chapter summarizes present knowledge on how bacterial endophytes can establish induced systemic resistance (ISR) within the host plants and their mechanisms. The concerns regarding the biotechnological application of living organisms, such as endophytes, their bioformulation development and its commercial success will also be discussed.

4.2  Endophytes providing disease resistance and mode of action Endophytes are renowned for their ability to protect plants against devastating diseases, which affect both crops and woody perennials (Swain et al., 2019). The biocontrol ability of endophytes against diseases were reviewed by various researchers from time to time (Latha et al., 2019; Tewari et al., 2019). For example, Botrytis cinerea, a most widely studied necrotrophic phytopathogenic fungus can be controlled by the use of endophytic microorganisms. The endophytic bacteria induce biocontrol through indirect mechanisms such as (1) antagonism against phytopathogens by siderophore production; (2) antibiotic production; (3) lytic enzyme production, and (4) the ability to induce systemic resistance in host plants against pathogens. Examples of bacterial endophytes, such as Arthrobacter, Bacillus, Burkholderia, Enterobacter, Micrococcus, Pantoea, Pseudomonas, Rhizobium, Serratia, Streptomyces, etc., are used nowadays as biocontrol agents against various plant pathogens. Among this,

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Bacillus and Pseudomonas are the well-studied group and indirectly promotes the growth and development of many plants (Santoyo et al., 2012; Tiwari et al., 2019). Bolívar-Anillo et al. (2019) described the use of various endophytic microorganisms as biological control agents in controlling B. cinerea. Eljounaidi et al. (2016) summarized different case studies regarding bacterial endophytes antagonistic to pathogens causing vascular wilt diseases. The endophytic colonization and antagonistic properties of some selected isolates are represented in Figs. 4.1 and 4.2, respectively. Plant

Figure 4.1  Scanning electron microscopic image showing the presence of bacteria in root tissues of cow pea plant.

Figure 4.2  Dual culture technique showing in vitro antagonism by different PGPR strains.

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Figure 4.3  Plant growth-promoting and biocontrol traits exhibited by different endophytes from O. sativa. (A) Indole acetic acid production; (B) ammonia production; (C) HCN production; (D) siderophore production; and (E) volatile production.

growth-promoting and biocontrol traits exhibited by various endophytes isolated from rice plants (Oryza sativa) are shown in Fig. 4.3.

4.2.1 Siderophores Iron (Fe) is the fourth common element in the earth’s crust and all living organisms require iron for their growth and metabolism. Although abundant in the earth crust, iron is not readily available and under aerobic conditions, where the free ferrous iron (Fe2+) is oxidized to the ferric iron (Fe3+) forming oxyhydroxide polymers, which are not very easily soluble (Braun and Winkelmann, 1987). The term siderophore stands for “iron carriers” or “iron bearers” in Greek. They are low molecular weight compounds ranging from 500–1500 daltons and produced by many PGPR, including endophytes. Those molecules serve as iron chelators, which increase the availability of iron in the root region, thereby inhibiting the colonization of pathogens by not providing them adequate iron. These molecules can be categorized into three groups depending upon the moiety, which donates oxygen ligands for Fe3+: (1) catecholates or phenolates, (2) hydroxamates or carboxylates, and (3) the mixed types. Structures of siderophores produced by different bacteria are

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Figure 4.4  Examples of siderophores. I, enterobactin; II, pyochelin; III, alcaligin; IV, staphyloferrin A; V, heterobactin B; VI, mycobactin T; VII, rhizobactin 1021. Moities involved in the Fe+ coordination are highlighted as follows: catecholates are in red, phenolates are in violet, hydroxamate in green, and carboxylate in yellow. All structures were drawn using ChemDraw Pro12.0 Cambridgesoft, USA.

illustrated in Fig. 4.4. Examples of siderophore molecules containing catecholate groups are enterobactin (Streptomyces), pyochelin (Pseudomonas aeruginosa), and vibriobactin (Vibrio cholerae). The siderophores with hydroxamate groups include alcaligin (Alcaligenes denitrificans) and staphyloferrin (Staphylococcus spp.), whereas examples of mixed types are mycobactin (Mycobacterium tuberculosis) and petrobactin (B. anthrasis). Many endophytes are reported as siderophore producers, where Pseudomonas sp. are identified as one of the major groups. Recently, two new hydroxamate siderophores, namely legonoxamines A and B, were isolated from a soil bacterium, Streptomyces sp. MA37 (Maglangit et al., 2019). In another study, draft genome sequence was employed for the identification of a new desferrioxamine-like siderophore, FW0622 from a rare marine actinomycete Verrucosispora sp. FIM060022 (Zhao et al., 2019). The biosynthetic pathway of the strain showed that the nonribosomal peptide synthetase—independent synthetase pathway based on the putative biosynthetic siderophore gene cluster provided an insight into the diversity of the metabolites. The siderophores produced by fluorescent pseudomonads are pyoverdine, pyochelins, salicylic acid (SA), quinolobactin, azotobactin, and pseudomonine. SA is not only a compound with siderophore activity but also serves as a precursor in the biosynthesis of catechol type siderophores, such as yersiniabactin, mycobactin, pyoverdine, and pyochelin in bacteria. These iron-chelating molecules can also trigger the systemic resistance pathways in direct or indirect ways.

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Table 4.1  Endophytes reported for siderophore production. Endophyte

Host plant

Mode of action

Diazotrophic bacteria P. aeruginosa LSE 2 Bradyrhizobium LSBR 3 Streptomyces sp. KLBMP1213 Bacillus sp.

Rice Soybean

Plant growth promotion Ji et al. (2014) Plant growth promotion Kumawat et al. (2019)

S. sporocineres

References

Plant growth promotion Qin et al. (2015) Jatropha curcas L. Maize Plant growth promotion Ribeiro et al. (2018) and nutrient uptake under low P Rice Biocontrol Zeng et al. (2018)

Siderophores produced by microorganisms are usually in a reverse correlation with iron concentrations, that is, siderophore production is promoted under iron-limited conditions while suppressed under iron-rich conditions. In a study conducted in rice endophyte S. sporocinereus OsiSh-2 could excrete considerably more siderophores than a rice blast pathogen Magnaporthe oryzae Guy11 under iron-restricted conditions and displayed higher Fe3+ reducing activity during iron-supplemented conditions (Xu et al., 2017). Genomic analysis also revealed that nearly threefold siderophore biosynthetic genes and sixfold siderophore recognizing and transporting genes compared to the pathogen. Some of the endophytic bacteria reported well for siderophore production are represented in Table 4.1.

4.2.2  Antibiotic production Antibiotic production is an efficient mechanism employed by many endophytes in controlling soil borne pathogens. This ability can be correlated to the induction of systemic resistance in plants due to the underlying antimicrobial mechanism because of the presence of biosynthetic genes, as antibiotics can act as determinants, triggering the response (Xu et al., 2019). Other than antifungal activities, endophytes from a few plants have also been reported for the production of antiviral, anticancer, antioxidants, antidiabetic, and immunosuppressant compounds (Alvin et al., 2014; Matloub et al., 2019). Therefore the exploration of novel bioactive secondary metabolites from different endophytic microorganisms including actinomycetes, bacteria, and fungi is a valuable alternative to combat with the increasing levels of drugs resistance in various pathogens (Gouda et al., 2016). The most abundant antibiotic producing Grampositive bacterial endophytes found within diverse environments are Bacillus and Streptomyces species (Ek-Ramos et al., 2019). These secondary metabolites can be classified into various groups, such as alkaloids, benzopyranones, flavonoids, phenolic acids, steroids, quinones, saponins, tannins, terpenoids, tetralones, xanthones, and so on (Gouda et al., 2016). Endophytic Bacillus also have a great ability to produce different compounds such as fengycins, iturins, and surfactins (Hardoim, 2018). Bioactive metabolites produced by endophytes were best reviewed by various researchers (Hardoim, 2018; Matsumoto and Takahashi, 2017). Some of the bioactive

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Table 4.2  Selected examples of antibiotics produced by endophytic bacteria. Antibiotics

Producing organisms

Target organism/s

References

Fengycins and iturins Phthalic acids

B. amyloliqueaciens

Alvarez et al. (2012)

HCN

P. fluorescens P. aeruginosa Choromobacterium violacecum Streptomyces sp. BCC72023

Sclerotinia sclerotiorum F. oxysporum f. sp. lycopersici S. rolfsi R. solani

Macrolides, polyethers

B. subtilis EPC016

Colletotrichum gloeosporioides, C. capsici

Ramyabharathi and Raguchander (2014) Xu et al. (2017)

Supong et al. (2016)

compounds produced by different endophytes are shown in Table 4.2. Among these endophytes, Nocardiopsis sp. GRG 2 isolated from microalgal samples was able to produce a compound 1, 4-diaza-2, 5-dioxo-3-isobutylbicyclo[4.3.0]nonane (DDIBN) against uro pathogens (Rajivgandhi et al., 2018). In another study, B. subtilis EPC016 associated with cotton plants and B. cereus NRL2 isolated from Azadirachta indica were able to produce bioactive phthalic acids with antifungal activity against F. oxysporum f. sp. lycopersici (Ramyabharathi and Raguchander, 2014). In our earlier work, bioactive metabolites, namely, 1-hydroxyphenazine, pyocyanin, and phenazine-1-carboxamide were purified and characterized from a plant growth-promoting rhizobacterial strain, PM 105 (Emrin et al., 2015). Similarly, Li et al. (2013) reported six cyclic dipeptides isolated from an endophytic bacterium P. brassicacearum subsp. Neoaurantiaca from Salvia miltiorrhiza Bunge having antagonistic activities against pathogens responsible for Danshen rot disease. Some cyclic structured compounds produced by endophytic bacteria are illustrated in Fig. 4.5. Paenibacillin A, a 2(1H)-pyrazinone ring-containing natural product with anticancer property was isolated from an endophytic bacterium Paenibacillus sp. Xy-2 (Bian et al., 2016). Recently, a polyketide, chartreusin was isolated from Streptomyces sp. SKH1-2, an endophyte of Musa roots with antibacterial properties (Kuncharoen et al., 2019). Toxoflavin with strong antifungal activity was identified from an endophytic bacterium, Burkholderia gladioli HDXY-02, an inhabitant of a medicinal plant Lycorisaurea (Li et al., 2019). Another compound named (2E,5E)-phenyltetradeca2,5-dienoate was also isolated from an endophytic bacteria P. aeruginosa strain UICC B-40 (Pratiwi et al., 2017). Recently, five indole derivatives and two diketopiperazines along with a dihydro cinnamic acid were isolated from Pantoea ananatis VERA8, an endophyte of Baccharoides anthelmintica roots (Rustamova et al., 2019). In an investigation, a bioactive compound named (S) -2-hydroxy-N-((S)-1-((S)-8- hydroxy-1oxoisochroman-3-yl)-3-methylbutyl)-2-((S)-5-oxo-2,5-dihydrofuran-2-yl) acetamide was isolated and purified from an endophytic B. amyloliquefaciens RWL-1, isolated from O. sativa seeds (Shahzad et al., 2018).

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Figure 4.5  Bioactive compounds produced by different endophytic bacteria.

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4.2.3  Production of lytic enzymes Endophytic bacteria could produce lytic enzymes such as chitinases, cellulases, glucanases, lipases, pectinases, and proteases that can parasitize disease causing fungal strains (Abdallah et al., 2019). These bacteria can destroy phytopathogenic fungi through the inhibition of spore germination and germ-tube elongation. The production of such extracellular cell wall degrading enzymes has been associated with biocontrol abilities of the enzyme producing bacteria (Bouizgarne, 2013). These enzyme activities enable the plant to get protected from the biotic stress through pathogen elimination. The enzyme activities can be detected by measuring the degradation of specific substrates, such as cellulose, chitin, laminarin, and pectin, respectively, for cellulases, chitinases, glucanases, and pectinases and analyzed by various methods (Chenniappan et al., 2019; Tan et al., 2019). It is observed that the cell wall degradation by cellulase and pectinase enzymes occur when bacteria colonise the root epidermis and this enzymatic activity cannot be seen after colonization in the intercellular spaces of the root cortex. This suggests that endophytic bacteria induce the production of cellulase and pectinase enzymes only for its penetration into the host plants (Gupta et al., 2019). Many endophytic as well as epiphytic bacteria were reported for the production of lytic enzymes, which can inhibit many phytopathogens. Studies on inhibition of basidiospores germination of Moniliophthora perniciosa causative organism of cacao Witches’ broom by phylloplane actinomycetes revealed that the strains were able to produce chitinases on a liquid mineral medium with colloidal chitin, glucose, or cell walls of M. perniciosa as carbon source (Macagnan et al., 2008). In this study, the spore germination inhibition was higher when they were cultured using glucose as carbon source, which is followed by colloidal chitin and cell walls. Tang-um and Niamsup (2012) reported an endophytic actinomycete strain designated as P4 was studied for chitinase production and its association with the growth inhibition of F. oxysporum f.sp. lycopersici by Streptomyces sp. P4. In another report, a Gram-positive cellulose-decomposing endophytic bacterium Lysinibacillus xylanilyticus Chi-04 was isolated from medicinal plant Chiliadenus montanus exhibited cellulase activity of 0.18 U/min (Yousef et al., 2019). A similar study reported 45 endophytic bacterial isolates from Ammodendron bifolium plant resulted 40% of isolates positive for amylase and cellulase, whereas 13.3% and 53.3% isolates positive for protease and lipase activity, respectively (Zhu and She, 2018). Overexpression of eglS gene which encodes an endo-β-1,4-glucanase in B. amyloliquefaciens resulted in the increased bacterial population in the Chinese white cabbage plant tissues. This confers that endo-β-1,4-glucanase of B. amyloliquefaciens is required for the optimal endophytic colonization in the plant tissue (Fan et al., 2016). Passari et al. (2016) reported that 86.5%, 84.6%, and 90.3% of endophytic bacterial isolates obtained from an ethnomedicinal plant Clerodendrum colebrookianum showed significant activities for cellulase, amylase, and protease production, respectively. Achari and Ramesh (2019) represented the ability of an eggplant endophyte, B. cereus strain designated as XB177R could produce endoglucanase and pectinase enzymes, which is an advantage for systemic endophytic colonization of eggplant (Solanum melongena). Even though many endophytes are reported for the ability to produce lytic enzymes, the ideal mechanism of lytic

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Table 4.3  Lytic enzymes produced by endophytic bacteria. Enzyme

Producer

Target phytopathogen

Host plant

References

Chitinases

L. sphaericus

R. solani

Rice

Cellulase and amylase

Streptomyces sp. Streptomyce ssp Bacillus sp. Burkholderia sp. Streptomyces sp. B. pumilus

X. oryzae C. sublineolum R. solani

Rice Citrus Rice

Shabanamol et al. (2017) Hastuti et al. (2012) Quecine et al. (2008) Unpublished (from this research group) Ren et al. (2013)

Bacillus sp.

C. graminicola

Zeamays L (Corn)

Szilagyi-Zecchin et al. (2014)

S. griseus, Strep- Sclerospora tosporangium graminicola roseum

Pearl millet

Jogaiah et al. (2016)

Cellulase and protease Cellulase, amylase, and pectinase Protease

C. chrysosperma Populus

enzymes those act as inducers of systemic resistance in controlling pathogens is still unknown. Representative examples of lytic enzymes produced by some endophytic bacteria are shown in Table 4.3.

4.2.4  Induced systemic resistance Plants exhibit two types of resistant mechanisms based upon the external stimuli, namely, ISR and systemic acquired resistance (SAR), which can be differentiated based on the nature of the elicitor and the regulatory pathways involved. SAR develops on a plant upon the infection with a necrotizing pathogen and is more efficient in developing defense mechanism second time as the pathogen attack. ISR is another mechanism that is induced by an infection and increases the chemical or physical barrier of host plant rather than killing the pathogen. This protects plants against further attack by pathogenic microbes and herbivorous insects. As stated by Pieterse et al. (2014), many plant species could develop induced resistance in response to infection by pathogens, herbivorous attack, colonization in the roots by beneficial microorganisms, and after application of certain chemicals. ISR is expressed locally at the site of induction and systemically in plant parts that are spatially separated from the inducer, hence the term ISR. As summarized by Kloepper and Ryu (2006), some of the endophytic strains elicit systemic resistance that may be dependent on SA pathway and independent of ethylene or jasmonate pathways. Therefore ISR cannot be separated from SAR based on signal transduction pathways. Furthermore, it was described that these endophytic bacterial strains elicited systemic protection against pathogens, where the application of endophytes to one part of the plant exhibited a significant reduction in the incidence of a disease even though the pathogen is inoculated to another part of the host plant.

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4.2.4.1  Mechanism of ISR mediated by endophytes Endophytes bring about ISR through changing the physiological and biochemical reaction of the host leading to the synthesis of defense chemicals against the pathogenic challenge. Normally, ISR is activated by specific proteins known as pathogenrelated proteins (PR proteins), synthesis of polyphenols, flavonoids, phytoalexins, or induction of signal transduction pathways triggered by jasmonate/SA or ethylene (Ramamoorthy et al., 2001). The PR proteins inhibit the pathogen progression and facilitate protection against the pathogen and to induce biotic stress tolerance, the reactive oxygen species, which in turn regulate the function of important signaling molecule (Savatin et al., 2014). Investigations were carried out to determine whether the induction of PR-1a gene promoter was associated with systemic resistance, induced by bacterial strains (Park and Kloepper, 2000). A study indicated that when paddy plants inoculated with endophytic P. aeruginosa and P. pseudoalcaligenes showed the production of phenolics and flavonoids and induction of PR proteins like enzymes β-1,3-glucanase and catalase in plants even in the absence of pathogen Pyricularia grisea, responsible for fungal blast (Jha, 2019). The production of such enzymes due to endophytic bacteria recorded reduction of its autophagy dependent cell death as observed with reduced red fluorescence in bacterized plant cell. Karthikeyan et al. (2005) reported that the induction of PR proteins, aggregation of phenolics, and defense-related enzymes engaged in phenylpropanoid pathway played a vital role in defending the invasion of a pathogen Macrophomina phaseolina in black gram roots. Recently, an endophytic bacterium B. halotolerans Y6 isolated from Verticillium wilt-resistant cotton, which possesses strong antagonistic abilities to inhibit V. dahlia and the enzyme activity assay, heterologous expression and gene knockdown confirmed the virulence factor is β-glucanase Bgy6. In this study, Bgy6 overexpression has enhanced the resistance to Verticillium wilt with a disease index of 8.33 in upland cotton plants treated with OY6, which was lesser than both the plants treated with wild type Y6 or control without bacteria (Zhang et al., 2019). Other than pathogenesis-related proteins, the role of defense-related enzymes such as l-phenylalanine ammonia lyase (PAL), peroxidase (POX), polyphenol oxidase (PPO), and (chitinase and β-1,3 glucanase) in disease resistance have been reported by different researchers. In one of our studies, the development of ISR was verified through split root experiment, where individual treatment of beneficial bacteria could induce resistance in plants though there was no direct contact with the pathogen (Dutta et al., 2008). Similarly, Chung et al. (2015) reported a Bacillus strain YC7010T isolated from the roots of rice and developed as a novel biocontrol agent against bacterial blight through the induction of resistance in rice. In our study, Bacillus sp., and Streptomyces sp. isolated from the root, pseudostem and leaves of rice plants cultivated in the below sea water level rice cultivating areas of Kuttandu were developed as bioformulation for plant growth promotion and yield enhancement. The analysis of biocontrol effect against the sheath blight disease caused by R. solani confirmed that in the bacterial treated plants, the level of defense-related enzymes and total phenolics compounds were observed in higher amount (Fig. 4.6).

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Figure 4.6  Induction of systemic resistance in rice plants due to endophytic bacteria through defense-related enzymes and phenolics. (A) PAL; (B) POX; (C) PPO; and (D) total phenol content.

Some studies also evaluated the potential of endophytic Streptomyces spp. to trigger systemic resistance and alleviate oxidative stress in chickpea plants against the causative organism, Sclerotium rolfsii. The defense-related enzymes such as PAL and PPO were higher in the bacterial treated plants, along with the increase in total phenolics and flavonoids in greater amount. In plants inoculated with S. rolfsii showed that the defense pathway in chickpea is triggered by endophytes due to the ability to synthesize various enzymes, subsequently led to an induced resistance against the pathogen (Singh and Gaur, 2017). One of our studies reported that the application of two Pseudomonas strains, such as RRLJ 134 and RRLJ 04, exhibited the induction of systemic resistance in tea plants under split root conditions against brown root rot and charcoal stump rot. The activity of defense-related enzymes were increased rapidly up to 30 days after treatment in pathogen alone plant cuttings after which a decrease in their level was monitored, whereas in the bacterial treated plants, the enzymatic level was higher till the last day of observation (Mishra et al., 2014). Both the strains were isolated from the rhizosphere soils, which shown endophytic ability in our screening

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studies (unpublished data). Joe et al. (2012) reported that the rice plants treated with an endophytic Achromobacter xylosoxidans AUM54 followed by inoculation with rice blast fungus M. oryzae exhibited a significant increase in the activities of defenserelated enzymes such as PPO, POX, PAL, and chitinase. Similarly, Karthikeyan et al. (2005) reported endophytic P. fluorescens strains Endo2 and Endo35 for induced systemic disease protection against dry root rot of black gram (Vigna mungo L. Hepper) plants caused by a plant pathogen, M. phaseolina when tested under glasshouse conditions. Here, the activities of defense-related enzymes such as POX, PPO, and PAL were stimulated in addition to accumulation of phenolics and lignin. Likewise, an investigation reported a novel endophytic bacterium B. oryzicola YC7007 with antagonistic activity against F. fujikuroi, a causative agent of bakanae disease in rice via ISR and antibiotic production (Hossain et al., 2016). The root inoculation of tomato plants with Micromonospora strains isolated from nodules effectively reduced leaf infection by the fungal pathogen B. cinerea and the gene expression analyses shown that Micromonospora stimulated the plant capacity to activate defense mechanisms through the induction of jasmonate-regulated defenses upon pathogen attack (Martínez-Hidalgo et al., 2015). A wheat seed endophyte Paenibacillus displayed prominent biocontrol activity against F. graminearum, a causative agent of wheat head blight (Herrera et al., 2016). Abdallah et al. (2017) isolated five Bacillus spp. with antagonistic activities against fusarial wilt were found to produce SA, with the greatest production recorded for B. subtilis SV41. Accordingly, SV41-based treatments displayed a slight upregulation in acidic PR-1 and PR-3 expression genes in tomato plants inoculated or not with F. oxysporum f. sp. lycopersici. In some plants, the elicitors such as SA, jasmonic acid, ethylene, and abscisic acid were not required to confer defense mechanisms. Rashid et al. (2017) investigated that treatment with an endophytic B. velezensis YC7010 significantly ISR to the green peach aphid leaves via strongly expressing senescence promoting gene PAD4 (phytoalexin deficient 4) while suppressing the expression of BIK1(botrytis-induced kinase1). Similarly, a native olive root endophyte, P. fluorescens PICF7, antagonist to Verticillium wilt of olive, was able to trigger a wide range of defense responses in root tissues. Root colonization by this endophytic bacteriuminduced genes coding for lipoxygenase 2, catalase, 1-aminocyclopropane-1-carboxylate oxidase, and PAL and the computational analysis also revealed that different transcription factors (JERF, bHLH, WRKY) were upregulated in olive aerial tissues (Gómez-Lama Cabanás et al., 2014). Zhu (2019) reported that endophytic bacteria such as Staphylococcus, Kocuria, Bacillus sp. from A. bifolium plants promoted host seed ethylene release during germination endophytic bacteria–plant interaction, and increased osmotic stress tolerance in A. bifolium. Similarly, the inoculation of endophytic Methylobacterium oryzae CBMB20 in salt-stressed rice plants improved photosynthesis and reduced stress volatile emissions due to mellowing of ethylenedependent responses and activating vacuolar H+-ATPase (Chatterjee et al., 2019). An investigation evaluated the additive potential of an endophytic PGPR, B. pumilus INR7, and a chemical inducer benzothiadiazole to induce systemic resistance against Xanthomonas axonopodis pv. vesicatoria causative of bacterial spot in pepper plants under field conditions. It was found that the addictive effect was accompanied

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by the expression of defense marker genes in pepper such as CaPR1, CaTin1, and CaPR4 to a greater extent than treatment with either agent alone (Yi et al., 2013). Romero et al. (2019) reported two endophytic bacteria such as B. amyloliquefaciens and P. fluorescens individually and in combination investigated for the modulation of defense-related genes and systemic resistance against Alternaria alternata in Withania somnifera plants and enhanced the expression of SA and jasmonic acid responsive genes in the stressed plants. In this study, bacterial treated plants exhibited improved respiration, transpiration, photosynthesis, and stomatal conductance in presence of pathogen. An investigation demonstrated an endophytic P. viridiflava from apoplastic fluids obtained from canola leaves and analyzed their potential for inhibiting Xanthomonas campestris, Sclerotinia sclerotiorum, and Leptosphaeria maculans, where the protective effect could be due to the induction of resistance mediated by salicylic and jasmonic acid signaling pathways. Few studies have been suggested to use other molecules as signaling molecules such as melatonin, which increased the protective effect of melatonin against many phytopathogens (Liu et al., 2019). Likewise, the concept of using plant pathogenic toxins as inducers to induce disease resistance in plants has received increasing attention, and a protein Arthrinium phaeospermum toxin was isolated from a bamboo blight pathogen (Li et al., 2019).

4.3  Endophytes providing ISR against wilt diseases Many plants including both annual crops and woody perennials undergo death due to wilt diseases, which occur as a result of the attack by bacterial or fungal pathogens. During the infection, the pathogens make entry into plants via water conducting xylem vessels where they multiply, and thereby blocking the water and mineral transportation. These pathogens continue to propagate internally through xylem till the entire death of the plant (Pietro et al., 2003). It is difficult to diminish wilt diseases for several reasons. First, the infected plants cannot be restored due to the lack of efficient treatment methods. Second, many of the pathogens are soilborne and capable to produce resistant spores so that are able to survive in the soil for long duration even without host plants. Third, some of these pathogens are capable to infect a wide range of host plants and therefore control measures like crop rotation found not very effective. Hence, the use of genetic resistance was found to be an effective strategy to control vascular wilt diseases. This relies on the fact that vascular wilt pathogens live deep in the interior of their host plants, such studies into their biology are complicated. In general, the major vascular wilt pathogens coming under the category: Ceratocystis, Ophiostoma, Verticillium, and Fusarium (Yadeta and Thomma, 2013). Several studies showed endophytic bacteria were able to suppress the growth of wilt producing pathogen in different crop plants. A study confirmed that fusarial wilt (Panama disease) in banana plants caused by F. oxysporum f. sp. cubense was effectively controlled in planta by an endophyte, Burkholderia cenocepacia 869T2. Furthermore, B. cenocepacia could decrease the disease incidence of fusarial wilt in treated banana

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plants comparing to noninoculated plants infected in the field trials within a period of 7 months. Furthermore, the diversity of endophytes within banana plants were investigated and B. cenocepacia 869T2, and found that a pyrrolnitrin and pyrroloquinoline quinone potential producer can be used as a biological control agent against in banana plants (Ho et al., 2015). Abdallah et al. (2016) reported an endophytic B. cereus S42, recovered from Nicotiana glauca stem possess antagonistic activity against tomato pathogen, F. oxysporum f. sp. lycopersici. This endophytic culture supernatant and whole cell suspensions had significantly suppressed fusarial wilt by 87%–96% and enhanced tomato growth by 39%–79% compared to pathogen inoculated and untreated control through the production of phthalic acids and dibutyl esters. In another report, endophytes as well as rhizobacteria were screened against Ralstonia solanacearum, which causes bacterial wilt in eggplants. The treatment with bacterial cells of P. mallei (RBG4, ET17) and one Bacillus spp. (RCh6) reduced wilt incidence by 83% compared to control (Ramesh and Phadke, 2012). In our earlier studies, fluorescent pseudomonads strain RB 8, isolated from rhizoplane of a crop plant has lowered the number of wilted chick pea plants in F. oxysporum f.sp. ciceris infected soil. The disease suppression and growth enhancement along with the root colonization by these organisms indicate their possibility to use as biocontrol agents against chick pea wilt (Dileep Kumar, 1998). In another investigation, a P. aeruginosa RRLJ 04, and a B. cereus BS 03, were tested individually and in combination with Rhizobium RH 2 for their ability for plant growth promotion and nodulation in pigeon pea (Cajanus cajan). These treatments were shown a reduction in the number of wilted plants, as they grown in soil infested with F. udum. Seed bacterization with drug mutants of RRLJ 04 and BS 03 proved their ability to colonize the roots (Dutta et al., 2014). Recently, an antagonistic Streptomyces luteosporeus NIISTA32 was found to induce systemic resistance against wilt disease in mung bean plants under split root conditions when infected with F. oxysporum ITCC 4814. Another approach to control vascular disease is the use of genetic resistance, and it has been reported in various crops (Yadeta et al., 2014). An investigation on B. subtilis CBR05 induced Vitamin B6 biosynthetic genes in tomato confronted with X. campestris pv. Vesicatoria confirms the elicitation of ISR regulated by de novo pathway (Chandrasekaran et al., 2019). Introgression of wilt resistance was established with interspecific cross with genes from resistant wild species C. oxyacantha and C. palaestinus into susceptible cultivated species (C. tinctorius) and simple sequence repeat markers linked to wilt resistance in safflower caused by F. oxysporum f.sp. carthami (Anjani et al., 2018). In an observation, genes involved in the early defense response of L. usitatissimum (flax) against the fungus F. oxysporum were identified, using high throughput sequencing and also changes in the expression of genes encoding pathogenesis-related proteins and reactive oxygen species production (Dmitriev et al., 2017). Furthermore, the identified genes that were upregulated specifically in flax genotypes resistant to fusarial wilt and implies that the identified genes in resistant cultivars and BC2F5 populations exhibiting induced expression in response to fungal infection. Defenserelated genes, such as genes involved in SA and jasmonate signaling pathways,

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pathogenesis-related genes, mitogen-activated protein kinase, chitin elicitor receptor kinase1, and ethylene responsive transcription factor was identified by transcriptome comparison of sweet potato challenged with F. oxysporum f. sp. batatas (Fob) from a study that performed de novo transcriptome assembly and profiling of digital gene expression (Lin et al., 2017).

4.4  Optimization of bioactive metabolite production by endophytes through statistical approach Manipulation of nutritional environment is one of the approaches for the improvement of secondary metabolite production by beneficial bacteria. As the effectiveness of biocontrol formulations depend on the amount of cells of the antagonist at the colonization site, the nutritional additives may result in a stimulation of antagonist growth and better colonization (Nunes et al., 2001). Therefore statistical approach through RSM helps in the evaluation of relationship between the dependent (antibiotic production) variable and independent (medium components) variables. In a study, a high yield of actinomycin D was achieved with an endophytic S. parvulus Av-R5 in glucose soybean meal broth media after optimization through central composite design (Chandrakar and Gupta, 2019). Here, RSM has been used to optimize the nutrient composition and the fermentation conditions for enhanced production of antimicrobial compound active against multidrug resistant pathogens, and the reaction was done under submerged fermentation. In our study, statistical optimization using one-factor-at-a-time (OFAT) approach and finally Box-Behnken design (BBD) were employed for the enhanced antimicrobial metabolite, which resulted in an increase of 86.66% antibacterial production from a forest isolate S. nogalater designated as NIIST A30 (Jacob et al., 2017). The fermentation conditions such as pH, temperature, and incubation time were optimized through OFAT method and the nutrient composition in the fermentation media was optimized via RSM. In another investigation, OFAT followed by BBD was studied for a 3.30 fold increased production of granaticinic acid, an antimicrobial metabolite from an endophytic isolate S. thermoviolaceus NT1 (Roy et al., 2016). Here, various media, glucose concentration, initial pH, incubation temperature, incubation period, and inoculum size were optimized in the OFAT approach and the RSM analysis revealed a multifactorial combination such as 0.38% glucose, under pH 7.02, and 36.53°C yielded maximum antimicrobial compounds. In a study, static fermentation conditions of Aspergillus wentii EN-48, an endophytic fungus was optimized, employing RSM to enhance the production of an antitumor agent, asperolide A. The effect of initial pH, salinity, and incubation time on the production of the compound were further optimized based on the results obtained from the single-factor approach and finally, the optimized conditions resulted in a 13.9-fold yield enhancement, as per the predicted value and the optimized conditions were successfully used in scale up for the production of asperolide A (Xu et al., 2017). An investigation studied for the optimized production of antifungal metabolites produced by B. subtilis V26 and evaluated its efficiency in suppression of B. cinerea, the main

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cause of tomato fruit rot disease using a three-level three-factor Box-Benken design (Kilani-Feki et al., 2016). In this study, the optimum antifungal metabolite production of 6200 AU/mL was achieved at standardized amounts of soybean, potato extract, and culture medium volume and this, in turn, recorded more than 93% improvement in bio-fungicide production over the basal medium.

4.5  Bioformulation of endophytes Endophytic microorganisms can be formulated in various solid carriers, such as talc, peat, lignite, clay, etc., while liquid formulations were prepared in solvents such as water, oil, and organic solvents. Solid formulations include granules, microgranules, wettable powders, wettable/water-dispersible granules, and dusts. Solid formulations have shorter shelf life, susceptibility to environmental conditions, high contamination, and low field performance, while liquid formulations offer longer shelf life, that is, up to 2 years with high purity, carrier-free activity, ease in handling and application (Dhir, 2017). The microbes are present in dormant spore form which gives rise to active cells upon application in the field, and this helps increase its shelf life for more than one year. Liquid formulations possess high selectivity to target pests, safety to humans and nontarget organisms, and suitability for organic niche in contrast to chemical pesticides that possess broad spectrum and affect nontarget organisms including predators, parasites, as well as humans. These formulations are also known as flowable or aqueous suspensions and are composed of biomass suspensions in water, oils, or as emulsions. A classical liquid formulation consists of 10%–40% microbes, 1%–3% suspended ingredients, 1%–5% dispersants, 3%–8% surfactants, and 35%–65% carrier liquid, which is either oil or water (Mishra and Arora, 2016). Recently, we developed an endophytic bacterial bioformulation (Fig. 4.7) from a consortium of Peanibacillus elgii NIIST B578, B. subtilis NIIST B580 and B. subtilis NIIST B595 named as Endophytic bacterial formulation—PLANT TONIC, which increased plant growth and yield enhancement in rice and several other crop plants besides soil fertility improvement. The combined application of these three endophytes in the management of R. solani in rice confirmed the ISR through the enhanced production of defense-related enzyme such as PAL, POX, and PPO production and phenolics in rice plants compared to control up to 70 days after treatment (DAT). An observation evaluated the protective effects of endophytic bacterial strains (B. subtilis; EPCO16; and EPC5) and rhizobacterial strain (P. fluorescens; Pf1) against chilli wilt disease caused by F. solani by inducing systemic resistance by enhanced activities of POX, PPO, PAL, β-1,3 glucanase, chitinase, and also phenolics involved in the phytoalexin production (Sundaramoorthy et al., 2012). In another work, endophytic bacterial strains, Stenotrophomonas maltophila H8, P. aeruginosa H40 and B. subtilis H18 were evaluated for antagonistic activity against R. solani in cotton seedlings under nursery conditions in which the beneficial bacterial strains were applied as either talc based bioformulation or soil drench in R. solani infected and noninfected soil (Selim et al., 2017). They found that the soil drench application was performed

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Figure 4.7  Development of an endophytic bioformulation named “PLANT TONIC.”

better than talc-based bioformulation and the significant increase of seed reduction in disease severity was achieved in the treatment. Additionally, an apparent induction of the defense-related enzymes POX, PPO was observed and the GC-MS analysis shown the presence of various bioactive metabolites, which can induce systemic resistance in the pathogen-infected saplings.

4.6  Challenges related to the development of endophytic formulation Even though endophytes exhibit strong antagonism and ability to benefit their host plants through different ways as described earlier, several challenges have to overwhelm when developing them as bioformulation. One of the most vital issues of a bioformulation is the success or failure as a commercial product under field conditions

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even though they performed very well under laboratory and nursery trials. Endophytes can encounter challenges from various microorganisms when applied to the plant parts, depending upon the variation in population densities those are affected by different environmental conditions. The population of the introduced endophytes can be enhanced by the early establishment and multiplication of the endophytic community on plant root rhizosphere and their host tissues (Verma and Gange, 2013). Additionally, the poor performance of the microbes was due to their low colonization ability, lesser viability during storage, lethality to untargeted organisms, or nonsuitability to large-scale applications due to the very low yield in cultures. Moreover, some human-pathogenic bacteria are also reported among endophytes especially in immuno-suppressed individuals, which is a possible health threat (Hallmann and Berg, 2006). Hence, identifying endophytes that are potentially pathogenic is crucial, so they should not be considered for applications. It is known that various opportunistic human bacterial pathogens including Burkholderia, Enterobacter, Herbaspirillum, Ochrobactrum, Pseudomonas, Ralstonia, Staphylococcus, and Stenotrophomonas have been identified as colonizers of the plant rhizosphere. The biocontrol ability of endophytic microbes is due to the production of various bioactive metabolites, hence the whole organism may not be needed for the trials. However, some argue that when these microbial crude metabolites when applied to crop plants, many pathogens may become drug resistant as they have done with synthetic compounds (Verma and Gange, 2013). Hence, the researchers insisted to develop bioformulation simply diluting the entire microbial cell without any treatment. Many times, bioformulation with individual organisms may not be helpful in controlling the diseases, hence a consortium of bacteria may help in the process. According to Jeyanthi and Kanimozhi (2018), many plant growth-promoting bacterial genera have been commercialized so far, namely, Agrobacterium, Azospirillum, Azotobacter, Bacillus, Burkholderia, Delfitia, Paenibacillus, Pantoea, Pseudomonas, Serratia, Streptomyces, and Rhizobia. Even though most of the products are available in the market, the scenario is not good as only 7% of total bioformulation made per year is reaching in the hand of farmers (Kumari et al., 2019). When dealing with commercialization, a product should satisfy farmer’s requirements such as repeated results, safety and stability, cheap price, easy handling, and prolonged shelf life. However, a bioformulation has specific problems of viability loss, contamination, and reduced effectiveness against pathogen or pest during storage time (Vurukonda et al., 2018). Recently, the researchers across the world had focused on implementation of new technologies for the development of potential bioformulations. The latest techniques in molecular genetics include cutting-edge systems for genome editing and the use of RNA inhibition and knocking out the expression of selective genes. Another improvement in bioformulation like fluidized drying by fluid bed dryer (FBD) give hardening of inoculants to stress so that it can perform very well upon application on harsh soil conditions. FBD formulations can be applied as seed inoculation, soil inoculation, seedling dip, fertigation, and foliar spray (Sahu et al., 2018). Lyophilization can also be applied to store the bacterial inoculants for long term, where the water content is lowered and due to this, bacteria in formulation remain inactive, resistant to various stresses, and insensitive to contamination. This technique is applied to polymeric

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preparations such as alginate inoculants used to makes dry formulations (Bashan et al., 2016). Even though there are many developments in the field of microbial inoculants, a full understanding of the practical use of endophytes as bioformulation in agriculture is essential. Yet, there is an urgent need to improve communication between the public and researchers for efficient usage of bioformulation. It is therefore essential to increase the awareness among the farmers, regarding effective usage of products for a particular pathogen.

4.7  Future prospective in endophytic research Current research in endophytes attempts to screen putative endophytes, through in vitro experiments under optimized conditions, field experiments under different environmental conditions are needed to develop successful commercial biocontrol agents. Field experiments should investigate physiological and ecological aspects of endophytic biocontrol agents with crop plants and/or host plants and also their environmental effects in the field. Further, more research is essential for the commercialization of biocontrol formulations, as these will reduce economic and environmental costs significantly. This can be accomplished with novel strategies using molecular techniques (e.g., metagenomics), ecological dynamics, and statistical advances. Future works could be integrated with screening, testing antagonistic ability in green house and field trials, coupled with biomass production, following the commercialization of biocontrol formulation that is ultimately essential to ensure global food security. There are various procedures to monitor bacterial endophytes to enhance our knowledge of mechanisms behind antagonism, and thus we can have a better control over various plant diseases. Different approaches such as in vivo expression technology and recombination in vivo expression technology can provide an awareness into genes responsible for bacterial entry, competence, colonization in the plant, and the suppression of pathogens. Colonization in various plant parts is essential for assessing the competence of potential microbial biocontrol agents because it act as the delivery system for the endophytic microbes and/or their products (Czelleng et al., 2006). Investigating the endophytic colonization process through monitoring their entry site via fluorescence experiments can be very useful in the evaluation of their potential as biocontrol agents. Moreover, with the availability of GFP tagging, gene mutants with individual colors, which allow various microbes labeled with GFP and its derivatives to be monitored at the same time. Moreover, the construction of gene mutants in specific bacterial endophytes and evaluating their colonization and biocontrol accomplishment in planta may reveal the facts behind various biocontrol mechanisms. Maldonado-González et al. (2015) reported mechanisms in the construction of specific P. fluorescens PICF7 phenotypes and assessing their capability of colonizing olive roots and their performance in biocontrol efficiency against Verticillium wilt. Quantitative genetic tools, such as quantitative trait loci mapping allow the identification of genes or genetic loci underlying important biological traits of any organism

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of interest. This technology generates improved crop varieties via genetic engineering or traditional plant breading approaches (Qiu et al., 2019). The whole host genome sequencing could provide the basis to generate plants harboring an improved microbiome. Anticipation of CRISPR/Cas9 technology can hold great potential to improve or induce the ability of plants to preferentially recruit beneficial microbiota (Barakate and Stephens, 2016). However, the current ability to harness the plant microbiome in agriculture and to manipulating microbiomes in situ remain limited, and more studies and trials are needed to increase our understanding of the nature and mechanisms, underlying the microbiome–plant relationship before such an approach can be applied and commercialized.

4.8 Conclusion In conclusion, endophytes have excellent potential as biocontrol agents against plant pathogens, primarily due to their strong mechanisms of antagonism, as well as their ability to provide benefits to host plants such as growth promotion and induced host resistance. Despite obvious challenges, it is important to note the use of endophytes for growth promotion, disease management and yield enhancement is still new and biotechnological development for both is really just a beginning. There is really a great potential for mutualistic symbionts to make significant contributions toward cheaper sustainable pest/pathogen and pollutant control. Improvements in the delivery of endophytes and introduction to host plants can be made so that the advantage of endophyte precolonization and adaptation in host plants is fully exploited. Biotechnological innovations in bioformulations, application optimization, and elucidating useful bioactive compounds from endophytes can all contribute to strengthen the role of endophytes for the control of crop diseases. Although many bioformulations have been developed, very few products are found to be promising. So, there arise a crucial urge to develop the plant inhabiting bacteria such as endophytes to be studied in detail such as selection of organism, production method, delivery system, application technology, factors affecting bioformulation development, persistence in the environment, and release to the market.

Acknowledgments The authors are thankful to Dr. Ajayaghosh A., Director, CSIR-National Institute for Interdisciplinary Science and Technology (NIIST), Thiruvananthapuram for his permission to carry out this work. JJ thank Department of Science and Technology (DST), Government of India, for inspire fellowship (IF 130648). GVK and DT acknowledge University Grant Commission (UGC) and Council of Scientific and Industrial Research (CSIR), New Delhi, respectively, for providing Junior Research fellowships. Funding from Kerala State Council for Science, Technology & Environment (KSCSTE), Thiruvananthapuram, Kerala is highly acknowledged.

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Ajay Kumara, Samir Drobya, James Francis Whiteb, Vipin Kumar Singhc, Sandeep Kumar Singhc, V. Yeka Zhimoa, Antonio Biasia a Agriculture Research Organization, Volcani Centre, Rishon LeZion, Israel; bDepartment of Plant Biology, Rutgers University, New Brunswick, NJ, United States; cCentre of Advance Study in Science, Department of Botany, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India Chapter outline head 5.1 Introduction  107 5.2 Types of seed priming  109 5.2.1 Hydro-priming  109 5.2.2 Halo-priming  110 5.2.3 Osmo-priming  110 5.2.4 Solid matrix priming  110 5.2.5 Hormonal priming  110 5.2.6 Chemo-priming  111 5.2.7 Thermo-priming  111 5.2.8 Bio-priming  111

5.3 Factors affecting seed priming processes  112 5.4 Role of endophytes in seed priming  112 5.5 Future perspectives  117 5.6 Conclusion  118 References  118

5.1 Introduction The continuous rise in global population is one of the most severe problems that influence agriculture sectors and creates pressure to researchers as well as farmers to meet human demand for food in limited resources of land and changing climatic conditions. In this context improved quality of seed to fulfill the higher demand of agriculture has been recognized as a major challenge globally. Slower growth of developing seedlings under various abiotic factors (harsh environmental conditions) or biotic factors (pathogenic microorganisms) limits the growth and yield of crops; development of techniques for fast and homogeneous growth of seeds could be a sustainable approach for better agricultural productivity (Osburn and Schroth, 1988). In this aspect, improving Microbial Endophytes. http://dx.doi.org/10.1016/B978-0-12-819654-0.00005-3 Copyright © 2020 Elsevier Inc. All rights reserved.

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the quality, germination, and establishment of seed through “seed priming” is a sustainable approach to enhance yields and performance of plants (McDonald, 2000). Interestingly, primed seeds have been demonstrated to withstand the numbers of abiotic and biotic stresses leading to enhance seed emergence along with crop productivity. The term “seed priming” first proposed by Heydecker et al. (1973) was described as an effective technology for the enhancement in growth and vigor of plants through uniform emergence and better establishment of seed. Generally, during seed priming, seed has been dipped inside the limited amount of water, inorganic solutions, or osmoprotectants for a particular period of time that leads to significant changes in the physiological or metabolic profile of seedlings as well as increase the capacity of seedlings to withstand stress exposure (Tanou et al., 2012; Hussain et al., 2016). Various reports show beneficial effect of seed priming via uniform or early germination, increased nutrient extraction, reduced seed dormancy, etc (Taylor and Harman, 1990; Bruce et al., 2007; Hill et al., 2008; Farooq et al., 2009, 2019). According to the published studies, it has been broadly mentioned that priming of seeds mitigates the adverse impact of various biotic (such as, phytopathogens, plant diseases; van Hulten et al., 2006), and abiotic (drought, salinity, flooding) stress factors, that affect the physiology and metabolism of plants via different mechanisms (Kausar and Ashraf, 2003; Basra et al., 2005; Guan et al., 2009; Chandra Nayaka et al., 2010; Sharma et al., 2014; Kumar et al., 2016). In some cases priming of seeds modulates their biochemical status through improving α-amylase activity even in the low range of temperature (Anaytullah, 2007). Currently, different priming techniques such as hydro-priming, osmo-priming, solid matrix priming (SMP), nutria-priming (Majda et al., 2019), chemo-priming, thermo-priming, and bio-priming (Panuccio et al., 2018) are being used in order to improve seed characteristics, plant productivity as well as alleviate many environmental stresses (Paparella et al., 2015). Each technique has certain limitations and advantages, many reports are also available regarding seed priming that showed beneficial or deleterious effects of priming (Tarquis and Bradford, 1992). The selection of a particular methodology to induce seed germination is determined by the selected plant species, seed attributes, and procedures employed for priming (Ellis and Butcher, 1988; Hill et al., 2008; Ibrahim, 2016; Paparella et al., 2015). The use of microbes for seed priming is a viable and promising approach in the context of improvement in seed characteristics under changing environmental conditions. Seed bio-priming involves the integration of beneficial microbes including bacteria and fungi for improved plant growth and development. In addition, the microbe- or plant-derived secondary metabolic products like phytohormones and plant extracts (Panuccio et al., 2018) have also been documented for seed priming (Hamayun et al., 2010). Some of the commonly reported microbes for seed priming are species of Trichoderma, Enterobacter, Pseudomonas, and Bacillus (Raj et al., 2004). Positive influences of vegetable seed bio-priming based on treatment with strains of Trichoderma harzianum over other bacterial and fungal species has been reported by Ilyas (2006). It has been established that many of the applied bacterial, fungal, and mycorrhizal species (Balestrini et al., 2018) develop intimate endophytic association with seed of interest for priming and help considerably in overcoming the negative consequences of imposed by various stress factors (Waller et al., 2005). To date, various endophytic

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bacterial (Joe et al., 2012) and fungal strains have been identified and applied as plant or seed inoculants to enhance the growth and yield of crops. The intimate symbiotic interaction of endophytic bacterial species with host plants offers vast applicability as a potential tool in the agriculture sector in a sustainable manner to improve the crop growth and yield (Prasanna et al., 2012). Most importantly, endophytic bacterial strains equipped with nitrogen-fixing capability may be well-suited candidates for agricultural improvement strategies because they may provide plants with additional nitrogen (Reinhold-Hurek and Hurek, 1998). Such diazotrophic bacteria, have also been documented for their growth promotion ability including synthesis of phytohormone, siderophores, solubilization of phosphate, restriction of ethylene biosynthesis, and induction of resistance against different plant pathogens (Jha and Kumar, 2009), indicating their suitability for crop growth and yield improvement. Further, the capacity of some root endophytic microbes to alternate between root and soil phases and vector nutrients (nitrogen and soil minerals) to plants from the soil in the rhizophagy cycle (White et al., 2018) (Chapter 1), makes these endophytes good candidates for overall enhancement of the nutritional status of crops. Rhizophagy cycle microbes have also been shown to be important in stimulation of seedling development (Verma et al., 2017); seeds without these endophytes often possess roots that fail to show proper gravitropic response, where roots lay on the surface of soils, and do not form root hairs (Verma et al., 2017). It is evident that plants naturally depend on endophytic microbes for proper development, nutrient acquisition, growth, and health (Verma and White, 2019). Seed priming with endophytic microorganisms due to inherent properties of growth promotion, such as phytohormone production, induction of resistance, and tolerance to various abiotic and biotic stresses, could be utilized as an important eco-friendly approach to mitigate the problem of resistance development in plant pathogens as well as for the improvement in crop productivity. However, there are certain limitations of field application of primed seeds in management of crop productivity. The present chapter has been designed with the aim to explore the potential ability of diverse endophytic microbes in seed priming to achieve the goals of sustainable agriculture, methods to overcome limitations associated with field applications along with future perspectives.

5.2  Types of seed priming There are various types of seed priming techniques; some of them are discussed in further sections.

5.2.1 Hydro-priming This technique used for initiating seed germination without emergence of radicle that involves continuous or successive addition of limited amount of water to the seeds under the temperature ranging in between 5 and 20°C (McDonald, 1999). The hydropriming technique is generally used at those agricultural sites where climatic conditions are adverse (McDonald, 2000).

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5.2.2 Halo-priming In this technique, seeds have been immersed in various solutions of salts such as NaCl, KNO3, CaCl2, CaSO4, etc., to start the germination of seed even in the adverse environmental conditions and this treatment enhanced the salinity tolerance as reported in case of wheat by Basra et al. (2005). The priming with potassium nitrate and sodium chloride solutions, causing reduced water potential and increased water absorption by accumulating within the seeds (Parera and Cantliffe, 1994). Similarly, working on Vigna radiata L. seeds, Jisha and Puthur (2014) transformed the NaCl tolerant variety into more salt and drought tolerant by priming the seeds. Further, their study also showed better and enhanced tolerance capacity of sensitive variety against NaCl and polyethylene glycol (PEG) stress. This simple and economic halo-priming technique has been used to acclimate plants under various stresses (Sedghi et al., 2010).

5.2.3 Osmo-priming In this technique, seeds are dipped in the sugar, PEG, glycerol, CaCl2 solutions for a fixed interval of time (Tabassum et al., 2017) sorbitol, or mannitol followed by air drying before sowing. Normally, after dipping, water enters inside the seed that may lead to progressive accumulation of reactive oxygen species (ROS) and oxidative damage of cellular components. Osmo-priming checks oxidative injury caused by ROS by delaying the entry of water (Heydecker and Coolbear, 1977; Taylor et al., 1998). Osmo-priming appears beneficial for the germination of seed as well as crop performance under both saline and nonsaline conditions (Tabassum et al., 2017). In a study Hur (1992) reported treatment of Italian ryegrass (Lolium multiflorum) and sorghum (Sorghum bicolor) seeds with 20% PEG-8000 for 2 days at 10°C enhanced the rate of germination, under waterlogged, cold-stress, or saline conditions. Thus, osmo-priming may contribute to improved germination rate in part by increasing various enzyme activities.

5.2.4  Solid matrix priming It is also known as “matri-priming” or “matri-conditioning” technique. This technique has been also used as an alternative of osmo-priming because of less expense, lower volume of osmotic solution and also of temperature and aeration control. In solid matrix priming, seeds have been mixed with solid materials of organic or inorganic origin such as calcium silicate, calcined clay, and vermiculite with known water proportion, all these procedures carried out in a sealed container that permits air circulation and avoids excessive evaporation (Harman and Nelson, 1994; Rogis et al., 2004; Hacisalihoglu, 2007; Ermis¸ et al., 2016).

5.2.5  Hormonal priming This is the priming technique in which the seeds have been pretreated with different hormones such as salicylic acid, gibberellic acid, and kinetin, which promote

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the growth and development of the seedlings. There are various reports that show the effectiveness of hormonal priming (Hamada, 2001; Hussein et al., 2007; Afzal et al., 2006).

5.2.6 Chemo-priming In this technique priming of seed is caused by adding conventional disinfectants such as sodium hypochlorite (NaOCl), HCl, and also some agrochemicals to prevent microbial contaminations in the priming solutions (Parera and Cantliffe, 1990). Although treatments with NaOCl and HCl solutions reduce losses in germination in comparison to losses caused by pathogens. Furthermore, researches have been carried out to critically evaluate the benefit or losses caused by chemo-priming.

5.2.7 Thermo-priming In this technique seed has been treated at various time intervals before sowing. It is already proved that the treatment of seed with altering temperature results in better seed germination as compared to regular constant temperature (Shin et al., 2006; Markovskaya et al., 2007). Altering temperature can break seed dormancy and this technique is popular in improving germination efficiency under adverse climatic conditions (Huang et al., 2002). Alternating temperature of pre-sown treated seed of cucumber and melon, enhances the productivity of plants (Markovskaya et al., 2007). The variation in the ambient temperature influences thermosensory pathway that results in changes in flowering time (Franklin, 2009). The nature of the temperature such as hot (Khalil and Rasmussen, 1983) and cold (Runkle et al., 1999; Garner and Armitage, 2008) also influence or modulate the time of flowering.

5.2.8 Bio-priming It is the latest technique in which seeds have been treated with biological means like beneficial microorganism to protect the seed from diseases or also enhanced the growth and seed germination by modulating various growth hormones (Farooq et al., 2017). The term bio-priming was first used by Callan et al. (1990), during the experiment, in which they coated sweet corn seeds with a bacterial strain. Later on numerous reports have been published that used this technique to improve seedling growth. Bio-priming plays an important role in improving germination and viability of seed, growth, and yield of plants (Prasad et al., 2016; Bhatt et al., 2015). Like other priming techniques, this helps in improving physiological processes at the pre-sowing phase and also in the multiplication of applied microbes at the area of seed surrounding (Taylor and Harman, 1990). It has been found that bio-primed seed is able to provide a better level of protection against various diseases of plants compared to seed treated with various pesticides. The application of endophytic microbes such as bacteria or fungi through bio-priming techniques has potential benefits over traditional priming techniques because it has sufficient time and environment for the successful colonization inside the seeds.

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5.3  Factors affecting seed priming processes The priming of seed may be influenced by various factors such as aeration, quality of seed, temperature, light, and duration (Parera and Cantliffe, 1994; Bray, 1995). Aeration is one of the prime factors responsible for the emergence of seedlings and viability of seeds (Heydecker et al., 1975; Bujalski and Nienow, 1991). However, the impact of aeration varies with plant species. In a case study of onion, aeration of the PEG solution enhanced the rate of germination in comparison to nonaerated treatments (Heydecker and Coolbear, 1977; Bujalski et al., 1989) whereas no impact was found in case of lettuce germination in the aerated and nonaerated K3PO4 (Cantliffe, 1981). Temperature is also one of the important factors that influence seed priming. The range of temperatures during priming varies between 15 and 30°C, but in most plant species priming at 15°C improves overall performance of seed (Bradford, 1986; Basra et al., 2005). The lower range of temperature generally slows down the rate of germination (McDonald, 2000). Similarly, the impact of light on seed priming is also one of the factors that influences the rate of dormancy. In a study Khan et al. (1977) reported that illumination at the time of priming of celery seeds may reduce the rate of dormancy, whereas Cantliffe (1981) reported germination of lettuce seed in the dark enhances performance.

5.4  Role of endophytes in seed priming Nature harbors a large diversity of microbial communities, among them endophytes have received increasing attention worldwide because of their promising hidden potential against various biotic and abiotic stress factors, and also their potential applications in growth promotion of plants via modulating growth hormones, nutrient availability, siderophore production, etc. Devoid of their endophytic microbes, seeds of plants do not have the capacity to withstand the stresses caused by various abiotic and biotic factors. The uses of endophytic microorganisms in priming could serve as a viable option to circumvent the limitations associated with seed. Priming has been supposed to induce cellular metabolic processes, hence exposure to any detrimental environmental factors would allow them to respond rapidly and nullify the stresses in an effective manner as compared to nonprimed seeds. Further, seed priming with beneficial endophytic bacterial and fungal species may enhance the crop productivity significantly as described in Table 5.1. Application of root endophytic fungi Piriformospora indica belonging to the class basidiomycetes in order to enhance the disease resistance, tolerance to salinity stress, and increase in grain productivity of barley (Hordeum vulgare L) is documented (Waller et al., 2005). Endophyte-mediated induction of disease resistance was observed to be systemic in nature. The improved defense responses were demonstrated to result from the enhanced antioxidative behavior conferred by ascorbate-glutathione cycle, leading to increase in grain productivity. The fresh shoot weight of 4-week-old endophyte infested barley was recorded to be 1.65-fold higher compared to the control

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Table 5.1  Impact of endophytic microbes on the seed priming. Endophyte

Plant

Condition

Response

Acremonium loliae

Lolium perenne and Festuca arundinacea

Greenhouse Seeds from infected Clay (1987) plants of both species exhibited a higher rate of germination Lab Increased rate of ger- Waqas et al. mination, germina(2012) tion index, shoot and root length, and vigor index

Chrysosporium Glycine max L. pseudomerdarium, Aspergillus fumigatus and Paecilomyces sp. Increased plant seed Neotyphodium Festuca arundi- Field weight, number of coenophinacea Schreb., seeds per plant and alum and Festuca pranumber of panicles Neotyphodium tensis Huds. uncinatum Neotyphodium Festuca sinensis Greenhouse Improve seed germination and plant growth Increase germination Serratia plymu- Cucurbita pepo Field rates and suppress thica S13 L. symptoms of desiccation Field Improve the producPseudomonas Triticum tivity and grain sp. MN12 Aestivum biofortification Lab Enhance growth of Pseudomonas Solanum potato shoots and sp. IMBG294; tuberosum L. plant became reMethylobacsistance toward the terium sp. soft rot disease IMBG290 Photoperiod Enhance growth of Achromobacter Helianthus anchamber seedlings under xylosoxidans; nuus L. water stress, Bacillus produce SA, and pumilus inhibit growth of pathogenic fungi Reduction of pathoFusarium oxys- Solanum lycop- In vitro gen growth porum Fo47 ersicum L. In vitro Increase rice germiAchromobacter Oryza sativa nation and seedling xylosoxidans, vigor index AUM54 Field Promoted growth of Phomopsis Oryza sativa rice liquidambari

References

Majidi and Mirlohi (2016)

Peng et al. (2013) Fürnkranz et al. (2012)

Rehman et al. (2018) Pavlo et al. (2011)

Forchetti et al. (2010)

Aimé et al. (2013) Joe et al. (2012)

Chen et al. (2013) (Continued)

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Table 5.1  Impact of endophytic microbes on the seed priming. (Cont.) Endophyte Beauveria bassiana and Purpureocillium lilacinum Neotyphodium coenophialum Peronospora indica

Plant Gossypium hirsutum

Condition Response Greenhouse Plant growth enhanced

References Lopez and Sword (2015)

Festuca arundinacea Nicotiana attenuate

Lab

Van Hecke et al. (2005) Barazani et al. (2005)

Gaeumannomyces cylindrosporus

Zea mays L.

Enhance plant nutrient supply Pot Enhanced seed germination, plant growth and increased stalk elongation Greenhouse Increased heavy metal tolerance; improved height, root length, and fresh weight of treated seedlings

Yihui et al. (2017)

group. The grain yield increase for two different barley cultivars, that is, “Annabell” and “Ingrid” was found to be 11% and 5.5%, respectively and was attributed mainly to the rise in number of ears per plant. Thus, the increase in shoot fresh weight was directly correlated with the increase in grain yield. Interestingly, the endophyte was also capable of enhancing grain yield in soil systems receiving high nitrogen input. The easy in vitro cultivation of the Piriformospora indica without the requirement of host cells suggests the effective utilization of fungus for improving the resistance against plant diseases and enhanced grain yield. Induction of seedling growth in the wheat by inoculation with plant growth-promoting endophytic bacterium Bacillus subtilis strain 11BM is presented (Egorshina et al., 2012). The inoculation with endophyte was found effective in increasing root and shoot weight as compared to control sets. Wheat seeds treated with endophyte spores culminated into the transient rise of hormonal status of indole acetic acid (IAA) and indole butyric acid (IBA) in the seedlings of root as well as shoot. The considerable alteration in wheat plant hormones was considered as a prime mechanism responsible for induced seedling growth. There are reports of seed priming with endophytic fungus Epichloë (= Neotyphodium) on the growth of Festuca sinensis under greenhouse environment indicating beneficial impacts (Peng et al., 2013). The interactive investigations were conducted in combination with hydro-priming. Significant improvements were recorded for seedling germination, seed vigor index, above ground biomass, number of leaves, radicle, and coleoptile length as well as dry weight of seedlings. The species endophyte Epichloë have been reported to enhance the seed germination of Festuca arundinacea (Pinkerton et al., 1990), Lolium perenne (Clay, 1987),

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and Achnatherum inebrians (Li, 2007) under water-stressed condition. The probable mechanisms of stress tolerance were attributed to the reduced seed germination time and increase in germination rate conferred by the endophyte. In addition, the endophyte may also modulate antioxidant defense system based on enzymatic activities of superoxide dismutase and peroxidase causing increased tolerance to ROS produced under stress condition (Zhang and Nan, 2007, 2010). Moreover, high-seed germination rates could provide competitive benefits to surviving species as compared to those not treated with endophytes (Gundel et al., 2006). Experimental investigations have shown that the tall fescue (Festuca arundinacea) endophyte Epichloë coenophiala significantly altered the chemistry of the root (Malinowski et al., 1998) without direct involvement leading to increased root biomass and could be envisaged as a mechanism for providing tolerance to host system under water stress environment. The fungal endophyte (Epichloë coenophiala) association with host (tall fescue) has been demonstrated facilitate the rapid uptake of mineral nutrients and transfer to shoot system leading to improved growth (Malinowski et al., 2000). This growth enhancement is likely the result of Epichloë-induced stimulation of increased root exudation, with resultant increased nutrient mining by microbes in the rhizophere, followed by increased rhizophagy cycle activity and oxidative nutrient extraction from microbes in root cells (White et al., 2018). It is common in grasses of many species infected by Epichloë endophytes that the fungus colonizes only aerial parts of plants, but stimulates symbiosis with soil microbes in roots, which results in increases in plant size. Vázquez-de-Aldana et al. (2013) has assessed the potential of endophyte Epichloë festucae on seed germination of two different lines of Festuca rubra in arsenic contaminated soil. The study revealed the positive influence of endophyte on radicle length of germinated seeds as compared to those free from endophytes, suggesting that the symbiotic association with the host system could be exploited for crop improvement even under metal contaminated sites. In another experimental investigation, regarding the effect of an endophyte in genus Epichloë on water stressed ecotypes of Festuca sinensis under controlled green house conditions, the study revealed the beneficial effects of endophytic association (Wang et al., 2017). The positive impact of endophyte association under water stressed environments was represented in terms of increased seedling biomass, plant growth, and stem diameter. Endophytic bacterial species equipped with plant growth promoting traits may induce tolerance to salt stress by modulating the morphological, physiological, and biochemical characteristics of plants (Mahmood et al., 2016), suggesting their utilization in crop enhancement under stress conditions. Priming of seeds from two barley genotypes (Haider-93 and Frontier-87) with endophytic bacterial strain Enterobacter spp. FD17 was performed to elucidate the role of bio-priming in alleviation of salt stress (Tabassum et al., 2018). Seed priming was helpful in improving grain yield, photosynthetic pigments, soluble protein accumulation, membrane stability, and osmolyte concentration. The increased osmolyte concentration under salinity stress may be the outcome of endophyte-induced enhanced expression of genes governing the synthesis of osmolytes (Miotto-Vilanova et al., 2016). Moreover, the bacterially synthesized osmolytes may act in synergistic fashion with plant-produced osmolytes to enhance

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the tolerance against the salinity stress (Dimkpa et al., 2009). The order of different priming technique was observed as osmo-priming > bio-priming > hydro-priming. In conclusion, endophytic bacterial association was positively associated with seedlings, biochemical, and physiological attributes under salinity stress. Effect of inoculation of two different endophytic bacterial strains namely, Burkholderia phytofirmans (PsJN) and Enterobacter sp. (FD17) in combination with biochar has been performed to alleviate the negative consequences of salinity stress in maize (Akhtar et al., 2015). The bacterial endophyte inoculated maize seeds when applied in combination with biochar were much effective in minimizing the detrimental effects of salinity by reducing the absorption of sodium ions in xylem or by maintaining the nutrient balance of plant system. The better activity (in terms of reducing sodium ion uptake) in biochar added soil was recorded for Enterobacter sp. (FD17) as compared to Burkholderia phytofirmans (PsJN). The inoculation with endophytes resulted into increase in stomatal conductance (gs), as well as photosynthetic rate (An) for both saline and nonsaline soil as compared to uninoculated soil system. The endophyte mediated enhancement in the selected photosynthetic parameter, that is, gs as well as An however, was much prominent for saline soil amended with biochar. Further, the bacterial strain PsJN was recorded as more potent endophyte in enhancing the photosynthetic parameters gs and An for maize leaves as compared to strain FD17 under saline soil conditions implemented with biochar. In contrast, there were no considerable differences between the two bacterial strains inoculated in nonsaline soils receiving the same quantity of biochar. Such improvements in plant physiological processes under saline environment could be employed for enhancing the crop productivity in a sustainable manner. Bu et al. (2019) explained the effects of endophyte Epichloë sinica on Roegneria kamoji seedlings under artificial drought stress rendered through PEG. The presence of endophyte was observed to enhance the germination potential as well as the rate of seedling emergence for seeds treated with increased concentrations of PEG. On the other hand, the seeds primed with endophyte had significantly lowered content of reactive oxygen species (ROS) in seedling leaves even in the presence of high content of PEG-6000. Moreover, considerable changes in root and shoot morphology was also noticed for seedlings harboring endophytes as compared to those not having endophytic association. The promising outcome with endophytic microbes could serve as a strong base for improving crop productivity under water stress conditions. The endophytic fungus Piriformospora indica-induced alternation in plant metabolites under drought stress is recently elucidated (Ghaffari et al., 2019). The barley seedlings primed with endophyte cell suspension under mild drought stress has been presented to have changes in abundance of total 145 plant proteins in contrast to 104 in untreated seedlings. On the other hand, under severe drought stress environment, plant proteins showing considerable changes in their abundance were recorded as 144 and 462, respectively for endophyte treated and untreated ones. The plant proteins showing changes were related to primary plant metabolic activities and documented to be involved in alleviating the negative effects imposed by oxidative stress under drought. Root colonization by endophytic fungi was associated with enhanced biological functions of photosynthetic machinery and electron transport pathway, in addition to induced build up of proteins with protective role in different biological processes

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including energy production, primary metabolic pathways, and autophagy. Resource redistribution in host cells along with maintenance of water channels (aquaporins) in endophyte inoculated plants as the effective mechanisms to cope up with the detrimental consequences of drought stress could be considered for managing agricultural productivity under changing environmental conditions. Inoculation effect of endophytic fungi together with the biocontrol agent Trichoderm viride in terms of plant growth and content of steroidal lactones (Withanolide A) in Withania somnifera (Indian Ginseng) has been emphasized (Kushwaha et al., 2019). The combined treatment with biocontrol agent and endophytic fungi Aspergillus terreus strain 2aWF (2aWF), Penicillium oxalicum strain 5aWF (5aWF), and Sarocladium kiliense strain 10aWF (10aWF) in W. somnifera resulted into an increase in shoot weight (65%–150%), root weight (35%–74.5%), and plant height (15%–35%) after 150 days of inoculation as compared to untreated plants. In combined treatment, the Withanolide A content was reported to be increased by 109%–242%. Interestingly, the Withanolide A content in root was observed to be raised by 19%–73%, in contrast to the no increase in those inoculated with T. viride alone. The combined treatment was effective in enhancing the chlorophyll a content up to 115%–164% as compared to control ones. Further, the coinoculation was also reported to influence the expression of genes participating in Withanolide biosynthesis. The up-regulation of resistance gene (NPR1) by 3−7-folds in combined treatment could be exploited for medicinal plants protection against the fungi causing root knot disease. The role of endophytic fungi Serendipita indica and Atractiella rhizophila (SMLTX-18) in seedling growth and nutrient acquisition under pot condition has been demonstrated for Quercus virginiana (Jin et al., 2019). After 60 days of treatment, the endophyte exhibited growth promotional effects on selected plants. The inoculated seedlings accumulated higher content of total nitrogen in above ground biomass, although the differences as compared to uninoculated were insignificant. The content of total potassium in treated seedlings was considerably improved under the influence of endophyte inoculation as compared to control ones. The positive influence of seedling treatment with endophytic microbes substantiates the significance of microbiomeassisted ecofriendly techniques for improving growth and development of seedlings.

5.5  Future perspectives The application of beneficial endophytes to enhance plant performance under natural environmental conditions is of immense importance in the area of agricultural sciences. Since, impacts imparted by endophytic microbes used for seed priming is greatly influenced by host plant as well as prevailing environmental conditions and critical investigations under field conditions should be performed to harness the potential of seed primed with endophytes in crop productivity enhancement. The interactive effects of other priming techniques with endophytic microbe-based bio-priming could provide better outcomes for the management of crop productivity. More studies under field conditions for widely distributed endophytic arbuscular mycorrhizal and other endophytic fungi could provide novel and beneficial approachs to combat the problem

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of reduced crop productivity imposed by abiotic and biotic stresses. The search for newer endophytic bacterial and fungal species would provide many opportunities for different plant species. Moreover, the application of nitrogen-fixing endophytic bacteria for seed priming have potential to enhance the seed characteristics in terms of seed vigor, germination rate, and overall crop productivity under changing environmental conditions. The identification and transfer of endophytic fungal and bacterial species genes are responsible for crop improvement, such as disease resistance and stress tolerance genes would be helpful in simultaneous improvement of seed quality and enhancement in agricultural productivity. In this context, the findings that the resistance genes harbored by uncultivable endophytes give a potential avenue for application in plant disease management. Further, the detailed investigations of improved plant disease resistance under the influence of microbial endophytes would help to unveil the precise molecular mechanisms of host protection.

5.6 Conclusion Application of endophytes for seed priming has several promising potentials in the field of seed technology and agricultural productivity. Available evidence has shown the positive influence of priming on seed quality, seedling growth, and crop productivity even under stressed conditions. Priming with endophytes has also shown increases in disease resistance, and tolerance to numbers of biotic and abiotic stresses. Few studies regarding the combined application of endophyte-based priming with other priming methods like osmo-priming and hydro-priming may have more conspicuous impacts on plant performance under natural environmental conditions as compared to those manifested by bio-priming alone. Extensive research on bio-priming using endophytes could give new insights and a better understating of endophyte and host relationships.

References Afzal, I., Basra, S.M., Farooq, M., Nawaz, A., 2006. Alleviation of salinity stress in spring wheat by hormonal priming with ABA, salicylic acid and ascorbic acid. Int. J. Agric. Biol. 8, 23–28. Aimé, S., Alabouvette, C., Steinberg, C., Olivain, C., 2013. The endophytic strain Fusarium oxysporum Fo47: a good candidate for priming the defense responses in tomato roots. Mol. Plant Microbe. Interact. 26, 918–926. Akhtar, S.S., Andersen, M.N., Naveed, M., Zahir, Z.A., Liu, F., 2015. Interactive effect of biochar and plant growth-promoting bacterial endophytes on ameliorating salinity stress in maize. Funct. Plant Biol. 42, 770–781. Anaytullah, B.B., 2007. Nitrate-hardened seeds increase germination, amylase activity and proline content in wheat seedlings at low temperature. Physiol. Mol. Biol. Plants 13, 199–207. Balestrini, R., Chitarra, W., Antoniou, C., Ruocco, M., Fotopoulos, V., 2018. Improvement of plant performance under water deficit with the employment of biological and chemical priming agents. J. Agric. Sci. 156, 680–688.

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Barazani, O., Benderoth, M., Groten, K., Kuhlemeier, C., Baldwin, I.T., 2005. Piriformospora indica and Sebacina vermifera increase growth performance at the expense of herbivore resistance in Nicotiana attenuata. Oecologia 146, 234–243. Basra, S.M.A., Afzal, I., Anwar, S., Shafique, M., Haq, A., Majeed, K., 2005. Effect of different seed invigoration techniques on wheat (Triticum aestivum L) seeds sown under saline and non-saline conditions. J. Seed Technol. 28, 36–45. Bhatt, R.M., Selvakumar, G., Upreti, K.K., Boregowda, P.C., 2015. Effect of biopriming with Enterobacter strains on seed germination and seedling growth of tomato (Solanum lycopersicum L) under osmotic stress. Proc. Nat. Acad. Sci. India Section B: Biol. Sci. 85, 63–69. Bradford, K.J., 1986. Manipulation of Seed Water Relations via Osmotic Priming to Improve Germination Under Stress Conditions. HortScience, USA. Bray, C.M., 1995. Biochemical processes during the osmopriming of seeds. Seed Development and Germination. Marcel Dekker, New York, pp. 767–789. Bruce, T.J., Matthes, M.C., Napier, J.A., Pickett, J.A., 2007. Stressful “memories” of plants: evidence and possible mechanisms. Plant Sci. 173, 603–608. Bu, Y., Guo, P., Ji, Y., Zhang, S., Yu, H., Wang, Z., 2019. Effects of Epichloë sinica on Roegneria kamoji seedling physiology under PEG-6000 simulated drought stress. Symbiosis 77, 123–132. Bujalski, W., Nienow, A.W., 1991. Large-scale osmotic priming of onion seeds: a comparison of different strategies for oxygenation. Sci. Horticul. 46, 13–24. Bujalski, W., Nienow, A.W., Gray, D., 1989. Establishing the large scale osmotic priming of onion seeds by using enriched air. Ann. Appl. Biol. 115, 171–176. Callan, N.W., Mathre, D., Miller, J.B., 1990. Bio-priming seed treatment for biological control of Pythium ultimum preemergence damping-off in sh-2 sweet corn. Plant Dis. 74, 368–372. Cantliffe, D.J., 1981. Priming of lettuce seed for early and uniform emergence under conditions of environmental stress. In: Symposium on Timing of Field Production of Vegetables. 122, pp. 29–38. Chandra Nayaka, S., Niranjana, S.R., Uday Shankar, A.C., Niranjan Raj, S., Reddy, M.S., Prakash, H.S., Mortensen, C.N., 2010. Seed biopriming with novel strain of Trichoderma harzianum for the control of toxigenic Fusarium verticillioides and fumonisins in maize. Arch. Phytopathol. Plant Prot. 43, 264–282. Chen, Y., Ren, C.G., Yang, B., Peng, Y., Dai, C.C., 2013. Priming effects of the endophytic fungus Phomopsis liquidambari on soil mineral N transformations. Microb. Ecol. 65, 161–170. Clay, K., 1987. Effects of fungal endophytes on the seed and seedling biology of Lolium perenne and Festuca arundinacea. Oecologia 73, 358–362. Dimkpa, C., Weinand, T., Asch, F., 2009. Plant–rhizobacteria interactions alleviate abiotic stress conditions. Plant Cell Environ. 32, 1682–1694. Egorshina, A.A., Khairullin, R.M., Sakhabutdinova, A.R., Luk’yantsev, M.A., 2012. Involvement of phytohormones in the development of interaction between wheat seedlings and endophytic Bacillus subtilis strain 11BM. Russ. J. Plant Physiol. 59, 134–140. Ellis, R.H., Butcher, P.D., 1988. The effects of priming and ‘natural’ differences in quality amongst onion seed lots on the response of the rate of germination to temperature and the identification of the characteristics under genotypic control. J. Exp. Bot. 39, 935–950. Ermis¸, S., Kara, F., Özden, E., Demir, I., 2016. Solid matrix priming of cabbage seed lots: repair of ageing and increasing seed quality. Tarım Bilimleri Dergisi 22, 588–595. Farooq, M., Basra, S.M.A., Wahid, A., Ahmad, N., Saleem, B.A., 2009. Improving the drought tolerance in rice (Oryza sativa L) by exogenous application of salicylic acid. J. Agron. Crop Sci. 195, 237–246.

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Éder de Vilhena Araújoa, João Guilherme de Moraes Pontesa, Stephanie Nemesio da Silvaa, Luciana da Silva Amaralb, Taicia Pacheco Filla a Institute of Chemistry, Universidade Estadual de Campinas, Campinas, Brazil; b Department of Chemistry, Universidade Federal de São Carlos, São Carlos, Brazil Chapter outline head 6.1 Introduction  125 6.2 Mechanisms used by endophytic fungal to colonize plant tissues  127 6.2.1 Fungal colonization aspects  127 6.2.2 Fungal colonization characterization  131

6.3 Mechanisms used by endophytic fungi to promote growth of plant  133 6.3.1 Direct growth promotion mechanism  134 6.3.2 Indirect growth promotion mechanisms  137

6.4 Increase of resistance of plant to biotic and abiotic stresses  138 6.4.1 Biotic stress tolerance  138 6.4.2 Abiotic stress tolerance  143

References  147

6.1 Introduction Every single plant on earth is colonized by complex microbial communities (Brader et al., 2017). The microorganisms that colonize the host plant may play different roles concerning plant development, growth, and health. Whereas some microorganisms assume pathogenic characteristics leading to an aggressive behavior and consequently, plant diseases, others can promote beneficial associations with the host enabling growth and tolerance to biotic and abiotic stresses via a multitude of mechanisms (Brader et al., 2017). This impressive microbial community includes endophytes (fungal and bacterial) that are defined as microbes that have the ability to colonize different internal regions of host plant, without causing any apparent harm symptoms to the host (Cabral et al., 1993; Petrini, 1991). The endophytic microorganisms represent, both as individuals and collectively, a continuum of mostly variable associations: mutualism, commensalism, latent pathogenicity, and exploitation (Cabral et al., 1993). However, they differ somewhat in their modes of colonization. Bacteria primarily colonize intercellularly though they have also been found intracellularly, and they are frequently Microbial Endophytes. http://dx.doi.org/10.1016/B978-0-12-819654-0.00006-5 Copyright © 2020 Elsevier Inc. All rights reserved.

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found in the vascular tissues of host plants (Hurek et al., 1994). The colonization by fungi may be inter- or intracellular throughout the root, leaves, stems, bark, petioles, and reproductive structures (Torres et al., 2011). In this chapter, we will focus on the chemical interactions and colonization mechanisms of endophytic fungi only. These endophytic microorganisms are considered ubiquitous, and have been encountered in many plant species worldwide, growing under specific natural conditions of each environment (Rodrigues et al., 2009). Data from the reference analysis suggested that some environmental conditions, such as temperature, humidity, illumination, geographic location, and vegetation significantly affect the distribution pattern of endophytic fungi (Suryanarayanan et al., 2005). In addition, there is evidence that endophytic fungi may exhibit some level of host adaptation. Interestingly, a few fungal species have been described to be isolated from the same host over time and geographic space (Rajamanikyam et al., 2017). Approximately, it has been estimated that more than one million different endophytic fungal strains inhabit about 300,000 various plant species (Huang et al., 2007). Although they were described in the past decades, in recent years considerable attention has been given to these class of fungi, with the conduction of a large number of studies involving a variety of approaches to obtain new bioactive molecules for several applications, in pharmaceuticals, chemical industries, and agriculture (Azevedo et al., 2000). The endophyte definition also includes latent pathogens that may live symptomless in their host tissues for a period of time before the outbreak of disease symptoms (Fisher et al., 1992). The presence of the phytopathogens Fusarium equiseti, F. oxysporum, and Phoma sorghina in healthy rice plants, clearly supports this hypothesis (Fisher et al., 1992). Apparently, symptomless endophytes may become symptomatic when the host is stressed; however, the chemical mechanisms and factors governing such fungal behavior are still not fully understood (Fisher et al., 1992). It is likely that in such cases the same individual undergoes one or more physiological alterations that switch it from a mutualistic or neutral to an antagonistic way of life (Fisher et al., 1992). The relationship between the fungus and the host plant should be regarded as flexible interaction, whose directionality was determined by slight differences in fungal gene expression in response to the host reaction, or conversely, by host recognition and response to the fungi (Moller et al., 2016). These interactions are a mode of communication between plants and microbes, which is initiated by secretion of different signaling molecules, and the endophyte-plant symbioses may be represented by a broad continuum of interactions, from strong antagonisms to obligate mutualisms (Sieber, 2007). Mutualisms are generally thought to have evolved from antagonistic interactions, mainly parasitic, and the same has been assumed for fungal endophytes of grasses and woody plants (Rajamanikyam et al., 2017). However, when the pathogen-host-endophytic interaction is imbalanced, it may result in disease, so the interaction between the endophyte and its host is a balanced antagonism (Schulz et al., 1999). Overall, literature postulates that pathogenicity and endophytism are a consequence of fine-tuned interactions between host defenses and the mechanisms used by the fungus to bypass such plant defenses. In addition, environmental conditions and other organisms living in the same habitat also seem important to dictate the characteristics of the fungal-host interaction (Schulz et al., 1999).

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The plant-pathogen-endophyte interactions depend on endophytic niche, and consequently the competition of ecological niche occurs for nutrition between diverse microorganisms, mainly endophytes and pathogens (Deng and Cao, 2017). Endophytic fungi are capable to colonize the host plant inter- or intracellular and are found in single cells (Gao et al., 2010; Katoch et al., 2014). The colonization of plant tissues by endophytes involves several steps as host recognition, spore germination, penetration of the epidermis, and tissue colonization (Azevedo et al., 2000; Petrini, 1991). The endophytes niche becomes established after successful colonization in host tissue, which is a reliable source of nutrition provided by the host plant and subsequently the endophyte may protect the host against other microorganisms (Dutta et al., 2014). In response to colonization, the plants produce lignin and other cell wall deposits to limit the growth of endophytes and as result, the cell wall becomes rereinforced after endophytic colonization, thus it becomes difficult for pathogens to infest (Dutta et al., 2014; Harman et al., 2004). In nutrient-limited conditions some endophytes showed predatory behavior and this microbial predation is a more general way to suppress plant pathogens (Gao et al., 2010; Ownley et al., 2010). Endophytic plant associations could also promote the growth of their host plants by fixing atmospheric nitrogen, producing phytohormones, controlling phytopathogens, or by enhancing the uptake of minerals. In this sense, there are many studies demonstrating the beneficial effects of endophytes (Deng and Cao, 2017) that will be discussed in detail in the next sections. Recent studies demonstrated that certain endophytes promote host plant growth through the synthesis of phytohormones such as indole-3-acetic acid (IAA), gibberellins (GAs), and cytokinins (CKs) (Khan et al., 2012). The ability to promote growth of the host plants by activating the expression of a certain enzymes and genes by endophytic fungi is also reported (Chen et al., 2005). Furthermore, the endophytic fungi are capable to produce bioactive compounds in order to enhance the resistance of host plant to biotic (pathogens, insects, and herbivores) and abiotic stresses such as drought, salinity, extreme temperatures, and heavy metal toxicity are severe threats to agroecosystems (Khare et al., 2018; Wang et al., 2003).

6.2  Mechanisms used by endophytic fungal to colonize plant tissues 6.2.1  Fungal colonization aspects Endophytic fungi are ubiquitous in terrestrial plants and can colonize all parts of the host. In nature, their transmission can occur in two manners: vertically and horizontally. The vertical transmission is related to the fungi movement from host to its progeny, for example, transmission through seeds, plant tissues, vegetative propagules, etc., whereas horizontal transmission occurs by the fungi transference through sexual and asexual spores or by insect inoculation (Verma et al., 2017). Most present, the endophytic transmission occurs mainly through airborne spores as inoculum source, seeds, and propagules by insects (Mohanan and Varma, 1988;

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Petrini, 1991); however, belowground the main inoculum source is the soil and other colonized roots (Wearn et al., 2012). When in contact with the plant, endophytic fungi can then colonize plant tissues inter- or intracellularly, sometimes restricted to the growing of the cell. The endophytes access new sources of nutrition through the colonization, where the plant provides the important substrates, while in exchange the endophytes may protect the plant (Gao et al., 2010). On the other hand, not all endophytic fungi have the ability to permanently colonize the plant, due to the impossibility of adapting to the nutritional mode of the plant (Garrido-Jurado et al., 2017). The colonization steps of plant tissues by the endophytes described in the literature are: host recognition, spore germination, tissue penetration, and tissue colonization/ multiplication (Dutta et al., 2014; Gao et al., 2010; Petrini, 1991). Each step is detailed as follows.

6.2.1.1  Host recognition Endophytic fungi have developed host recognition mechanisms similar to the biotrophic and necrotrophic fungi (Stone and Petrini, 1997). When the endophyte is specific to a determined host (host-specific endophytes), there is a singular recognition mechanism involved. However, if the endophytic recognition occurs for more than one host, then the recognition is specific for a determined plant response (Peters et al., 1998). Host-specific endophytes show specialized mechanisms of host recognition with coordinated communication and regulation with its hosts, and this subject is very little explored in literature, needing a greater clarification about the physiology of plant response to the endophyte (Stone and Petrini, 1997). In the endophyte-plant interactions there is a complementarity between the genetic systems of both symbionts that is a result from coevolutionary processes and biological adaptations, which comes due to a long period of seasons. The recognition is determined in the function of fungal gene expression and in relation to host response to the endophyte (Moricca and Ragazzi, 2008). Lahrmann et al. (2013) studied how the endophyte Piriformospora indica establishes the colonization process in barley and Arabidopsis through global characterization of fungal transcriptional responses in different symbiotic stages. They verified that 123 of the 216 genes that encode small secreted proteins—SSPs (effectors)— were related to barley or Arabidopsis responses. During the symbiosis, SSPs facilitate the colonization process and these can interact in a specific manner with each host. Furthermore, SSPs have important roles in the host recognition processes and signaling pathways in roots (Lahrmann et al., 2013).

6.2.1.2  Spore germination One of the strategies of endophytic colonization is the spore germination that occurs in seeds and during the plant development (Dutta et al., 2014). On the other hand, environmental factors influence the endophytic colonization pattern, as well as the fungal spore germination, growing, reproduction, and metabolism during the life cycle (Jia et al., 2016).

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There are techniques to induce the fungal spore germination (Beckman and Payne, 1983; Odell and Smith, 1982). Huang et al. (2018) used leaf disc assays with light microscopy and scanning electron microscopy and in planta assays to analyze the spore germination in leaves from Populus trichocarpa of three different endophyte classes (Dothideomycetes, Eurotiomycetes, and Sordariomycetes), which have the potential to reduce the severity of plant disease. Thus it was possible to observe the different stages of biological process (conidial germination, hyphae growing, and entry into stomata) for the three endophytes and compare the movement duration in different conditions as the humidity (Huang et al., 2018).

6.2.1.3  Tissue penetration The penetration is often performed by some pathogenic fungi through the production of enzymes that degrade the wax and the cell wall and allow the tissue penetration or can occur also through the natural opening as stomata, lenticels, or by the wounds (Kolattukudy, 1985; Saunders et al., 2010). It is assumed that endophytic fungi can adopt the same mechanical and enzymatic strategies than pathogenic fungi to penetrate the plant tissue (Fisher et al., 1992; Varma et al., 2004). Fungi developed special mechanisms to penetrate the host tissue. In general, when in contact with the plant surface the endophytic fungi produce enzymes such as the β-1,3-glucanases, chitinases, and cellulases to hydrolyze the plant cell wall as well as to suppress pathogen activities and degrade oomycetes (Chandra, 2012; Dutta et al., 2014). It is used in literature the denomination “plant cell wall-degrading enzymes” (PCWDEs or CWDEs) to specify the enzymes produced by the fungi that are used for nutrition acquisition and cell wall decomposition; however, this term is most often associated with pathogenic fungi. It is possible to access genomic data (substrate, category, number of genes, gene family, etc.) from CWDEs in database—Fungal PCWDE Database—http://pcwde.riceblast.snu.ac.kr/ (Choi et al., 2013). Katoch et al. (2014) studied the enzymatic activity of extracellular enzymes isolated (amylase, cellulase, protease, and lipase) of 26 endophytic fungi from Bacopa monnieri that showed amyolytic activity (100% of endophytes), lipolytic activity (98%), cellulolytic activity (28%), and proteolytic activity (31%). Besides, they verified the antimicrobial activity against Bacillus subtilis (79% of endophytes), Pseudomonas aeroginosa (21%), Salmonella typhimurium (58%), Escherichia coli (50%), Klebsiella pneumonia (100%), Staphylococcus aureus (90%), and Candida albicans (79%). Katoch et al. (2014) suggest that the production of PCWDE as the cellulase by endophytic fungi may already be performed before the association of the endophyte with the host and the fungi may have biologically developed this production capacity in accordance with plant’s requirements (Katoch et al., 2014). Knapp et al. (2018) sequenced the genome of two dark septate endophytes (DSE)—Cadophora sp. and Periconia macrospinosa. They verified that both DSE possess enzymes belonging to carbohydrate-active enzymes (CAZymes) class, which also include PCWDE. Furthermore, they verified through gene sequence and principal component analysis that Cadophora sp. showed the highest number of CAZymes and

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of PCWDE genes among the 32 species analyzed, an even larger number of PCWDE than the pathogenic fungi Colletotrichum spp. These results indicate that DSE possess plant cell wall degrading capacity and this corroborate with the fact that they are able to break down plant polysaccharides during colonization (Knapp et al., 2018). Fungi that infect insects (entomopathogenic fungi) can also penetrate the plant tissues via natural openings, or by enzymatic activity and mechanical pressure through the cell walls, resulting in an endophytic association with the host. Such fungi are described as “endophytic entomopathogenic fungi”, and they can colonize above- and belowground tissues (root) of their hosts. (Vidal and Jaber, 2015). Some examples of entomopathogenic fungi are: Lecanicillium lecanii found in scale insects of family Coccidae, Paecilomyces farinosus in Eligma narcissus (Lepidoptera: Noctuidae), and Cladosporium spp. in several species of insects. These fungi were found as endophytes in Araceae, bark of Cassia caroliniana and Ericaceae, respectively (AbdelBaky and Abdel-Salam, 2003; Gómez-Vidal et al., 2006; Mohanan and Varma, 1988; Vega et al., 2008). After the penetration of endophytic fungi in the tissues, the plants can develop a natural protection barrier that becomes more difficult the invasion of other pathogens (González-Coloma et al., 2016). Despite the beneficial effects of entomopathogenic fungi as biocontrol agent in the agriculture (Ownley et al., 2010), the consequences of their successful application are not yet fully understood and the issue has been discussed in literature in recent years (Bamisile et al., 2018; Jaber and Ownley, 2018; Vidal and Jaber, 2015).

6.2.1.4  Tissue colonization/multiplication The success of colonization is dependent on several factors such as cell wall thickness, plant phylogeny, defense compounds produced by hosts, and ability of colonization by fungi species (Saunders et al., 2010). Due to the diversity of endophytes that exist in nature, when it is necessary to isolate a specific fungus species for in vitro study, there should be considered some factors that can influence the fungal isolation such as species diversity, geographical locations, leaf age, tissue type, and seasonal effects (Arnold and Herre, 2003; Yadav et al., 2016). Plant tissues rich in antifungal defense compounds often are used for studies of endophytic colonization, due to the tolerance to these compounds showed by endophytes (Fisher et al., 1992; Saunders and Kohn, 2009; Suryanarayanan, 2013). Glenn et al. (2001) analyzed the tolerance of 29 Fusarium species in maize, and they verified that just 11 species showed some level of tolerance. They observed that the resistance of fungi in maize is related with the ability of the endophyte to detoxify antimicrobials compounds produced by the maize such as 6-methoxy-2-benzoxazolinone and 2-benzoxazolinone, metabolizing them to N-(2-hydroxy-4-methoxyphenyl) malonamic acid, and N-(2-hydroxyphenyl) malonamic acid, respectively, after 24 h. These results explain the success of colonization of an endophyte in a determined type of plant tissue. Guo et al. (2008a) studied age (1–3 years), type of tissue and the four seasonal effects in the colonization of endophytic fungi (10,659 isolates) from 16,200 tissue

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segments of pine (Pinus tabulaeformis) that were separated in four collections of 4,050 segments. They verified that the colonization rate in the needles increased with age that can be explained by the longer time of accumulation of endophytes and this is in accordance with the hypothesis of predominance of horizontal transmission of endophytes in trees, while in most cases there was no significant difference in the colonization rate in the xylem, because this is more protected by the bark and the composition of the xylem is less used by endophytes (Guo et al., 2008a). The isolation rate and endophytic colonization in needles were altered in different seasons in the following descending order: spring > winter > autumn > summer. This difference in the observed rate is due to the growth of needles in the summer and fungal propagules have had not enough time to invade the plant tissues (Guo et al., 2008a). In relation to chemical strategies that endophytic fungi use to establish the colonization in plants, there are studies concerning endophytes and their ability to perform biotransformation reactions in antifungal compounds produced by the host plant. The biotransformation allows the plant defense compounds to be less toxic to the endophyte and its development. Fill et al. (2018) analyzed through nuclear magnetic resonance spectroscopy and mass spectrometry the biotransformation process of antifungal substrates produced by the host Melia azedarach (Meliaceae) against the endophyte Penicillium brasilianum. In this case, the endophyte metabolizes 4-bromobenzoic acid and 4-chlorobenzoic acid to new brominated and chlorinated compounds that are less toxic to the endophyte assayed by antifungal tests. Thus the new compounds are produced through enzymatic reactions that promote conjugation of the halobenzoic acids with amino acids valine, proline, and isoleucine.

6.2.2  Fungal colonization characterization It is not yet fully clear in the literature the mechanisms of action that endophytic fungi use to colonize its hosts (Kharwar and Redman, 2009; Pawlowski and Hartman, 2016; Verma et al., 2017). However, there are studies concerning characterization methods used to detect the colonization or be able to analyze the effects caused by it (BernardiWenzel et al., 2010; Chow and Ting, 2019; Reyna et al., 2012). Generally, four approaches are used for endophytic fungi detection: microscopy; isolation; biochemical; and DNA-based methods (Bayman, 2007; Raja et al., 2016). Through spectroscopic techniques, it is possible to obtain the detailed composition of plant tissues (Chow and Ting, 2019). Some techniques used for this purpose are described as follow: wheat germ agglutinin Alexa Fluor (WGA-AF), confocal laser scanning microscopy (CLSM), and Fourier transform infrared spectroscopy (FTIR).

6.2.2.1  Wheat germ agglutinin Alexa Fluor The detection of some endophytic fungal structures (non-Clavicipitaceous endophytes) in roots tissues can be performed through WGA-AF 488 that interacts with fungal chitin and allows the detection of hyphae by the appearance of green coloration (Andrade-Linares and Franken, 2013). This technique accompanied by propidium iodide as costaining provides data about fungal hyphae (green) and the plant cell wall (red)

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in contrasting shades. Thus it is possible to monitor the behavior of hyphae during differential stages of colonization (Redkar et al., 2018). Hussin et al. (2017) used the WGA-AF 488 to study the colonization of the endophyte Piriformospora indica in roots from quinoa (Chenopodium quinoa) and also, they evaluated plant water relations and leaves gas exchange. After 48 h of Piriformospora indica inoculation in the root, it was possible to analyze the beginning of colonization with the spore germination and extracellular establishment. Furthermore, it was also acquired micrographs that show the inter- and intracellular endophytic colonization after 4 weeks under two conditions: sufficient water supply and water stress using WGA-AF 488 and photographed with fluorescence microscopy (Hussin et al., 2017). These results show the potential of the technique for monitoring the development of fungal colonization that may help to understand more about this biological process that until then is not fully detailed in the literature.

6.2.2.2  Confocal laser scanning microscopy Another technique used for monitoring plant colonization by endophytic microbes is the CLSM (Cardinale, 2014; Maciá-Vicente et al., 2009). The use of confocal microscopy allows access data of the cellular elements and macromolecules localization (proteins, DNA, RNA, etc.); also it is possible to obtain three-dimensional images; and to obtain information about the relation between structures and functions of cells, among many others (Sarala and Senthilkumar, 2012). Zhang et al. (2017) studied the Epichloë colonization in grass embryos through confocal microscopy images with the aim of better understanding the mechanisms involved in seed transmission because the Epichloë spp. infection can improve the plant resistance, which increases its commercial interest. In this study, it was analyzed five stages of fungal hyphae development as well as the distribution in different section of green seeds, and they were able to confirm previous studies (Philipson and Christey, 1986) that hyphae infection occurs in very early embryo. Furthermore, they show the CLMS as an alternative to current techniques used industrially (seed-squash method and the tissue print-immunoblot) because of that it allows reliability and speed in the analysis (Zhang et al., 2017).

6.2.2.3  Fourier transform infrared spectroscopy Recently, Chow and Ting (2019) studied by FTIR the compositional change in plant cell walls from oil palm in three sample types: control, plant tissue with putative endophyte inoculation (Diaporthe phaseolorum, Trichoderma asperellum and Penicillium citrinum), and plant tissue with pathogen inoculation (Ganoderma boninense, causing the basal stem rot). The infrared spectrum provides a “fingerprint” of different profiles of samples for comparison with databases (Chow and Ting, 2019). In this study, it was analyzed CWDEs activities such as cellulase, pectinase, xylanase, and laccase that are biochemical markers to initiate the colonization of both the pathogen and the endophytes and also it was evaluated the relative lignin/carbohydrate rate in cell wall as well as the mean percentage weight loss in ramets (Chow and Ting, 2019).

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All fungi analyzed were able to produce CWDEs especially Penicillium citrinum that produced the highest activity of cellulase, pectinase and xylanase, and Ganoderma boninense, responsible for highest activity of laccase after 28 days, that is related with a greater impact on the turgor of the plant and consequently their pathogenicity (Chow and Ting, 2019). These results show that FTIR together with experimental data can provide information about the effect of fungal colonization on the host. Spectroscopic techniques allied to databases can still be much explored for the study of fungal colonization; however, it is necessary to spend time for a good interpretation of the experimental results in relation to the biological issue.

6.3  Mechanisms used by endophytic fungi to promote growth of plant Endophytic fungi can provide some effects on their host plants after colonization, including measurable benefits. While the plant protects and ensures access to nutrients for the fungus, the latter promote plant growth and can act as biocontrol agents by producing bioactive substances (Carrol, 1988; Dai et al., 2008). The positive effects of this relationship were reported by Berta et al. (1989) who demonstrated increased branching of roots systems of Allium porrum colonized by the arbuscular mycorrhizal fungi (AMF) Glomus sp. Furthermore, Mucciarelli et al. (2003) inoculated Mentha piperita with a leaf fungal endophyte both in vitro and pot cultures, and they observed great effects on the host growth promoted by the endophyte, mainly, taller plants, bearing more expanded leaves with better dry biomass percentage over the total. Notably, other studies demonstrated that various endophytic fungi produce phytohormones such as GAs, auxins, and strigolactiones (SLs), which may exert pronounced effects as plant growth regulators (Akiyama et al., 2005; Spaepen et al., 2007; You et al., 2013). In terms of interaction, endophytic fungi initiate colonization in host tissue by secretion of different signaling compounds. The perception of the microorganism at the cell surface of the plant initiates defense responses to halt or slow microbial growth. These responses are associated with increased demands for energy, and they can include physical changes (e.g., callose deposition to reinforce the cell wall), biochemical responses including the production of several secondary metabolites or signaling molecules that perturb infection such as ethylene and salicylic acid (SA) (Bolton, 2009; Chisholm et al., 2006). Both pathogen-host and the endophyte-host interactions involve constant mutual antagonisms at least based on the secondary metabolites the partners produce (antibacterial, antifungal, and herbicidal) but when this interaction is imbalanced, it may result in disease that occurs between pathogen and its host. Whereas the endophyte-host interactions are a balanced antagonism to endophytic infection (Schulz et al., 1999) maintained by avoiding activation of the host defenses and activating

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resistance against toxic metabolites of the host (De Silva et al., 2019), the plant-endophyte interaction remains asymptomatic. After colonization of plant tissues have been established, the plant-endophyte symbiotic association induces significant changes in plant physiology that facilitates nutrients exchange and enzymatic activity (Khan et al., 2015) via different mechanisms displayed by endophytes and potentially contributes to the protection of plant host against pathogen and insects showing that these microorganisms may directly or indirectly act in plant growth-promoting (Hassan, 2017; Vujanovic and Vujanovic, 2007).

6.3.1  Direct growth promotion mechanism Several mechanisms of direct plant growth enhancement by endophytic fungi have been documented. They include mobilization of mineral nutrients such as nitrogen and phosphorus, increasing hormones production (e.g., auxins, and CKs), activation of the expression of specific enzymes and genes responsible for the synthesis or inhibition of biomolecules associated to growth, production of volatile compounds, and synthetic intermediates that are involved in plant development (Jia et al., 2016). Depending on the endophytic, more than one mechanism can be used to promote host plant growth. Moreover, many of these fungi possess several properties that enable them to facilitate plant development allowing endophytic to use different pathways oftentimes during the life cycle of the plant (Varma et al., 2004). However, only the most common mechanisms will be detailed here.

6.3.1.1  Signaling compounds Endophytic fungi can promote the growth of the host plant through the secretion or regulation of a vast array of phytohormones, plant signaling compounds that function as messengers to control plant growth, development, and to modulate plant responses to environmental changes (Dan et al., 2012; Lubna et al., 2019; Santner et al., 2009). Particularly, auxins can affect almost every aspect of plant development. By far the major auxin active in plants is IAA. In fact, IAA is known to play a role in cell division and differentiation in plants, promoting cell elongation and increasing the root length and root hair abundance. Accordingly, more root surface area become available. This modification increases plant ability to absorb more water and nutrients, which further promote plant growth (Gravel et al., 2007). Recently, Mehmood et al. (2018) demonstrated that Fusarium oxysporum could promote growth and proliferation of host plant maize by secreting IAA. Other important plant hormones are bioactive GAs, diterpenoid acids synthesized by the terpenoid pathway (Yamaguchi, 2008). They are involved in various processes of plant development, including seed germination, stem and bud’s elongation, leaf expansion, flowering, and fruit development (Davies, 2010). Recently, it has been reported that Aspergillus fumigatus and Fusarium proliferatum can promote the growth of mutant rice by produce phytohormones, particularly GAs. These plants exhibited higher chlorophyll content, root-shoot length, and biomass production (Bilal et al., 2018).

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Briefly, GAs modulate the expression of specific genes that act on the growth of different parts of the plant (Yamaguchi, 2008). Furthermore, Itoh et al. (1999) revealed that expression of Nty, a gene encoding tobacco gibberellin 3β-hydroxilase, was restricted to specific regions, which are consistent with the sites of GA action, suggesting that, in some cases, bioactive GAs are produced at the site of their action. Like other phytohormones, CKs are potentially important for plant growth promotion. They are N6-substituted purine derivatives with Isopentenyladenine (iP), zeatin (Z), and dihydrozeatin (DZ) being the predominant CKs found in higher plants (Werner et al., 2001). The symbiotic interaction between endophytic and host plant with production of CKs modify cell division and enlargement, favor outgrowth of lateral buds ordinarily suppressed by apical buds and thus modify apical dominance and help maintain chlorophyll levels in detached plant parts, influencing a wide variety of other growth responses (Sakakibara, 2006) as evidenced by Vadassery et al. (2008) through studies of Arabidopsis growth and reproduction by the endophytic fungus Piriformospora indica. The abscisic acid (ABA) is an essential plant hormone that belongs to a class of metabolites known as isoprenoids. It is biosynthesized both by the host plant and fungi but by different pathways. The ABA biosynthesis is via the 2-C-methyl-Derythritol-4-phosphate pathway in plants, whereas in fungi this metabolite is mainly biosynthesized by the MVA pathway (Hauser et al., 2012; Nambara and MarionPoll, 2005). Usually, phytopathogens may produce ABA to suppress the plant immune responses acting as signaling molecule during inter-species communication (Lievens et al., 2017). However, this compound could play important roles in promoting plant growth mediating seed dormancy and germination, bud growth, and adaption to the environment (Miyazono et al., 2009). In supporting of this, results of Waqas et al. (2015) showed that the production of ABA by Paecilomyces formosus is involved in promotion japonica rice plant growth. Another important mechanism of endophytic-host plant growth promotion is the regulation of ethylene (ET), an olefin produced by higher plants and microorganisms. Curiously, ethylene is stimulatory while in others it is inhibitory. It may activate a wide array of genes responsible for direct and indirect defenses. Ethylene is well known for its profound effects on plant developmental steps, being involved, for example, in seed germination, tissue differentiation, root formation and elongation, lateral bud development, flower opening and senescence, fruit ripening, production of volatile organic compounds responsible for aroma formation in fruits and leaf and fruit abscission (Arshad and Frankenberger, 2002; Bakshi et al., 2015). Especially in the case reported by Yuan et al. (2016), the endophytic fungus Gilmaniella sp. induced ethylene production in Atractylodes lance improving sesquiterpenoids accumulation in the plant. In turn, these compounds act as hormones and antifeedants, which improve the plant development (Chadwick et al., 2013). Moreover, commonly ethylene is synthesized by plants upon exposure environmental stress and is thought to initiate many plant effects, including senescence, chlorosis, and organ abscission, which affects roots elongation and root hair formation, reducing root surface area available for nutrient absorption (Glick, 2005; JohnstonMonje and Raizada, 2011). In this context, Barazani et al. (2007) evidenced the

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increasing growth of Nicotiana attenuata promoted by Sebacina vermifera through inhibiting ethylene signaling. They measured the ability of the fungus to induce a systemic down-regulation of plant’s ethylene biosynthesis due to lowered transcript levels of the ethylene synthesis genes promoting the host plant growth. In addiction those already mentioned, jasmonic acid, SA, and SLs are also important hormones produced or regulated by endophytic fungi related to the growth of the host plant (Schulz, 2006). Therefore, a large number of signaling compounds could be involved in plant enhancement by endophyte-host interaction that seems to play a central role in this process (Ismail et al., 2018).

6.3.1.2  Enhance the absorption of nutrient Endophytic-host association can improve the plant’s supply of important minerals required in the plant development, increasing the absorption of nutrients predominantly via extension of the host’s root length, penetration of substrates, and excretion of enzymes by infected roots and/or hyphae and the selectivity of ion uptake. Especially, phosphorus and nitrogen tends to be the key nutrient that limits plant productivity (Lambers et al., 2008). Nitrogen is the main nutrient responsible for the growth of plants acting in production of new cells and tissues. Essentially, N is a component of amino acids, proteins, nucleic acids, and chlorophyll; moreover, nitrogen metabolism shuttles amino acids into energy-generating pathways (Bolton, 2009). Even though this mineral is so important in several biological processes in plants, it needs to be fixed from the soil. Therefore nitrogen assimilation is one of the most important biological processes second only to photosynthesis (Masclaux-Daubresse et al., 2010). Only a few groups of microorganisms are capable to convert nitrogen into biologically accessible forms, including endophytic fungi (Rosenblueth et al., 2018) that contain the highly conserved gene nifH, which encodes the iron protein subunit of nitrogenase (Lambers et al., 2008). They use fixed carbon from plants to drive the energy-intensive, oxygen-sensitive process of reduction of N2, creating ammonia in a process known as biological nitrogen fixation (Johnston-Monje and Raizada, 2011). In this way, a study about the association between Phomopsis liquidambari and rice showed that this fungus assists its host with acquire soil nitrogen, especially in nutrient-limited soil. The authors believe that the colonization by Phomopsis liquidambari affects the diversity and abundance of ammonia-oxidizers and N-fixers in the plant rhizosphere, increase available soil N and improve N-uptake by rice (Yang et al., 2015), promoting the growth and yield in such crops (Yuan et al., 2007). Phosphorus is a necessary nutrient of plants because it is an essential constituent of adenosine triphosphate, the “molecular unit of currency” of intracellular energy transfer, and a constituent of genetic material such genes and chromosomes. Phosphorus is involved in cell multiplication inducing the development of seed, increases stalk and stem strength, flowering and enhancements in crop excellence. The deficiency of this mineral directly affects growth and productivity of the plants reducing, for example, the development of roots, shoots, and leaf as also delaying maturation (Rana et al., 2019).

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Although there is a great evidence of P great importance for plants, only a small amount is available for plant absorption. The concentration of soluble P in soil ranges from 0.05 to 10 ppm, whereas more than 80% of it remains as insoluble salts. In acidic soil, P occurs mainly as FePO4 and AlPO4 and fixed form in alkaline soils is tricalcium phosphate [Ca3(PO4)2] (Kapri and Laksshmi, 2010; Varma et al., 2004). In this sense, there are many studies that report the ability of several endophytic fungi to solubilize inorganic phosphorus compounds and make them available for plant uptake (Gasoni and Gurfinkel, 1997). According to Spagnoletti et al. (2017) the endophytic fungi Cochliobolus sp., Curvularia sp., Drechslera sp., Ophiosphaerella herpotricha, Ophiosphaerella sp., and Setosphaeria rostrata promote wheat growth releasing P from soil insoluble phosphates to crops, showing potential application of this endophytic as biofertilizers in different soils. Additionally, other micro and macrominerals such as potassium and zinc, in their unavailable form, can be also solubilized by endophytic fungi boosting plant growth and development. The study of Colla et al. (2014) investigated the effects on coating seeds with three arbuscular mycorrhizal (AM) endophytic fungi: Glomus intraradices, Glomus mossae, and Trichoderma atroviride, and it was demonstrated that this AM fungi improve soil structure, nutrient uptake by plants as P, K, and Zn, maintain and restore soil health and fertility. Thus the results evidence that several endophytic fungi have the potential to provide the required nutrients to plant host in sufficient amounts to enhance their productivity (Colla et al., 2008).

6.3.2  Indirect growth promotion mechanisms Since 1980s with Webber’s (1981) studies that report an example of plant protection giving by the endophytic fungus Phomopsis oblonga against the beetle Physocnemum brevilineum through the production of toxic compounds for the insect by the fungus, many examples have been reported in the literature about endophytic ability in controlling host diseases and insects pests (Fuchs et al., 2017; Gange et al., 2012; McGee, 2002), contributing indirectly for the host plant growth and development. In general, the term indirect promotion of plant growth is related to the endophytic capacity to decrease or prevent some of the effects of phytopathogens and herbivores in a process termed as biological control, enabling the development of the host (Patle et al., 2018). It can be possible through the activation of biologically active compounds production against pathogenic microorganisms by the endophyte, which compete for nutrients supply in colonization sites or induce resistance in the plant by the activation of its own defense system (Zabalgogeazcoa, 2008). Often, these mechanisms occur at the same time (Latz et al., 2018). In the great majority, indirect promotion of plant growth involves the production of secondary metabolites by endophytic fungi with antibacterial, antifungal, and insecticidal properties that strongly inhibit the colonization or growth of plant pathogenic organisms (Gunatilaka, 2006). For example, Musetti et al. (2006) demonstrated the completely inhibition of sporulation of the pathogen Plasmopara viticola by the endophytic fungus Alternaria alternata isolated from grapevine leaves, attributing this activity to the secretion of three antifungal diketopiperazines produced

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by A. alternata. In the case of Cheplick and Clay (1988), resistance to herbivorous insect S. frugiperda in grasses is attributed to the production of many alkaloids that are synthesized during plant interaction by the endophytic fungi (Clavicipitaceae and Ascomycetes). Besides diketopiperazines and alkaloids, recent studies have reported hundreds of natural products, including terpenoids, flavonoids, and steroids produced by endophytes (Guo et al., 2008b), which are involved in diseases control and increase resistance, contributing to the plant growth.

6.4  Increase of resistance of plant to biotic and abiotic stresses 6.4.1  Biotic stress tolerance Biological control using endophytic fungi as a new efficient method is being widely used for environmental remediation and killing insects or pathogens (Guo et al., 2008b).

6.4.1.1  Defense against insects In nature, insects and plants can be associated in diverse ways and plant feeding insects depend on the nutrients provided by plants for their growth and reproduction (Beyaert et al., 2010). To protect their host plant, some endophytic fungi in association with the host plant are able to produce toxic compounds to insects such as alkaloids (Clay and Schardl, 2002). Webber (1981) reported repellent action by the interaction of endophytic fungus Phomopsis Oblonga and the elm tree, against the beetle Physocnemum brevilineum. According to Johnson et al. (1985), the endophytic fungus Acremonium coenophialum in tall fescue (Festuca arundinacea) exhibited insecticidal activity against two aphid species Rhopalosihum padi and Shizaphis graminum, and both species were unable to survive in the host plant after confined to endophyte-infected tall fescue plants (Table 6.1). Kanda et al. (1994) reported the preference of larvae of the bluegrass webworm Parapediasia teterrella for diets with endophyte-free plants of L. perenne and F. arundinacea, to a point that the larvae would starve to death if only plants infected with Acremonium were available. Toxicity tests realized by Abraham et al. (2015) into crude ethyl acetate culture filtrate extracts from mangrove fungal endophytes (Aspergillus tamarii and Aspergillus versicolor) that showed insecticidal activity against Spodoptera litura larvae. The presented studies earlier shown that the presence of endophytes, even of those belonging to groups where some members are known to control insects, do not always result in an effective pest control. However, these bioinsecticides produced by endophytic fungi represent an alternative for safety and environmental issues surrounding the use of chemical insecticides (Vega, 2008).

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Table 6.1  Host plant with enhanced defense responses to biotic and abiotic stress conferred by endophytic fungi. Host plant

Endophytic fungi Type of stress

Cordia alliodora

Leucopoprinus gongylophorus

Ulmus Ulmacea

Phomopsis Oblonga

Festuca arundinacea

Acremonium coephialum

Lollium perene and Festuca arundinacea

Acremonium coephialum

R. mucronata, Sonneratia alba and Avicennia marina. Phoenix dactylifera

Aspergillus tamarii and Aspergillus versicolor

Cirsium arvense

Cucumis sativus

Achnatherum robustum

Beauveria bassiana, Lecanicillium dimorphum, L.cf. psalliotae Chaetomium cochliodes, Cladosporium cladosporioides, Trichoderma viride Chaetomium Ch1001

Epichloë gansuensis

Mechanisms

Produce some chemicals antagonistic to ants’ fungal symbiont Insect: Produce repellent Physocnemum action to beetle brevilineum Insect: Exhibited Rhopalosihum insecticidal padi and activity against Shizaphis aphid species graminum Insect: Exhibited Parapediasia insecticidal teterella activity against larvae of bluegrass webworm Insect: Exhibited Spodoptera insecticidal litura activity Insect: Atta colombica

References Bittleston et al. (2011)

Webber (1981) Johnson et al. (1985)

Kanda et al. (1994)

Abraham et al. (2015)

Insect: data palm Modulate the Gómezpests expression of cell Vidal division-related et al. proteins in host (2009) Insect: foliar feeding insects

Produce some Gange chemicals toxic to et al. pathogens (2012)

Insect: root-knot Produced abscisic Yan et al. nematode acid affecting (2011) Meloidogyne motility of the incognita second stage juveniles of insects Herbivores: Produce indoleGuerre livestock and diterpene alkaloid (2015) horses lolitrem B and the ergot alkaloid ergovaline tremorgenic compounds (Continued)

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Table 6.1  Host plant with enhanced defense responses to biotic and abiotic stress conferred by endophytic fungi. (Cont.) Host plant Schenodorus arundinaceos

Endophytic fungi Type of stress Herbivores: Neotyphodium grazing coenophialum livestock

Festuca arundinacea Schreb

Epichloë sp.

Herbivores: Sheep

Atractylodes lancea

Gilmaniella sp. AL12

Pathogenic fungi

Curcuma wenyujin

Chaetomium globosum L18

Pathogenic fungi

Mechanisms Produce alkaloid ergovaline that may cause fat necrosis Produce indole diterpene alkaloids as lolitrems Produce jasmonic acid inducing defense responses Produce some chemicals toxic to pathogens Produce trichothecin toxic to pathogens Produce cell wall-degrading enzymes to kill pathogenic fungi

Pathogenic fungi Maytenus hookeri Trichothecium roseum Pathogenic fungi Phragmites Choiromyces australis aborigium, Stachybotrys elegans, Cylindrocarpon sp. Produce cadinene Cassia spectabilis Phomopsis cassia Pathogenic fungi: sesquiterpenoids Cadosporium toxic to pathogens sphaerospermum, and C. cladosporioides Pathogenic Produce some Angelica sinensis Myxormia sp. fungi: chemicals toxic to Fusarium pathogens oxysporum and F. Solani Pathogenic fungi: Improve the Hordeum vulgare Acremonium Gaeumancompetence for var. disticum blochii, A. nomyces space inhibiting furcatum, graminis var. the colonization Aspergillus tritici of pathogens fumigatus, Cylindrocarpon sp., C. destructans, Dactylariia sp., Fusarium equiseti, Phoma herbarum, P. leveillei

References Guerre (2015)

Bush et al. (1997)

Ren and Dai (2012) Wang et al. (2012) Zhang et al. (2010) Cao et al. (2009)

Silva et al. (2006)

Yang et al. (2012)

MaciáVicente et al. (2008)

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Table 6.1  Host plant with enhanced defense responses to biotic and abiotic stress conferred by endophytic fungi. (Cont.) Host plant Endophytic fungi Type of stress Pathogenic Triticum aestivum Chaetomium sp, fungi: cv. Phoma sp. Puccinia recondite Trptergyium Cryptosporiopsis Pathogenic fungi: wiifordii cf. quercina Pyricularia oryzae Salt Glycine Penicillium angustifoli minioluteum Hordeum vulgare Piriformospora indica

Salt

Zea mays Mexicana Oryza sativa

Salt

Piriformospora indica Piriformospora indica

Salt

Theobroma cacao Trichoderma hamatum DIS 219b

Drought

Festuca arundinacea

Acremonium coenophialum

Drought

Lolium perenne

Epichloë clavipitacea

Drought

Dichanthelium lanuginosum

Curvularia sp.

Temperature— Heat

Arabidopsis thaliana

Paecilomyces formosus LHL10

Temperature— cold

Mechanisms Activate defense reactions of the plant

References Dingle and McGee (2003)

Produce cryptocin Strobel and cryptocandin et al. toxic to pathogens 1999) Produce endogenous abscisic acid and high salicylic acid Produce antioxidants due to activation of glutathioneascorbate cycle Upregulation of aquaporins Increased glycerol concentration Delayed droughtinduced changes in stomatal conductance and net photosynthesis Increase level of glutamine synthetase. Produce loline alkaloid toreduce stress Produce cell wall melanin that may dissipate heat along the hyphae and/or complex with oxygen radicals Accumulation of pigments and induced cold response pathway

Khan et al. (2011) Waller et al. (2005)

Gond et al. (2015) Jogawat et al. (2016) Bae et al. (2009)

Bacon (1993) West et al., (1993) Redman et al. (2002)

Su et al. (2015)

(Continued)

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Table 6.1  Host plant with enhanced defense responses to biotic and abiotic stress conferred by endophytic fungi. (Cont.) Host plant Helianthus annuus L.

Endophytic fungi Type of stress Heavy metal— Glomus Cd intraradices

Zea mays L.

Exophiala pisciphila

Heavy metal— Cd

Zea mays L.

Exophiala pisciphila

Heavy metal— Cd, Pb, and Zn

Mechanisms Cd induced guaiacol peroxidase activity in roots in both mycorrhizal and nonmycorrhizal plants Increase the activities of antioxidant enzymes and lowmolecular weight antioxidants Restricting the translocation of heavy metals ions from roots to shoots

References Andrade et al. (2008)

Wang et al. (2016)

Li et al. (2011)

6.4.1.2  Defense against herbivores Endophytic fungi are also important microorganism mediators of plant-herbivores interactions (Feath and Hammon, 1992; Gaylord et al., 1996). The associations between host plants and endophytes can produce compounds that can negatively affect vertebrate herbivores, suggesting a defensive mutualistic relationship between plant and endophytes (Clay, 2014). Alkaloids have been described to be toxic to herbivores. The alkaloids (ergotamine, ergosine, ergocosine, ergocryptine, and ergocristine) concentrations must reach a certain threshold in the plant (Schardl et al., 2004) in order to be biologically active and assist the plant defense system. Endophytes are not uniformly present in all plant tissues and the alkaloids are translocated, and consequently the sites of accumulation may or may not be the same sites of biosynthesis (Siegel and Bush, 1992). Herbivore toxicity is caused by several endophyte derived alkaloids, and these are of great agronomic and scientific importance (Guerre, 2015). Sometimes the bioactive compounds can be beneficial to protect the host plant from predation, but alkaloids as the indole-diterpene alkaloid lolitrem B and the ergot alkaloid ergovaline are tremorgenic mycotoxins for vertebrate herbivores, causing diseases such as ryegrass staggers and fescue toxicosis, respectively, to grazing livestock (Guerre, 2015). Once it had been established that the endophyte was the cause of animal toxicity, significant research was undertaken to readily identify each alkaloid, elucidate the biosynthetic pathway, and identify the genes and gene products required for alkaloid production (Bacon, 1993; Philipson and Christey, 1986).

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6.4.1.3  Defense against pathogens Fungal pathogens are one of the major causative agents of plant diseases that can infect most plant parts, including roots, stems, leaves, flowers, and fruits (Hyde et al., 2009). In this context, the endophytic fungi can protect the plant against pathogens through different strategies, such as competition with pathogens for colonization sites and nutrients, (Arnold et al., 2003; Clarke et al., 2006; Ownley et al., 2010; Strobel et al., 2001). Hallmann and Sikora (1994) reported that Fusarium oxysporum reduced the population of plant parasitic root knot nematode in tomato roots in Kenya. They suggested that culture filtrate of endophytic F. oxysporum were toxic to these nematodes causing inactivation and death. The potential of biological control of the endophytic fungi Trichoderma hamatum, Penicillium sp., Bacillus sp., Paecilomyces lilacinus, and Fusarium sp., against pathogen Drechslera tritici-repentis (Dtr) was evaluated by Larran et al. (2016). The results suggest that all endophytic fungi shown to be promising to control tan spot disease in cereals caused by Dtr. In several cases, tolerance to biotic stresses is correlated with the bioactive compounds produced by endophytic fungi in association with the host plant against pathogens (Saikkonen et al., 1998; Tan and Zou, 2001; Zhang et al., 2006).

6.4.2  Abiotic stress tolerance In their life cycle the plants are constantly being challenged for several abiotic stresses as salinity, drought, temperature, and heavy metal toxicity stress (Ghosh et al., 2017). Associations between endophytic fungi and host plants may mediate the effects of abiotic stresses adjusting, regulating, or modifying plant physiological, biochemical, and metabolic activities (Dastogeer and Wylie, 2017). Resistance strategies to abiotic stress can be a powerful tool to ensure productivity in the worldwide agriculture regarding the impacts of the global climate change and may reduce chemical input into the environment (Nagargade et al., 2017).

6.4.2.1  Salinity stress tolerance The soil salinization is a major process of land degradation that decreases soil fertility and is a significant component of desertification processes in the world’s dryland and can be result from the proximity of semi-arid sites to the sea, or due to saline ground water rising into the root zone and concentrating there when evaporation becomes excessive (Rabie, 2005; Thomas and Middleton, 1993). According to Marschner (1995) and Adiku et al. (2001), plants can be stressed by salinity in three ways: (1) low water potential of the root environment leads to water deficits in crop plants, (2) toxic effects of ions, mainly sodium (Na) and chlorine (Cl), and (3) nutrient imbalance caused by decreased nutrient uptake and/or transport to the shoot. Salt stress induces ionic and osmotic imbalance inside plant cells and the plant tolerance mechanism to salinity stress involves the alleviation of antioxidant enzymes like catalases or superoxide dismutases. Reactive oxygen species scavengers such as ascorbate, glutathione, and tocopherol act as antioxidants (Rouhier et al., 2008).

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In the reported study by Khan et al. (2011), it was possible to notice a significant growth and resistance to salt stress promoted by endophytic fungus Penicillium minioluteum in soybean plant. The potential of Piriformospora indica to induce tolerance to salt stress was evaluated by Waller et al. (2005) in to monocotyledonous plant barley and the host plants also shows increase of tolerance to salt stress in comparison with the control plants.

6.4.2.2  Drought stress tolerance Drought is the most common environmental (abiotic) stress, which limits almost 25% of the production of the world (Fathi and Tari, 2016). High evapotranspiration rate, water resource restriction, and other factors have led to an increasing attention to the studies of drought effects worldwide (Azad and Kaminskyj, 2016). Although plant species vary in their sensitivity and response to the decrease in water potential caused by drought, low temperature, or high salinity, it may be assumed that all plants have encoded capability for stress perception, signaling, and response (Bohnert et al., 1995). Fungal endophytes also have been shown to provide fitness benefit to plant when exposed to water-limiting conditions (Hubbard et al., 2014). To evaluate the drought tolerance phenomenon, solutes that are accumulated in tissues of endophyte-infected plant are compared to non-infected plants, or also to reduced leaf conductance and a decrease of the transpiration stream, or due to thicker cuticle formation (Malinowski and Belesky, 2000). There are some possible drought scenarios to the plant adaptations such as better water uptake from the soil by the root system; reduced transpiration losses; and water storage in the tissues of the host (Malinowski and Belesky, 2000). Bacon (1993) reported the benefits of association of tall fescue, Festuca arundinacea and endophytic fungus Acremonium coenophialum for tolerance to water stress. The results indicate that tall fescue infected by A. coenophialum was more tolerant to drought stress than uninfected plants. The benefits obtained through of association endophytic fungi and host plant was reported also by West et al. (1993) found that the growth advantage of infected tall fescue over uninfected fescue in experimental plots was significantly higher in water-stressed versus well-watered plots. Greater drought tolerance may result from altered stomatal behavior and osmotic adjustment, leading to better turgor maintenance (Bacon, 1993; West et al., 1993).

6.4.2.3  Temperatures stress tolerance Environmental stresses come in many forms and interactions between organisms are major forces that impact the development of organisms and have helped to shape species evolution (McLellan et al., 2007). High and low temperatures (heat and cold stresses) inhibit plant growth by destroying the photosynthetic apparatus and cell membranes (Chuansheng and Flin, 2010). Endophytes have been reported to protect host plants from the extreme temperature damages (Singh et al., 2011). Signaling by phytohormones especially ABA is one of the important defense strategies by higher plants under abiotic stresses as shown in Fig. 6.1. ABA significantly increases during abiotic stresses, like high temperature in rice seedlings (Raghavendra et al., 2010).

Figure 6.1  (A) Ergots alkaloids produced by fungi of the genus Epichloë in plants that in biotic stress situation enhances host resistance to insects and nematodes (Guerre, 2015); (B) The phytohormone abscisic acid as a strategy to signal the abiotic stress—drought and salt stress (Raghavendra et al., 2010); (C) Some phytohormones and/or signaling compounds produced by endophytes; (D) The strategy that Fusarium species uses to colonize maize through biotransformation of antimicrobials compounds 6-methoxy-2-benzoxazolinone and 2-benzoxazolinone to N-(2-hydroxy-4-methoxyphenyl) malonamic acid and N-(2hydroxyphenyl) malonamic acid, respectively (Glenn et al., 2001); (E) Enzymatic reactions that promote conjugation of the halobenzoic acids with amino acids performed by Penicillium brasilianum in Meliaceae (Fill et al., 2018).

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Heat stress cause upregulation of genes responsible for ABA biosynthesis, whereas it downregulates the genes involved in ABA catabolism (Toh et al., 2008). Dichanthelium lanuginosum plants inoculated with the fungal endophyte Curvularia showed tolerance to soil temperature at 50°C for 3 days and intermittent as high as 65°C for 10 days, whereas noninfected plants died at 50°C (Redman et al., 2002). In general, endophytic fungi may produce melanin, which might disperse heat along the hyphae or form a complex with oxygen radicals (Redman et al., 2002).

6.4.2.4  Heavy metal stress tolerance Since the past century, many regions worldwide suffer from heavy metals contamination due to anthropogenic activities and in the last years have received considerable attention of contemporary science (Su et al., 2014). Metals including lead, chromium, arsenic, zinc, cadmium, copper, and mercury can cause significant damage to the environment and human health as a result of their mobilities and solubilities (Dermont et al., 2007). According to Mulligan et al. (2001), remediating contaminated soils by heavy metals can be obtained by application of the conventional techniques as containment physical, encapsulation, vitrification, Ex situ treatment (physical separation, soil washing, and pyrometallurgical), In situ (reactive barriers, soil flushing, and electrokinetic). Generally, the application of these techniques is too expensive and often harmful to soil microbial activity (Ma et al., 1993; McGrath et al., 1995; Mulligan et al., 2001). The biological processes for decontamination is a challenging task because heavy metals cannot be degraded and hence persist in the soil (Lebeau et al., 2008). To overcome this scenario, phytoremediation in recent years has been a proposed method to decontaminate soil, water, and air pollutants without significantly affecting microbial activity and soil fertility (Raskyn et al., 1997; Salt et al., 1998). The phytoremediation is a cost-effective “green” technology based on the use of metal-accumulating plants to remove toxic metals, including radionuclides, from soil and water (Salt et al., 1998). Plants have evolved tolerance mechanisms to excessive metal concentrations such as metal efflux, reduced metal uptake by root immobilization or mycorrhizal action, intracellular chelation by metal complexes with phytochelatins, and compartmentalization in the vacuoles (Hall, 2002). AMF, such as Glomus mosseae, are important soil microorganisms forming symbiotic associations with most of the vascular plant species (Benedetto et al., 2005). Improved nutritional status and reduced or altered metal uptake are among the most related advantages of mycorrhizal association to host plants under metal stress (Andrade et al., 2008; Toler et al., 2005). As consequence of physiological changes, the plants have an improved performance under metal stress conditions (Parádi et al., 2003). Nevertheless, the overall mechanisms by which AM fungi alleviate metal toxicity in hosts are still not completely elucidated, considering controversial results depending on the specific plant/fungal/metal species interactions (Andrade et al., 2010). In a study reported by Andrade et al. (2008) the association between sunflower plant with G. intraradices showed to be less sensitive to Cd stress than nonmycorrhizal plants. Mycorrhizal sunflowers showed enhanced Cd accumulation and some

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tolerance to excessive Cd concentrations in plant tissues. In another example, Wang et al. (2016) evaluated the root-associated DSE, Exophiala pisciphila of Zea mays under soil Cd stress (0, 10, 50, and 100 mg kg−1) observing enhanced antioxidant enzyme activity. Three key genes involved in uptake, detoxification and transport: namely, ZIP was downregulated; PCS upregulated; and MTP was upregulated upon inoculation with DSE and exposed to subsequent high Cd concentrations. Alteration in the levels of ACC by Pseudomonas and Gigaspora can alter the tolerance of heavy metals directly through the manipulation of plant ethylene levels (Gamalero et al., 2010). The Table 6.1 summarizes several cases of the interactions between host plant and endophytic fungi to mitigate abiotic and biotic stresses.

Acknowledgments This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001 and Fundação de Amparo à Pesquisa no Estado de São Paulo (Grant Number FAPESP 2017/24462-4).

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Vinaya Chandran, Hitha Shaji, Linu Mathew School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, India Chapter outline head 7.1 Introduction  161 7.2 How do endophytes help in stress tolerance?  162 7.3 Plant growth promotion by the endophytes  163 7.3.1 Phytohormone production by the endophytes  163 7.3.2 Phosphate solubilization by the endophytes  163 7.3.3 Production of siderophores by the endophytes  164 7.3.4 Nitrogen fixation by the endophytes  164 7.3.5 Synthesis of biologically active compounds  164

7.4 Abiotic stress alleviation by the endophytes  164 7.4.1 Drought stress  164 7.4.2 Salinity stress  168 7.4.3 Temperature stress  170 7.4.4 Heavy metal stress  172 7.4.5 Role of endophytes in nutrient starvation  176

7.5 Biotic stress  177 7.5.1 Role of endophytes in biotic tolerance  177

7.6 Commercial applications of stress tolerant endophytes  180 7.7 Conclusion  181 References  182

7.1 Introduction With an ever-increasing global population, a quantum jump in crop production is the most desired challenge of the coming years. According to the studies of UN in 2017, the world population is expected to reach 8.6 billion by 2030 and 98 billion by 2050 (Union, 2017). As the population increases, the demand for healthy and nutritious food also increases. However, agricultural intensification by the use of new farm equipment, high-yielding crop varieties, intensive tillage, irrigation, chemical fertilizers, pesticides, and other manufactured inputs; has been going on, in parallel with the needs of growing population. But these approaches often detrimentally affect agricultural ecosystems resulting in considerable ruin of soil ecology and fertility, high irrigation needs, etc. The global ecosystem is also being threatened by other manmade activities such as accelerated conversion of forest lands into cultivated land, land cover change, disturbances of natural resources, and pollution. These trends pressurize the Microbial Endophytes. http://dx.doi.org/10.1016/B978-0-12-819654-0.00007-7 Copyright © 2020 Elsevier Inc. All rights reserved.

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environment and results in climatic variation. These climatic change and shrinkage of arable lands are the major threats to the agricultural productivity. Unfavorable environmental changes, such as drought, temperature extremes, or soil salinity, etc., leads to the loss of crop yield. Mooney et al. (2009) reported that climate change has emerged as an important threat to natural ecosystems and main source for creating stress. It acts as stressor both directly and through the interaction with other stressors (Staudt et al., 2013). Temperature, drought, salinity, and heavy metal pollution are major stress factors associated to climate change. The environmental stress is simply classified as abiotic and biotic stress based on their nature. Sometimes abiotic stress influence biotic stress and reduce crop productivity (Kumar and Verma, 2018). However, both stresses are either natural or human induced. The foremost effect of these stresses are the loss of soil microbial diversity, soil fertility, and competition for nutrient resources (Chodak et al., 2015) resulting in the reduction agriculture productivity. Plant growth and development is accomplished through cell division, cell enlargement, and differentiation. It involves genetic, physiological, ecological, and morphological events and their complex interactions. The quality of plant growth depends on various abiotic and biotic limiting factors. Crop plants needs to cope up with these factors for their growth, development, and productivity (Meena et al., 2017) and are able to cope with these adverse environmental conditions with their intrinsic metabolic capabilities (Simontacchi et al., 2015). Many times plants overcome these burdens of stresses with the support of the microbiome they inhabit (Turner et al., 2013; Ngumbi and Kloepper, 2016). Plant– microbe interactions are an integral part of the ecosystem that modulates local and systemic mechanisms in plants. They provide fundamental support to the plants in acquiring nutrients, disease resistance, and tolerating abiotic stresses (Turner et al., 2013). The beneficial microbes either form symbiotic associations at the surface or endophytic interactions inside the plant parts.

7.2  How do endophytes help in stress tolerance? Endophytes are nonpathogenic (fungi or bacteria) plant associated microbes that live inside host microenvironment (Schulz and Boyle, 2006). They colonize intracellular or intercellular spaces of plant compartments without causing significant morphological changes (Baroni et al., 2015). They occur in the surrounding environment of the host plant and are infected via the same pattern as the pathogenic microorganisms to enter into the plants (Wilson, 1995). Usually, endophytic microbes enter plants through natural openings such as hydathodes, stomata and lenticels, root hairs, and wounds caused by mechanical damage. Once they enter into the host, they successfully colonize the internal plant tissue and transmitted to the next generation via seed or vegetative plant parts. The interaction between endophyte and host plant is characterized as a symbiotic relationship. Currently, entophytes demand an alluring attention on agricultural productivity, because they promote host plant growth (Compant et al., 2005), improve nutrients uptake (Johnston-Monje and Raizada, 2011), control/ resist plant diseases (Hallmann and Sikora, 2011), and enhance tolerance to abiotic stresses (Forchetti et al., 2010). Moreover, they are an important factor influencing the response of plants to climate change.

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7.3  Plant growth promotion by the endophytes 7.3.1  Phytohormone production by the endophytes A set of phytohormones and related signaling networks are involved in mediating plant growth promotional activities and stress tolerance. Endophytes are capable of synthesizing plant hormones like indole-3-acetic acid (IAA), gibberellins (GAs), and cytokinins in plants (Glick, 2012). The gaseous plant hormone ethylene mediates a wide range of plant responses to their biotic and abiotic stresses (Abeles et al., 2012). It is a multifunctional hormone regulating growth and development and defense response (Jing et al., 2005; Masood et al., 2012). However, it also induces or inhibits plant senescence (Masood et al., 2012; Nazar et al., 2014). Moreover, excess ethylene concentrations leads to chlorosis and subsequent plant death. The peak of ethylene biosynthesis is related to environmental stress (Hyodo et al., 1991) and its regulating process depends on its concentration and complex interaction with other hormones (Iqbal et al., 2017). A number of endophytic bacteria are involved in lowering of plant ethylene concentration through the action of enzyme ACC deaminase (Glick, 2014), the enzyme that cleaves the compound ACC, which is the immediate precursor of ethylene in all higher plants. The endophytes also promote the synthesis of IAA (Lee et al., 2004). Under high salinity, IAA induces the ACS gene family enhancing the production of ethylene by effectively utilizing 1-aminocyclopropane-1-carboxylate. The endophytic fungi Phoma glomer, ata LWL2, and Penicillium sp. LWL3 significantly secrete phytohormones, namely, IAA and GAs (Waqas et al., 2012). Endophytic bacteria Pseudomonas putida W, 619 enhance plant growth through the synthesis of the plant auxin IAA (Taghavi et al., 2005). Endophytic bacterium, Sphingomonas sp. LK11, was isolated from the leaves of Tephrosia apollinea produces GAs and IAA (Khan et al., 2014). Drought triggers the production of the abscisic acid (ABA), which in turn causes stomatal closure and induces expression of stressrelated genes (Shinozaki and Yamaguchi-Shinozaki, 2007). ABA involved in pivotal physiological responses required for salt stress adaptation such as ion and water homeostasis, other phytohormones antioxidants and ROS scavengers’ production (Achard et al., 2006). Razem et al. (2006) documented that ABA and GA play antagonistic roles in controlling many developmental processes. The endophyte Burkholderia kururiensis promoted rice plant growth by production of the plant auxin, IAA (Mattos et al., 2008).

7.3.2  Phosphate solubilization by the endophytes Endophytes promote phosphate-solubilizing activity (Wakelin et al., 2004). Phosphorous exist abundantly in agricultural soils, but the majority of it remains unavailable as an insoluble form (Miller et al., 2010). Several endophytic bacteria and fungi release organic acids into the soil, which solubilize the phosphate complexes and convert it into ortho-phosphate for plant up-take and utilization (Yadav and Yadav, 2017).

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7.3.3  Production of siderophores by the endophytes Siderophores are the small molecular iron chelating compounds that can avail iron to plants and deprive pathogen of iron (Costa and Loper, 1994). Also, siderophores help iron deficient plant in fixing nitrogen since diazotrophs require Fe++ and Mo as cofactors for nitrogenase synthesis and functioning.

7.3.4  Nitrogen fixation by the endophytes Nitrogen is the most requiring nutritional element, but plants cannot reduce atmospheric nitrogen to ammonia. Several prokaryotic endophytes have the ability to fix atmospheric nitrogen (Yadav and Yadav, 2017). In addition, endophytes directly transport biologically fixed nitrogen to plants (James et al., 1994).

7.3.5  Synthesis of biologically active compounds Endophytic bacteria supply essential vitamins to plants (Rodelas et al., 1993). The production of auxin-like compounds (ACCS) increases seed production and germination (Clay, 1987). Also endophytes are good sources of phytochemicals (Nisa et al., 2015) and biologically active secondary metabolites (Brader et al., 2014), which impede plant pathogens and involved in various plant metabolism. Other effects of endophyte infection on the host plant include osmotic adjustment, stomatal regulation, modification of root morphology, and enhanced uptake of minerals (Belesky and Malinowski, 2000) (Table 7.1).

7.4  Abiotic stress alleviation by the endophytes The major abiotic stresses are drought (Chaves and Oliveira, 2004), low/high temperature, salinity, heavy metal stress, acidic conditions, excess light (Nakashima and Yamaguchi-Shinozaki, 2006; Bailey-Serres and Voesenek, 2008), anaerobiosis (Agarwal and Grover, 2006), and nutrient starvation (Hirel et al., 2007). As per the FAO reports about 20% of irrigated land in the developing world has been damaged by waterlogging or salinity. Approximately, 250 million people have been directly affected by desertification, and nearly 1 billion are at risk. Drought has affected 64% of the global land area, flood another 13% of the land area, salinity 6%, mineral deficiency 9%, acidic soils 15%, and cold 57% (Mittler, 2006; Cramer et al., 2011). Major abiotic stresses, its detrimental mechanism in plants, and role of various endophytes in abiotic stress tolerance are described further.

7.4.1  Drought stress Drought is a common but critical environmental stress to crop productivity. The severity of drought is unpredictable because it depends on many factors such as availability and distribution of rainfall, evaporative demands, and moisture storing capacity of

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Table 7.1  Endophyte-associated plant growth promoting activities. Host plant Endophyte species

Association benefit

Rice

Burkholderia sp., Herbaspirillum seropedicae

Beet

Bacillus pumilus, Chryseobacterium indologene, Acinetobacter johnsonii Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, Azospirillum amazonense,and Burkholderia sp. Enterobacter sp. herbaspirilum sp. Asospirillum brasilence, Burkholderia cepacia, Bacillus sbtilis, B. lentimorbus, Streptomyces sp. Azospirillum lipoferum Bacillus amyloliquefaciens, B. japonicum, Azospirillum brasilense

Increase in nitrogen Baldani et al. fixation (2000) Synthesis of indole3-acetic acid (IAA) Increased concentration of Shi et al. (2010) carbohydrates

Sugar cane

Cabbage Maize

Soybean

References

Nitrogen fixation, produce Oliveira et al. siderophores (2009) IAA synthesis, Phosphate solubilization

Growth promotion Nitrogen fixation; IAA synthesis

Production of siderosophores; antifungal activity; nitrogen fixation

Zakria et al. (2008) Ferreira et al. (2013)

Subramanian et al. (2015a).

soils (Wery et al., 1994). The absorption of water is necessary for all stages of plant growth. Drought stress is considered as a growth-limiting factor and its effects ranges from morphological to molecular level. Kaya et al. (2006) reported that draught severely reduce germination and seedling growth in sunflower. In Hordeum vulgare postanthesis drought stress reduced grain yield by reducing the number of tillers, spikes, and grains per plant and individual grain weight (Samarah, 2005). Reduced cell growth is one of the most drought-sensitive physiological processes due to the loss of turgor pressure. This is because of the reduction in water uptake permits decreased tissue water content that results loss of turgor pressure (Taiz and Zeiger, 2006). Also under draught stress, cell elongation of higher plants can be inhibited by interruption of water flow from the xylem to the surrounding elongating cells (Nonami, 1998). Scarcity of water decreases leaf water potential and stomatal opening (Osakabe et al., 2014), reduces leaf size, suppresses root growth, reduces seed number, size, and viability, delays flowering and fruiting and limits plant growth and productivity (Xu et al., 2016). Wahid et al. (2005) reported that the major effect of drought is reduction in photosynthesis, which arises by a decrease in leaf expansion,

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impaired photosynthetic machinery, premature leaf senescence, and associated reduction in food production. When the availability of soil water is limited the plants are pressurized to close their stomata (Cornic and Massacci, 1996). This limits the CO2 uptake into the leaves leading to the accumulation of reactive oxygen species, which induce oxidative stress. Accumulation of free radical induces changes in membrane function, protein conformation, lipid peroxidation, and finally cell death. Another important effect of draught is on the acquisition of nutrients by the root and their transport. Lowered absorption of the inorganic nutrients can result from interference in nutrient uptake and reduced transpiration flow (Peuke et al., 2002).

7.4.1.1  Mechanism of drought stress tolerance by plants The drought tolerance is a complex mechanism; involving in a number of physiological and biochemical processes at cell, tissue, organ, and whole-plant levels. The major mechanisms include reduction in water loss by increasing stomatal resistance, increased water uptake by developing large and deep root systems, accumulation of osmolytes, osmoprotectant synthesis, and smaller and succulent leaves to reduce the transpirational loss (Farooq et al., 2009). Phytohormones such as auxin, cytokinin, gibberilin, ethylene, and ABA play important role in plant to abiotic stress under stressful conditions (Skirycz et al., 2010). During water stress plants undergo a series of physiological and molecular changes, such as increased ethylene production, change in chlorophyll content, and damage of photosynthesis apparatus leading to the inhibition of photosynthesis (Lata and Prasad, 2011). Auxins have an indirect but key role in the draught stress tolerance by enhancing lateral root growth and formation. IAA is the most active auxin that regulates the vascular tissue differentiation, adventitious and lateral root differentiation, cell division, and shoot growth during drought stress (Goswami et al., 2016). ABA is the next important phytohormone, which ameliorates drought stress via regulating transcription of drought-related genes and root hydraulic conductivity (Jiang et al., 2013). It involved in the water regulation by controlling stomatal closure and signal transduction pathways (Yamaguchi-Shinozaki and Shinozaki, 1994). Another important drought stress tolerance of plants is associated with the accumulation of mineral nutrients (Samarah et al., 2004) and enhanced synthesis of osmoprotectants (Hare et al., 1998), which are the part of normal plant metabolism. Accumulation of these minerals helps in the water retention and the maintenance of the structural integrity of the cell membranes.

7.4.1.2  Drought stress alleviation by the endophytes It is well-documented that fungal endophytes confer some level of drought tolerance to plants (Clay and Schardl, 2002). It is involved in the adjustments in host osmolyte concentrations or stomatal activity (Malinowski and Belesky, 2000). Colletotrichum magna and C. protuberate 4666D confer significant drought tolerance to wheat, tomato, and watermelon plants (Redman et al., 2001). Association of Pseudomonas indica to the roots of Chinese cabbage promotes the growth of root and shoot and also lateral root formation. When allowed to colonize plants, experimentally exposed to polyethylene glycol (a chemical to mimic drought stress) the activities of antioxidants such as

Endophytic microbial influence on plant stress responses

167

peroxidase (POX), catalase (CAT), and superoxide dismutases (SOD) were increased in leaves within 24 h to prevent or repair the damage caused by reactive oxygen species (ROS). Thus, the endophytic fungus prevents the drought-induced decline in the photosynthetic efficiency and degradation of chlorophyll and thylakoid proteins. During drought stress, colonized plants express drought-related genes DREB2A, CBL1, ANACO72, and RD29 on their leaves. In addition, CAS protein a Ca2+ sensing regulator in the thylakoid membrane is elevated. Hence, in drought-stressed condition antioxidant enzyme activities, drought-related gene expression, and CAS are the three crucial target of P. indica in Chinese cabbage leaves (Sun et al., 2010). Waqas et al. (2012) examined the effect of endophytic fungi Phoma glomerata LWL2 and Penicillium sp. LWL3 to salinity and drought stress. The symbiotic-association alleviated stress by compromising the activities of reduced glutathione, CAT, peroxidase, and polyphenol oxidase. Under stress conditions, the endophyte-infection significantly regulated stress through down-regulated ABA, altered JA, and elevated salicylic acid (SA) in host cucumber plant. Under drought stress condion maize (Zea mays L) was inoculated with Azospirillum spp. a plant growth promoting bacteria (PGPB). Azospirillum lipoferum enhanced growth of maize by the production of phytohormones such as ABA, IAA, and GAs. The results suggest that both ABA and GAs produced by endophytic Azospirillum contribute to drought stress alleviation of host plants (Cohen et al., 2009). The adverse environmental conditions influence the symbiotic interaction between plant, Lolium perenne to its fungal endophyte Neotyphodium spp. When endophyte infections are present plants showed higher sensitivity to drought. The endophyte infection significantly promoted the development of reproductive tillers and seed production. These features provide a beneficial influence on plants to persist in sites where water is a growth-limiting factor. The endophyte-induced adaptation, likewise, increases the root dry weight and root/shoot ratio and helps the plant to withstand stress condition (Hesse et al., 2005). The association of fungal endophyte Acremonium coenophialum with tall fescue Festuca arundinacea Schreb resulted in a wide range of ecological efficiency to the plants. The colonization of tall fescue by the fungus follows the natural sequence of fescue seed germination, seedling and tiller growth. The tagged plants were more tolerant of environmental abiotic stresses than uninfected grasses. Tolerance to water stress is mediated by modulating the osmoregulatory system that produces an increased cellular turgor pressure in tissue. This mechanism help to lower the stomatal conductance thereby protecting the plant from water stress. In addition, the association of fungus also leads to the accumulation of insect deterrent, toxins, and their synergist that protect the plant from deleterious effect of insect pathogen. The efficiency of plants to trive on low nitrogen soil is also enhanced because fungal infected tall fescue contains polyps. The increased level of glutamine synthetase enables its competitive ability to survive on low soil nitrogen (Bacon, 1993). To improve the economic viability and environmental sustainability of Poplars, they are inoculated with a consortium of endophytes. The inoculated plants demonstrated an improved leaf physiology and reduced damage by ROS. Plant endophyte symbiosis mediates the production of phytohormones [SA, ABA, IAA, jasmonic acid (JA), gibberellins-3-acid, epibrassinolides], which aid in growth promotion and stress

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Table 7.2  Endophyte-mediated drought stress tolerance in plants. Endophyte

Plant hosts

Sinorhizobiummeli- Medicago sativa lotii Trichoderma Theobroma cacao hamatum DIS 219b Piriformospora indica

Brassica campestris ssp. chinensis

Pantoea agglomerans

Saccharum offincinarum cv. SP70-1143 Oryza sativa

Trichoderma harzianum

Physiological changes in plants

References

FeSOD and CU/ZnSOD are up-regulated Delayed droughtinduced changes in stomatalconductance and net photosynthesis Increased level of peroxidases, catalases, and superoxide dismutases IAA and proline production

Naya et al. (2007) Bae et al. (2009)

Upregulation of aquaporin, dehydrinandmalonialdehyde genes

Pandey et al. (2016)

Sun et al. (2010) Vargas et al. (2014)

tolerance in host plant. The associated microbial strains contain microbial genes involved in the biosynthesis of trehalose, (R, R)-butane-2, 3-diol, and acetoin. These gene activities encode beneficial effects to host plant against drought stress (Khan et al., 2016) (Table 7.2.)

7.4.2  Salinity stress Salinity is one of the most common abiotic stress factors limiting crop productivity with adverse effect from seed germination and throughout plant life cycle (Munns and Tester, 2008). More than 45 million hectares of irrigated land of the world have been affected by salinity, and 1.5 million hectares are taken out of production each year as a result of high salinity levels in the soil (Munns and Tester, 2008). Salt accumulate in the soil through irrigation, fertilizers, weathering of minerals, or sometimes migrate upward from ground water (Shrivastava and Kumar, 2015). Most of the crop plants are sensitive to high salt concentration in the soil. At low salt concentrations, crop yields are mildly affected or not affected at all (Maggio et al., 2001). As the concentrations increase, the crop growth and yields move toward zero. The salt is found in soil as charged ions such as Ca2+ (calcium), Na+ (sodium), K+ (potassium), Cl− (chloride), and NO3− (nitrate) mostly under different conditions (Shrivastava and Kumar, 2015). Many salts are plant nutrients; high salt levels in the soil can upset the nutrient balance in the plant or interfere with the uptake of some nutrients (Blaylock, 1994). For example, plants show a significant reduction in Phosphorus (P) uptake in saline soil because phosphate ions precipitate with Ca ions (Bano and Fatima, 2009).

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High salinity level affects plants in various ways: causes water stress in soil leading to osmotic stress, ion toxicity (N, Ca, K, P, Fe, Zn), nutritional disorders, alteration of metabolic processes, membrane disorganization, reduction of cell division and expansion, genotoxicity (Hasegawa et al., 2000; Munns, 2002), changes in enzyme activity (Seckin et al., 2009), DNA, RNA, and protein synthesis, and mitosis inhibition (Tabur and Demir, 2010; Javid et al., 2011). Together, these effects reduce plant growth, development, and survival. For example, soil salinity results in poor microbial activity due to toxic effect of ions leading to nutrient deficiency in soil. Moreover, it causes low water potential in soil resulting in osmotic stress so that it is difficult for plant to uptake water and nutrients from soil. In addition it causes adverse effect on nitrogen fixation trough the reduced production of nitrogenase enzyme which is responsible for the nitrogen fixation. Salinity also affects photosynthesis; mainly through a reduction in chlorophyll content and stomatal conductance, and through a decrease in photosystem II efficiency (Netondo et al., 2004).

7.4.2.1  Plant mechanisms to tide over soil salinity Plants usually try to avoid high saline environments by keeping sensitive plant tissues away from high saline zone or by exuding ions from roots or compartmentalize ions away from the cytoplasm of cells (Silva et al., 2010). This mechanism strictly limits the influx of salts into the shoots, but suffer from very much reduced growth. Next is osmotic adjustment, achieved mainly by accumulation of high levels of sodium and chloride in the shoots, accompanied by synthesis of substantial amounts of the compatible solute glycine betaine. Tolerance to salinity stress warrants the maintenance or quick adjustment of both osmotic and ionic homeostasis within the cells. Salinity, restrict the growth, and productivity of plants worldwide. Salt stress in plants results into a wide variety of physiological and biochemical changes. Plants subjected to high salt concentration such as rice (Oryza sativa L.) variety GJ-17 when inoculated with endophytic baterium Pseudomonas pseudoalcaligenes resulted in accumulation of proline and glycine betaine-like quaternary compound for alleviating salinity stress (Jha et al., 2011). These osmoprotectants produced against salinity play an important role in protecting subcellular structures and mediating osmotic adjustment in stressed plant (Ashraf and Foolad, 2007).

7.4.2.2  Salinity stress alleviation by the endophytes The association of two endophytic fungi Glomerata LWL2 and Penicillium sp. LWL3 reprogrammed the growth of host cucumber plant during saline stress condition. The microbial-assisted phytohormones GAs and IAA significantly promoted the plant biomass and related growth attributes. These host tagged endophyte has the potential to assimilate essential nutrients like potassium, calcium, and magnesium as compared to untagged plants. This study examined that during stress condition Endophyticassociation promoted the host-benefit ratio in cucumber plants by reducing the sodium toxicity in soil. This symbiotic-association mitigated stress by down-regulating the elevated concentration of ABA, JA, and SA in host plants (Waqas et al., 2012) (Table 7.3).

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Microbial Endophytes

Table 7.3  Endophyte-mediated salt stress tolerance in plants. Physiological changes in plants

Endophyte

Plant hosts

Pseudomonas pseudoalcaligenes along with Bacillus pumilus P. pseudoalcaligenes

O. sativa

Accumulation of glycine betaine Jha et al. (2011) like compounds, decline in proline content by 5%

Arabidopsis thaliana Tomato

Regulation of Na+ and K+ homeostasis ACC deaminase activity has the potentialto facilitate plant growth Upregulation of aquaporins

Pseudomonas fluorescens YsS6 and P. migulae 8R6 Piriformospora indica Zea mays mexicana P. indica O. sativa

References

Abdelaziz et al. (2017) Ali et al. (2017)

Lata et al. (2018)

Increased glycerol concentration Jogawat et al. (2016)

Maize plants inoculated with arbuscular mycorrhizal fungi (AMF) enhanced plants antioxidant capacity. It is proposed that AMF association enhance efficiency of photosystem II and stomatal conductance in plants. When compared to nonmycorrihizal plant the accumulation of hydrogen peroxide, and the membrane electrolyte leakage in AM associated plant was lowered. In addition, ROS production and photorespiration were also decreased. The activation of antioxidant enzymes (SOD or CAT) protects the maize from oxidative stress and helps to overcome salt stress (Estrada et al., 2013). Tomato (Solanum lycopersicum) plants treated with endophytes Bacillus pumilus AM11 and Exiguobacterium sp. AM25 showed high rate of antioxidant enzymatic activities such as POX, polyphenol oxidases (PPO), and CAT in response to stress. CAT catalyze the conversion of H2O2 into water while POX and PPO help in defense mechanism by oxidizing quinones. The high rate of antioxidant enzymes significantly reduced ROS-based adverse effects such as lipid peroxidation, CAT, and peroxidase activities. Inoculation of bacterial endophytic strains improve plant-growth-promoting parameters (biomass), photosynthetic rate, pigment accumulation compared to untagged plants (Waqas et al., 2017).

7.4.3  Temperature stress Climate change is a major cause of temperature stress. Both cold and/or hot temperature is major cause of crop loss (Koini et al., 2009; Nagarajan and Nagarajan, 2009). The effect of high temperature on plants is primarily on photosynthetic activity (Mei and Flinn, 2010). Besides, it is responsible for changes in plasma membrane and water content (transpiration), impaired photosynthesis, enzyme dysfunctioning, reduced cell division, and plant growth. As a result, stressed plants show low germination rates, growth retardation, reduced photosynthesis, and they often die (Kai and Iba, 2001). Heat stress alters metabolism in plants and accordingly modulates the production of antioxidants, secondary metabolites, hormones, osmoprotectants, and many other

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essential biomolecules, which help to defend against high temperature impacts (Nahar et al., 2010). For example the concentration of JA increase many fold during stress condition. Plants have complex regulatory mechanism for heat tolerance. Plants execute various beneficiary mechanisms to overcome heat stress, which includes production and accumulation of enzymes and osmolytes such as heat shock proteins (HSP20, HSP 60, HSP70, HSP 90, HSP100), and ROS-scavenging enzymes such as ascorbate peroxidase and CAT (Kotak et al., 2007; Qu et al., 2013). Cold temperature is also a major factor limiting the productivity and geographical distribution of many species, including important agricultural crops. Cold-hardy plants increase their freezing tolerance upon exposure to low, nonfreezing temperatures by a phenomenon known as cold acclimation (Thomashow, 1999). Through this, certain species of plants have acquired an ability to tolerate super-cooling or freezing temperatures by increasing their antifreezing response within a short photoperiod (Thomashow, 2010).

7.4.3.1  Heat stress alleviation by the endophytes Endophytic fungus Aspergillus japonicus EuR-26 having heat stress alleviation potential was isolated from the weed Euphorbia indica L. The isolated strain was then inoculated into soybean and sunflower seedlings. Plants association with A. japonicus displayed higher concentrations of SA, IAA, flavonoids, and phenolics accumulation; which enhanced the immune response of seedling under thermal stress condition. It is proposed that the production of phytohormones and secondary metabolites by endophytic association helped to mitigate heat stress by negotiating the activities of ABA, CAT, and ascorbic acid oxidase in both soybean and sunflower. This study was an attempt to explore endophytic fungi that have a potential to improve plant biomass and other growth features under high temperature stress (40°C) in comparison to endophyte-free plants (Hamayun et al., 2018). A plant growth-stimulating bacterium Burkholderia phytofirmans PsJN, capable of epiphytic and endophytic colonization of grapevine potato and tomato (Sessitsch et al., 2005), could protect the plants against cold and heat stress (Barka et al., 2006). The host plantlets showed significantly increased levels of starch, proline, and phenolics. PsJN is also reported for the higher expression of ACC deaminase which hydrolyses the ethylene precursor 1-aminocyclopropane-1-carboxylate into ammonia and a ketobutyrate, thereby reducing the destructive effects of temperature and drought by lowering the production of ethylene in plants (Theocharis et al., 2012). The Ascomycetous mitosporic endophytes SMCD 2206, 2210, and 2215 were reported as the most effective at promoting both heat and drought tolerance in wheat seed germination. They are able to survive and confer combined tolerance to both heat and drought stresses (Hubbard et al., 2014)

7.4.3.2  Cold stress alleviation by the endophytes Frost injury is common in cold sensitive plants due to the presence of epiphytic bacteria present on the leaf surface causing inability to supercooling. Supercooling is a process by which the membrane damage due to crystal ice formation is prevented.

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Microbial Endophytes

Table 7.4  Endophyte-mediated temperature stress tolerance in plants. Endophyte

Plant hosts

Serratia marcescens strain SRM

Summer squash (Cucurbita pepo)

Pseudomonas vancouverensis OB155-gfp P. fredriksbergensis OS261-gfp

Dichanthelium languinosum

Physiological changes in plants

References

Influence seedling Selvakumar et al. growth at cold (2008) temperatures. Solubilize P and produce IAA Codes for proteins Subramanian et al. Solanum that protect (2015b) lycopersicum Mill. cells against cold/chilling stress. Reduced membrane damage and ROS level. Tomato lipoxynase Curvularia species Increases host heat Yuan et al. (2010) tolerance

Ice nucleating activity (ice+ strain) of endophytic bacteria is one of the core reasons for frost injury, which can be impeded by increasing the number of nonice nucleation active bacteria strains. Pseudomonas syringae, an endophytic bacterium having ice+ strain is the reason of frost injury in many cold sensitive plants and the frost injury caused by these bacteria can be hindered by preemptive competitive inclusion of ice– strain bacteria. This is possible, as the younger as well as tender leaves and flowers have a low number of P. syringae and competition with non-ice bacteria can lower the microbial load thereby preventing frost injury by aiding supercooling (Cambours et al., 2005) (Table 7.4).

7.4.4  Heavy metal stress Metals found in soils, are referred to as essential micronutrients for normal plant growth (Fe, Mn, Zn, Cu, Mg, Mo, and Ni) and nonessential elements (Cd, Sb, Cr, Pb, As, Co, Ag, Se, and Hg) causing reduction or inhibition of plant growth (Rascio and Navari-Izzo, 2011; Wuana and Okieimen, 2011; Schutzendubel and Polle, 2002). Most of the agricultural land area in the world is slightly or moderately contaminated by metal toxicity. This is due to continued industrialization, intensive agricultural practices including long-term use of fertilizers, sludge application and bad irrigation, and anthropogenic activities (Passariello et al., 2002). These heavy metals have severe impact not only on plants but also on human health. Heavy metals are nondegradable metallic elements that have a higher density than 4 g/cm3, and also poisonous at minute concentration (Duruibe et al., 2007). The regulatory limit of cadmium (100 mg/kg) in agricultural soil (Salt et al., 1995) is continuously exceeding because of several human activities. Plants exposed to high

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levels of Cd causes drastic reduction in water and nutrient uptake and photosynthesis leading to growth inhibition, chlorosis, browning of root tips, and finally death (Wojcik and Tukiendorf, 2004; Mohanpuria et al., 2007). In addition to Cd, zinc toxicity (150–300 mg/kg) also found in contaminated soil, which also shows same effect as Cd (Ebbs and Kochian, 1997). Excess Zn is also responsible for manganese, copper (Ebbs and Kochian, 1997), and phosphorus (Lee et al., 1996) deficiencies in plant shoots. Industrial and mining activities have contributed to the excess amount of essential micronutrient copper (Cu) in soil which plays a cytotoxic role, induces stress, and causes injury to plants leading to retarded plant growth, leaf chlorosis (Lewis et al., 2001) and oxidative stress (Stadtman and Oliver, 1991). Lead is another abundant toxic element leaches out into the soil by the disposal of sewage sludge, smelting and mining activities, lead containing paints, paper, pulp, gasoline, and explosives. It exerts adverse effect on of enzyme activities, cause water imbalance, alter membrane permeability, and disturbs mineral nutrition (Sharma and Dubey, 2005). Cobalt (Co) toxicity is caused by the deposition from the burning of fossil fuels, wearing of Co containing alloys and spreading of sewage sludge and manure (Barceloux and Barceloux 1999). Chatterjee and Chatterjee (2000) reported that high level Co restricted the concentration of Fe, amount of chlorophyll, and protein and CAT activity in leaves of cauliflower. Nickel (Ni) contamination is also found in certain areas by human activities such as mining works, emission of smelters, burning of coal and oil, sewage, phosphate fertilizers, and pesticides (Gimeno-García et al., 1996). Excess of Ni2+ causes various physiological alterations and toxicity symptoms such as chlorosis and necrosis in different plant species (Zornoza et al., 1999; Pandey and Sharma, 2002; Rahman et al., 2005). Mercury (Hg) is the next toxic metal that has become a critical environmental concern due to their potential adverse ecological effects. Among different forms (HgS, Hg2+, and Hg) the ionic form (Hg2+) is predominant in agricultural soil (Han et al., 2006). Toxic level of Hg2+ is strongly phytotoxic to plant cells and induces visible injuries and physiological disorders in plants (Zhou et al., 2008). Excess Hg2+ also interfere the mitochondrial activity and induces oxidative stress by modulating the generation of ROS leads to the disruption of membrane lipids and cellular metabolism in plants (Israr and Sahi, 2006; Cargnelutti et al., 2006). It is well-documented that Cr is a toxic agent for the plant growth and development (Panda and Choudhury, 2005). Cr is transported and accumulated mainly in roots via carrier ions such as sulfate or iron (Singh et al., 2013a). Cr drastically reduces the development of stems and leaves during plant early growth stage (Nematshahi et al., 2012). Chromium toxicity inhibits the cell division and elongation of plant roots, results in the shortening of the overall length of roots (Shanker et al., 2005). Iron and manganese are essential nutrients and have many important biological roles in the plant growth and development. But both are toxic when it accumulates to high levels in soil. High Mn concentration in plant tissues may alter activities of enzymes and hormones (Kitao et al., 1997). Excess iron causes free radical production that impairs cellular structure and damages membranes, DNA, and proteins (Arora et al., 2002). Physical barriers are the prior mechanism defense in plants against metals (Emamverdian et al., 2015) such as some morphological structures like thick cuticle, biologically active tissues like trichomes, and cell walls. As a first step toward dealing with

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Microbial Endophytes

metal intoxication, plants adopt avoidance or preventing metal entry in to plant root (Viehweger, 2014). This may be achieved by immobilization of metals by mycorrhizal association or metal sequestration by exuding organic compounds from root (Dalvi and Bhalerao, 2013). If heavy metals ions overcome biophysical barriers and enter tissues and cells, plants initiate several tolerance mechanisms as the next step. This includes the induction of metallochaperones or chelators, such as nicotianamine, spermine, putrescine, mugineic acids, organic acids, glutathione (Bricker et al., 2001) phytochelatins, and metallothioneins or cellular exudates, such as flavonoid and phenolic compounds, protons, heat shock proteins, and specific amino acids, such as proline and histidine, and hormones such as SA, JA, and ethylene (Prasad, 2004; John et al., 2012). Biosynthesis of these diverse cellular biomolecules effectively tolerates or neutralizes metal toxicity (Memon et al., 2001).

7.4.4.1  Metal stress alleviation by the endophytes Micrococcus yunnanensis SMJ12, Vibrio sagamiensis SMJ18, and Salinicola peritrichatus SMJ30 are endophytic bacterial population isolated from Spartina maritima tissues showed significant metal tolerance. A high proportion of these bacteria exert tolerance to one or several heavy metals and metalloids including As, Cu, and Zn—the most abundant in plant tissues and soil. In addition, these strains also shows multiple enzymatic properties (amylase, cellulase, chitinase, lipase, and protease) as well as plant growth promoting properties, including nitrogen fixation, phosphate solubilization, IAA, and ACC deaminase synthesis, and production of siderophores (Mesa et al., 2015). Experimentally engineered endohytes can improve phytoremediation of organic pollutants and toxic metals. Bioengineered endophyte Burkholderia cepacia VM1468 is inoculated to its natural host yellow lupin. The biological engineered strain processes two important characters (1) pTOM-Bu61 plasmid, coding for constitutive trichloroethylene (TCE) degradation and (2) ncc–nre Nickel (Ni) resistance determinant system. Inoculation of Burkholderia cepacia has a positive effect plant growth in the presence of toxic metals and a 30% increase in root biomass was observed. It also reduced the amount of trichloroethylene via transpiration and increased the uptake of Ni. Inoculation with B. cepacia VM1468 resulted in decreased Ni and TCE phytotoxicity (Weyens et al., 2010). Phytoremediation of recalcitrant pollutants from the environment presents a significant problem as it result in phytotoxic effects in plants. The inoculation of plants with strain Pseudomonas putida VM1441 (pNAH7) resulted in the protection of the host plant from the phytotoxic effects of naphthalene. This strain was found to be an efficient root colonizer. The NAH7 plasmid present in this strain has ability to utilize naphthalene as the sole carbon source. Endosymbiotic association of plants with this strain facilitated higher seed germination, plant transpiration, and naphthalene degradation compared with uninoculated plants in contaminated soil (Germaine et al., 2006). The heterologous expression of ncc-nre encoded nickel resistance gene in Burkholderia cepacia L.S.2.4 and Herbaspirillum seropedicae LMG2284 was accompanied by nickel removal from shoots and roots of Lolium perenne (cv Atlas). The capacity of this endophytic bacteria to remove nickel through sequestration or

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bio-precipitation processes offer interesting benefits to the associated host plants. When inoculated in to the host plant, they alter the nickel speciation and therefore decrease the accumulation of toxic ions in plant metabolism system. The inoculation of Lolium perenne (cv Atlas) with H. seropedicae LMG2284 having nickel resistance gene ncc-nre resulted in a significant decrease of the nickel concentration in the roots and shoots (Lodewyckx et al., 2001). Polycyclic aromatic hydrocarbon degrading bacteria Microbacterium sp. F10a was evaluated for promoting the growth of wheat and phenanthrene and pyrene removal from heavy metal contaminated soil. The strain has a cell surface hydrophobicity that increases solubility of aqueous polycyclic aromatic hydrocarbon consequently promoting its uptake by roots and translocating them to various aerial parts of plants. This strain produced plant growth promoting factors—IAA, siderophore, and 1-aminocyclopropane-1-carboxylate deaminase and has the capacity to solubilize inorganic phosphate. Inoculation of wheat with the strain was found to significantly increase the growth and phenanthrene and pyrene removal from soils in a low-temperature environment (Sheng et al., 2009). Pseudomonas fluorescens G10 and Microbacterium sp G16 are two lead-resistant endophytic bacteria isolated from rape (Brassica napus) roots grown in heavy metalcontaminated soils. The two strains colonize the interior of rape root after inoculation. These two strains exhibit production of IAA, siderophores, and 1-aminocyclopropane1-carboxylate deaminase activity. An increase in water-soluble Pb uptake and biomass production was seen in bacteria inoculated plants. Once inside the plant, pytochelatin binds to Pb and aids in detoxification mechanism (Sheng et al., 2008) (Table 7.5). Table 7.5  Endophyte-mediated metal stress tolerance in plants. Endophyte

Plant hosts

Neotyphodium uncinatum

Elymus dahuricus

Bacillus sp. SLS18

Sorghum bicolor Solanum nigrum and Phytolacca acinosa Brassica juncea

Staphylococcus arlettae

Pseudomonas koreensis AGB-1

Physiological changes in plants Antioxidative enzymes activities (AEA) and proline content increased Accumulation of root tillers and biomass

Arsenic reductase activity. Increased soiled hydrogenase, phosphatase, and available phosphorus Miscanthus sinensis High tolerance to metal (loid) concentrations through extracellular sequestration, increased catalase and SOD activities in plants

References Zhang et al. (2012) Luo et al. (2012)

Srivastava et al. (2013)

Babu et al. (2015)

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7.4.5  Role of endophytes in nutrient starvation Diazotrophic endophyte Klebsiella pneumoniae 342 (Kp342), nitrogen (N)-fixing bacteria that inhabit the interior of grasses without causing any disease are inoculated into wheat (Triticum aestivum L.). The association of Kp342 relieved the plant from nitrogen (N) deficiency symptoms and increased total N concentration. Production of nitrogenase reductase in the intercellular space of the root cortex, by Kp342 resulted in more root growth. This allow the Kp342-inoculated plants to absorb a majority of the N present in the soil thereby increasing the availability of N content in plant tissue. In contrast, uninoculated plant had very small roots. Moreover, Kp342 inoculation increased the dry weight of shoots and chlorophyll concentration. Thus, the Kp342– Trenton association relieved the wheat plant from nitrogen-deficiency symptoms (Iniguez et al., 2004). Strains of endophytic bacteria belonging to the genera, Enterobacter, Rahnella, Rhodanobacter, Pseudomonas, Stenotrophomonas, Xanthomonas, and Phyllobacterium associated with sweet potato plants [Ipomoea batatas (L.) Lam.] are able to fix nitrogen, produce IAA, and exhibit stress tolerance. These host-associated strains were tested for checking the above ability. The IAA producer grew well in the presence of nitrogen-free medium and had the nitrogenase subunit gene, nifH. The IAA aids in increased root production there by increasing the uptake of nitrogen from soil. These studies indicate that endophytic associations are beneficial for plant growth and crop improvement (Khan and Doty, 2009). In Andisols of southern Chile, crop production is limited due to the poor availability of P and high concentration of toxic aluminum (Al). Bacterial consortium of five endophytic strains, that is, Klebsiella sp. RC3, Stenotrophomonas sp. RC5, Klebsiella sp. RCJ4, Serratia sp. RCJ6 and Enterobacter sp. RJAL6 having Al-tolerant and growth-promoting factors are inoculated in to the roots of ryegrass grown in acidic Chilean volcanic soil. The strains were selected based on their ability to administer multiple growth promoting traits like—P solubilization, 1-aminocyclopropane-1-carboxylate deaminase activity, IAA production and exudation of organic acid anions. This bacterial consortium is capable of mitigating Al stress from the volcanic soil by forming a special structure called Al3+–siderophore complexes. This complex elevated the phosphate P concentration in shoots. Thus, the combination of native Al-tolerant bacteria to the roots of ryegrass was effective in decreasing Al toxicity and thereby promoted plant growth (Durań et al., 2016). Endophytes play important roles in phosphorus (P) solubilization in red acidic soil. Endophytes Pantoea dispersa isolated from cassava (Manihot esculenta Crantz) root has the potential to dissolve insoluble phosphorus when inoculated into red soil. The solubilizing process is accompanied by the production of two special organic acids, namely, SA and benzeneacetic acid. P. dispersa produce this acid to dissolve insoluble phosphorus present in soil. This microbial phosphorus solubilizing property can trigger microbial community and thereby making the soil favorable to plant growth (Chen et al., 2014a). Pyroverdines produced by Pseudomonas fluorescens C7R12 on association with Arabidopsis (Arabidopsis thaliana) promote plant growth under iron-deficient conditions. Pyroverdines are siderophores (high-affinity ferric iron chelators)

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Table 7.6  Endophyte-mediated nutrient uptake in plants. Endophyte

Plant hosts

Physiological changes in plants

Antioxidant enzyme activities, photosynthetic pigments, and low lipid peroxidation Siderophore, IAA, Gibberellic acid Giberrelic acid used by bacteria to O. sativa solubilize phosphate Triticum aestivum

References

Pseudomonas sp. Bacillus, Lysinibacillus

Sgroy et al. (2009)

Prosopisstrom bulifera (halophyte)

Reginato et al. (2012)

excreted by bacteria in the soil to acquire iron. This chemical modulates the expression of genes related to development and iron acquisition (Trapet et al., 2016) (Table 7.6).

7.5  Biotic stress Biotic stress in plants is caused by living pathogens including viruses, bacteria, fungi, nematodes, insects, arachnids, and weeds (Madani et al., 2019). Unlike abiotic stressing agents biotic stress agents directly and rapidly damage their host of its nutrients leading to reduced plant vigor (Fitter and Hay, 2012). Biotic stress affect at any phase of their life cycle: during seedling establishment, plant maturation, or grain or fruit setting. Singla and Krattinger (2016) reported that biotic stress is a major cause of preand postharvest losses of crop plants. However, the measure of biotic stress factors to cause crop yield or quality loss depends on the environment and thus varies from region to region and from one agroecology to another (Angessa and Li, 2016). The major detrimental effects of these biotic factors include imbalanced hormonal regulation, nutrient imbalance, and physiological disorder. The defenses to biotic stress include morphological physical barriers, chemical compounds, and proteins and enzymes. These confer tolerance to biotic stresses by protecting plant parts and by giving them strength and rigidity. Also, in response to pathogen attack plant initiates a series of metabolic actions including ROS generation and oxidative bursts to limit pathogen spread (Atkinson and Urwin, 2012). Also, a plant increase cell lignification to blocks invasion of parasites and reduces host susceptibility. The phytohormones ABA and etheylene exerts a positive effect on biotic stress resistance (Rejeb et al., 2014). During biotic stress condition, ABA acts antagonistically with ethylene and protect the plant against disease attack. Also the steroid hormones including JA and SA play a central role in signal transduction and defense mechanism (Verhage et al., 2010; Bari and Jones, 2009).

7.5.1  Role of endophytes in biotic tolerance Endophytes play a special role to maintain the health of plants, as they can protect or prepare the plant against biotic stresses and help in enhancing growth and

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yields (Tanaka et al., 2005; Lata et al., 2018). Endophytic bacteria Pseudomonas fluorescens associated with eggplant effectively inhibit a wilt pathogen Ralstonia solanacearum. In a greenhouse experiment seedling treated with PGPBEs showed an increased growth and 70% reduction in wilt incidence. The disease suppression in colonized plants is enhanced by the production of 2, 4-diacetylphloroglucinol (DAPG) an antimicrobial metabolite produced by Pseudomonas isolates. The production of siderophores and IAA were also recoded indicating that endophytic bacteria Pseudomonas were involved in biocontrol and growth promotion in eggplants (Ramesh et al., 2009). Symbiotic interaction between fungal endophytes Neotyphodium spp and cool-season grasses provide greater resistance to mammalian and insect herbivores, pathogens, and nematodes. This association of endophytes with host produce a wide range of alkaloids or stimulate the host grass to synthesize alkaloids and other secondary metabolite that protect the symbiotics. The fitness enhancements documented to host is due to the accumulation of four groups of alkaloids: lolines, peramine, ergot alkaloids, and lolitrems (Malinowski and Belesky, 2000). Black pepper root-associated endophytic strain Bacillus megaterium (BmBP17) protect the host from wide range of pathogens .The antimicrobial activity of BmBP17 was attributed by pyrazine 2-ethyl-3-methyl group of chemicals. These are volatile chemical compounds belonging to, heterocyclic hydrocarbons, sulfoxides and esters. They were predominantly present in solvent extracts of BmBP17. The antimicrobial property of pyrazine can be exploited for crop protection (Munjal et al., 2016). Endophytic Pseudomonas aeruginosa strain BP35 associated with black pepper show significant protection against infections headed by Phytophthora capsici and Radopholus similis. The efficiently colonized PaBP35 produce chemical metabolites rhamnolipids and phenazines inside the shoots of black pepper. The antimicrobial properties of the compounds protect the pl + ant from pathogen attack (Kumar et al., 2013). Phenazines and rhamnolipid-biosurfactants are antimicrobial metabolites produced by Pseudomonas aeruginosa PNA1 in response to pathogens. This ability of endophtic strain was used as a biological control strategy against Pythium splendens and Pythium myriotylum on bean (Phaseolus vulgaris L) and on cocoyam (Xanthosoma sagittifolium L Schott). The root colonization of Pseudomonas aeruginosa PNA1with bean and cocoyom showed a disease-suppressive effect in the host plant. The microscopic analysis revealed that phenazines and biosurfactant leads to the disintegration of Pythium hyphae. Henceforth, it is concluded that these two metabolites are acting synergistically in the control of Pythium spp. (Perneel et al., 2008). Endophytic fungi Fusarium oxysporum isolated from the cortical tissue of surface sterilized tomato roots produced secondary metabolites that are highly toxic to nematode Meloidogyne incognita. Anti-nematode activities of secondary metabolites (bikaverin, 3-O-methyl-8-Om + ethyl fusarubin, 8-O-methyl fusarubin, anhydrofusarubin, and fusarubin) are highly effective toward sedentary parasites. This antibiotic properties of secondary metabolites control plant parasitic nematodes and plant pathogenic fungi (Hallmann and Sikora, 1996) (Table 7.7).

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Table 7.7  Endophyte-mediated biotic stress tolerance in plants. Host plant

Endophyte species

Association benefit

References

Cucumber

Pseudomonas aeruginosa PW09

Pandey et al. (2012)

Black pepper (Piper nigrum L.)

Pseudomonas putida (Pseudomonas EF568932), and IISRBP 17 as Bacillus megaterium (B. megaterium EU071712 Bacillus pumilus strain SE34

Alleviate stress induced by Sclerotium rolfsii infection Antagonistic to Phytophthora capsici causing foot rot disease

Pea (Pisum sativum L.) Bananas (Musa paradisiaca spp.)

Pseudomonas fluorescens (Pf1) and endophytic Bacillus spp. (EPB22)

Oil palm

Burkholderia cepacia (B3) and Pseudomonas aeruginosa (P3) Eggplant Pseudomonas (Solanum fluorescens, melongena L.) Burkholderia cepacia Ginger

P. aeruginosa and P. monteilii

Coleus forskohlii

Pseudomonas monteilii and Glomus fasciculatum

Sugar beet, tobacco, Arabidopsis

B. amyloliquefaciens, B. subtilis, B. pasteurii, B. cereus, B. pumilus, B. mycoides, and B. sphaericu Achromobacter xylosoxidans

Rice

Aravind et al. (2009)

Phenolic compound released Benhamou et al. promote resistence to (1996) Fusarium oxysporum sp. Pathogenesis-related (PR) Harish et al. proteins, chitinase and (2009) β-1,3-glucanase and defense-related proteins, are activated Banana bunchy top virus (BBTV) Inhibit spread of Basal stem Sapak et al. rot (BSR) caused by (2008) Ganoderma boninense 2,4-diacetylphloroglucinol (DAPG inhibit wilt caused by Ralstonia solanacearum Antimicrobial activity against Pythium myriotylum Drechsler and Phyllosticta zingiberi Hori. Inhibit Root rot and wilt, caused by Fusarium chlamydosporum and and Ralstonia solanacearum Act against crown-rotting fungal pathogen, rootknot nematodes, and a stem-blight fungal pathogen Inhibit mycelial growth of Magnaporthe oryzae which cause rice blast

Ramesh et al. (2009)

Chen et al. (2014b)

Singh et al. (2013b)

Kloepper et al. (2004)

Joe et al. (2012)

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7.6  Commercial applications of stress tolerant endophytes The root-colonizing facultative endophyte Piriformospora indica has the potential for use in agriculture, horticulture, and floriculture (Ansari et al. 2014; Unnikumar et al., 2013; Johnson et al., 2014; Gill et al., 2016). The filamentous fungus P. indica was originally isolated by Verma et al. (1998) from the rhizosphere of several xerophytic plants located in the Indian Thar dessert in northwest India. It has numerous host benefits, such as the promotion of plant growth, particular in conditions of nutrient stress, and tolerance to a wide range of abiotic stresses as well as biotic (root and foliar pathogens) stresses. It also provides other beneficial effects such as enhanced nitrate and phosphate assimilation, (Bajaj et al., 2014) promotion of adventitious root and root hair formations (Oelmüller et al., 2009), alteration in the secondary metabolites (Das et al., 2012), hardening of tissue cultured plants (Sahay and Verma, 1999) and their preparation are suitable for field conditions (Zuccaro et al., 2011; Varma et al., 2012). Original inoculation experiments showed P. indica has the ability of to colonize plant root of broad host range, forming symbiotic root interactions with many crop plants, including barley Hordeum vulgare, Triticum aestivum (Serfling et al., 2007) and Oryza sativa (Jogawat et al., 2016), Saccharum officinarum (Varma et al., 2012), and Chinese cabbage (Sun et al., 2010), etc. P. indica showed an enhanced tolerance to various root and foliar pathogens such as maize (Kumar et al., 2009), tomato (Fakhro et al., 2010), wheat (Rabiey et al., 2015), and barley (Waller et al., 2008). Moreover, P. indica is able to extract, mobilize, and transport nutrients from soil and further its efficient translocation to aerial parts (Shahollari et al., 2007). The facultative endophyte Epicoccum nigrum from sugarcane is capable of producing compounds that inhibit the in vitro growth of sugarcane pathogens including Fusarium verticillioides, Ceratocystis paradoxa, Colletotrichum falcatum, and Xanthomomas albilineans. It is also known for its biocontrol activity against pathogens, such as Sclerotinia sclerotiarum in sunflower, Pythium in cotton, phytoplasma bacteria in apple, and Monilinia spp. in peaches (de Lima Fávaro et al., 2012). Peramine, a pyrrolopyrazine, a potent insect feeding deterrent, is synthesized by the epichloë group of fungal endophytes in association with their grass hosts (Lane et al., 2000; Clay and Schardl, 2002). It acts against both larvae and adults of Argentine stem weevil (ASW), Listronotus bonariensis, a major pest of perennial ryegrass (Rowan, 1993). Redman et al. (2011) reported that some class 2 fungal endophytes could enhance salt and drought tolerance to two commercial rice varieties, which were not adapted to these stresses. Furthermore, these endophytes reduced water consumption by 20%– 30% while increasing growth rate, reproductive yield, and biomass of greenhouse grown plants. Germaine et al. (2006) examined genetically tagged version of endophytic bacterium from poplar trees, which naturally possess the ability to degrade 2, 4-D. This strain was inoculated into pea for examining their signs of 2, 4-D toxicity and its translocation to the plants’ aerial tissues. The inoculation of pea plants with an endophytic strain Pseudomonas putida strain POPHV6 possessing the ability to degrade 2, 4-D

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protected this broad-leaved plant from the toxic effects of high levels of the herbicide. This allowed inoculated plants to increase their biomass, and thereby increasing the plant uptake of 2, 4-D from the soil. Once within the plant tissues, the 2, 4-D was quickly degraded by the root- and stem-colonizing endophytic biofilms and micro colonies of P. putida VM1450. These biofilms and micro colonies were responsible for the degradation of the 2, 4-D within the rhizosphere, root, and stem/leaf tissues. Hence, this strain allowed the pea plants to maintain their growth and increase xenobiotic removal through a high level of colonization and competence within the plant. Endophytes have been applied at various scales to treat hydrophobic pollutants [benzene, ethylbenzene, toluene, and xylene (BTEX) compounds], chlorinated solvents, nitrotoluene ammunition wastes, and excess nutrients. A number of endophytic bacteria found in poplar trees have the potential to enhance phytoremediation. They show the ability to degrade BTEX compound. BTEX is a highly volatile contaminant, with high solubility, and toxicity. Barac et al. (2004) demonstrated an engineered endophytic bacteria equipped with the appropriate degradation pathway improve the in planta degradation of toluene. The recombinant strain was made by the introduction of pTOM toluene-degradation plasmid of Burkholderia cepacia G4 into B. cepacia L.S.2.4, a natural endophyte of yellow lupine. Thereafter, the recombinant engineered endophytic bacteria successfully inoculated into surface-sterilized lupine seeds. These endophytic strain strongly degraded toluene, resulting in a marked decrease in its phytotoxicity. A wide variety of entomo-pathogenic fungi were able to colonize different plant species and confer protection against plant pathogens and also insect pests (Jaber and Ownley, 2018). They are important group of fungi with hardcore industrial applications. They showed (e.g., Beauveria bassiana and Metarhizium anisopliae) a promising potential for biocontrol of insect and pathogen pest in lab and field trials (Williams et al., 2013). Various techniques can be used for the external application endophytes into the host plant. Seed inoculation is the most suitable method than other techniques such as spraying on the surface of plant, soil drenching, etc. However, success of the inoculation technique depends on the nature of endophytes.

7.7 Conclusion Endophytes are nonpathogenic naturally colonized bacteria or fungi in almost all plant species. They symbiotically colonized within the plant tissues for their life cycle. Such host-associated endophytes in plant improve plant growth, yield, and health under normal environmental conditions. Also they involved in different metabolic activities and induce tolerance under detrimental conditions such as drought, heat, high salinity, poor nutrient availability and various biotic stresses. Furthermore it plays a key role as biological trigger to solve these detrimental climatic effects. Environmental stresses are a major constraint for crop product yield and quality to date. Plant associated endophytes not only helpful for adapting critical environmental conditions but also promoting crop growth and health. Based on their impact on plant stress tolerance beneficial endophytes may use as an ecofriendly approach for crop yield. It may also be a good tool for improvement of soil quality.

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Microbial bioformulation-based plant biostimulants: a plausible approach toward next generation of sustainable agriculture

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Mohd Aamir, Krishna Kumar Rai, Andleeb Zehra, Manish Kumar Dubey, Sunil Kumar, Vaishali Shukla, Ram S. Upadhyay Laboratory of Mycopathology and Microbial Technology, Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi, India Chapter outline head 8.1 Introduction  195 8.2 Concept of bioformulation, composition, and microbial metabolites  198 8.3 Production and marketing constraints  200 8.4 Bioformulation as biocontrol agents  206 8.5 Formulation and application methods  207 8.5.1 Solid Bioformulation  207 8.5.2 Liquid formulations  208

8.6 Microbial bioformulation-based plant biostimulants  209 8.6.1 Bacterial-based plant biostimulants  209 8.6.2 Fungal-based Biostimulants  211

8.7 Mechanisms implicated in plant biostimulatory effects on crop productivity  213 8.8 Current Scenario/Market Trends  215 8.9 Regulatory framework  216 8.10 Conclusion  217 References  218

8.1 Introduction Human population is growing swiftly and is expected to cross nine billion by 2050. The current trend of agricultural food production is insufficient to support global food demand. According to one study, it has been estimated that within the coming next 40 years the agricultural food production must be increased by 60% (Berger et al., 2018). However, the arable land will increase by 5% up to 2050. The extensive uses of agrochemicals for fulfilling the global food requirement have degraded 25% of the productive agricultural land that results in arid and nonfertile soils. Furthermore, rapid globalization trend and modern industrialization have diminished the arable lands for agricultural purposes and Microbial Endophytes. http://dx.doi.org/10.1016/B978-0-12-819654-0.00008-9 Copyright © 2020 Elsevier Inc. All rights reserved.

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call for intensifying the agricultural production through sustainable routes. The current trend of intensifying the agricultural food production is abundant use of chemical fertilizers, agrochemicals, use of improved hybrids, seed varieties, resistant cultivars, or other technological innovations, such as improved irrigation methods, all of which have supported the increased food production to some extent. However, the commercialization of such methods have greater repercussion on soil health, and also hazardous environmental concerns, damaged ecosystems or altered the delicate balance between various components of ecosystem. One of the most hazardous impacts of such agrochemicals in the soil ecosystem is the loss of beneficial microbial populations from the soil (Seneviratne and Kulasooriya, 2013). Additionally, in a rapidly changing climatic scenario, modern agriculture faces several challenges, including drought, temperature stress, soil salinity, depletion of mineral nutrients, and evolutionary pressure of pest and pathogens have affected the global productivity at the economic level. In this context, we need to enhance the agricultural production in a sustainable manner through the use of efficient agroecosystem, which considers the entire agroecosystem biochemical diversity with their potential to ameliorate the drastic effects of abiotic stress, depletion of soil mineral nutrients, pest, and pathogens (Tilman et al., 2011; Timmusk et al., 2017). The most common approach used in controlling the pest and pathogens, and thereby managing plant diseases is the use of chemical pesticides, removal of diseased plants, and controlling the growth of weed hosts. The conventional practices including the use of resistant varieties, expensive agrochemicals, and modification of cultural practices have been reported with hazardous effects, and now there is well-known evidence that some chemical pesticides do pose a potential risk to humans and other life forms with an unintended change in the environment. The microbial population associated with soil ecosystem is highly diverse both in their metabolic performance as well as in their functional aspects and acts as a main source of inoculum for the rhizosphere, the thin layer of soil adhering to and influenced by plant roots. The global food securities through sustainable practices invite the metabolic capabilities of microbiomes to conjugate stable yield with reduced impact on the agroecosystem (Crecchio et al., 2018). The molecular basis of interaction in between the microbes and plant system, and how such interaction affects the plant health and development is an important parameter for selection of microbes and their exploitation to field level. Furthermore, characterization of the biological activities of the important plant-probiotic members from soil microbiome is a key prerequisite for translational applications. Today, microbialbased bioproducts employed under the different trade name including biostimulants, bioinoculants, biofertilizer, and biopesticides type bioformulations have been considered as essential components of ecological sustainability and improved the crop productivity to a greater extent in an ecofriendly manner (Singh et al., 2016). Today, the biostimulant market across the globe is estimated about $2.0 billion and would be expected to expand $3.0 billion by 2021 with an annual growth rate of 10%–12% (Dunhamtrimmer.com, 2018). Furthermore, microbial-based bioformulations that increase the biological performance is greatly needed, and particularly, those bioformulations that exhibit complementary and synergistic effects with mineral fertilization. The biofertilizer and biocontrol formulations of the first generation prepared from plant growth-promoting rhizobacteria (PGPRs) are increasingly applied in sustainable

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agriculture but their application to field level has not been successful in most cases due to hampering in their action and generally do not fulfill expectations of the applier. Additionally, fungal biological control has been found to have greater impact on plant productivity and disease management and their efficacy is highly dependent on the formulation of that particular bioagents. The most commonly used bioformulated fungal bioagent is in solid form (contain only conidia) and in liquid form with a shelf life of 6 months at 5°C and 3 months at 25–35°C. However, the efficacy of fungal bioproduct could be increased with various adjuvants, surfactants, and oils. The addition of nutrients to a spore spray of fungi could improve the effectivity of biocontrol agent, compared with spores applied in water alone (Hall, 1982). In contrast, the fungal formulations developed in oils rather than water suspension has been found to have a greater infectivity. For example, Verticillium lecanii formulated with arachnid oil showed significantly better control of powdery mildew than without the addition of oil (Verhaar et al., 1999). Furthermore, in case of mass production of entomopathogenic fungi like Metarhizium anisopliae, a number of naturally occurring carrier cum growth media have been evaluated (Fogal, 1986; Quintela and McCoy, 1997). Furthermore, the most important factors delimiting the efficient application of these bioproducts are quality, reliability, and performance, hampering their progress in the market. Due to these factors, the success of microbe-derived products in market is limited and therefore new avenues and directions have to be explored to remove the associated problems and instigate belief among the end users/farmers. The past decade has seen the emergence of technological tools developed to promote sustainable agroecosystems. The enhancement of plant tolerance to numerous abiotic stresses is increasingly being supported by biostimulant products, as preferred alternatives to expensive agrochemicals. Biostimulants include living microorganisms, namely, plant growth-promoting fungi and PGPR) (Bhattacharyya and Jha, 2012). PGPRs are currently thought to be an effective tool for the biostimulation of plant growth (Calvo et al., 2014; Kumar et al., 2016a, 2016b). Recent advancement made in microbial biotechnological tools has helped in understanding the microbial diversity, functional attributes, metabolic route, and genetic potential of soil microbes, and therefore the development and commercialization of efficacious microbial products (biostimulants, biopesticides, and biofertilizers) with improved crop yield and adaptation against ongoing climatic changes (Umesha et al., 2018). Generally, microbes associated with plant system enhance the growth through multiple biochemical pathways (categorized as direct or indirect mechanisms) that may include manipulating the plant hormonal signaling, nutrient release and uptake, amelioration of abiotic stresses, preventing the pathogenic challenge (Jacoby et al., 2017; Kumar et al., 2016a, 2016b, 2018; Mendes et al., 2013; Van der Heijden et al., 2008; Verbon and Liberman, 2016). Besides having extensive diversity, eminent genetic potential, flexibility for the genomic changes, and multifunctional role in soil ecosystem, the ecofriendly microbial bioproducts have not taken the dominant role in the agro market because of constraints associated with their application. The most common constraints of these bioformulations include maintenance of the microbial population, vigor, erratic performance in the field, low organic carbon in the soil, inconsistence performance, and the poor shelf life. The performance and potential of the rhizospheric microbial population in field conditions

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greatly depends on application strategy, strain persistence, inoculum used in the rhizospheric soil and plant system. It has been noted that the efficacy of microbial system in field condition decreases than under in vitro conditions. However, the inconsistency in application of microbial bioproducts could be eliminated by their application in soil, seeds, roots and foliar spray. In this way, an efficient delivery plays a crucial role in performance of microbial bioproducts under the field condition (Vidhyasekaran and Muthamilan, 1999). The ongoing research has been focused for the development of formulations that are more reliable and consistent. The future bioformulations will target the incorporation of microbial metabolites along with microbes and will definitely have multiple beneficial roles along with efficient efficacy in sustainable agriculture.

8.2  Concept of bioformulation, composition, and microbial metabolites Bioformulation are defined as any biologically active substances derived from microbial biomass or product containing microbes and their metabolites that could be used in plant growth promotion, nutrient acquisition, and disease control in an ecofriendly manner. Basically, bioformulation is a mixture of an active ingredient in a formulated product made with inert (inactive) substances (http://npic.orst.edu/factsheets/ formulations.html). Bioformulated products offer green alternatives to conventionally used chemical fertilizers and pesticides for plant growth promotion, suppression of phytopathogens, maintaining the fertility of soil and disease suppression (Arora and Mishra, 2016). The actual definition of bioformulation is not uniformly available, and researchers define the term accordingly. For example, it was considered that the bioformulated products aim to preserve the microbes enhance their potential, delivering their mixture to the target and further improve their activities (Burges and Jones, 1998). The bioformulation constitutes the preparation(s) of microbes or their active gradient that could be utilized as substitutes for chemical pesticides/fertilizer. However, actual bioformulated product must contain an active ingredient and comprised of living microbe, spores, or their products, and must be in living state for successful development of formulation. The most common inert active substances include peat, talc, vermiculite, carboxymethylcellulose, and polymers specially xantham gum and diatomaceous earth. The inert carrier-based bioformulations have been found useful for incorporating the antagonistic microbial cells in both rhizospheric region and plant system for longer duration and could be applied through both foliar and soil application. Additionally, one more added advantages of such carrier-based bioformulations are the stability of the adduct, including antibiotics, siderophores, and phytohormones, volatile metabolites hydrolyzing enzymes in contact with the plant (Ardakani et al., 2010; Jorjani et al., 2011). The harsh environmental condition in bioformulated products is protected through some additives including gum, silica gel, methyl cellulose, and starch, which also contribute in improving the physical chemical and nutritional properties of bioformulated products (Schisler et al., 2004). In this context, bioformulated derivatives are the need of today's agriculture to counteract the effect

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of phytopathogens and disease prone agricultural zone. Generally, microbial species employed for bioformulated products contain beneficial rhizospheric microbes that affect the plant growth and development either directly or indirectly in their natural environment (Lugtenberg and Kamilova, 2009). The most common microbial species employed in microbial bioformulations includes bacterial genera Rhizobium, Bradyrhizobium, Mesorhizobium, Azotobacter, Bacillus, Pseudomonas, and most commonly used fungal genera belonging to Trichoderma spp. It has been suggested that various PGPRs and fungal genera secrete metabolites, including antibiotic and antifungal activities. Some of the most common antibiotics secreted by fluorescent pseudomonads include pyoluteorin, oomycin A, phenazines, and diacetylphloroglucinols (Haas and Defago, 2005). Similarly, species of Bacillus genera are well known producers of antibiotics such as zwittermycin and kanosamine (Raaijmakers et al., 2002). All these metabolites have been reported to have antiphytopathogenic activities. The utilization of such crucial metabolites in bioformulations will definitely open new avenues to control phytopathogens, and therefore helpful in controlling plant diseases. Cell-based bioformulations generally contain PGP microbes. Due to limitations in their application purposes now the bioformulated derivatives are developed along with microbial metabolites and other additives, which ensure the prolonged shelf life, quick delivery, and stability of the system. For example, the role of flavonoids in rhizobia-legume interaction is well known and has been used for rhizobial inoculation for facilitating the nodulation. Now it has been clearly demonstrated the role of flavonoids in nodulation, nitrogen fixation, and mitigation of abiotic stresses in legume crops (Oldroyd, 2013). Lipo-chitooligosachharides (LCOs) or nod factors are signaling molecules involved in rhizobia-legume association. The effect of LCOs on crop productivity showed positive response even in the absence of rhizobia inoculants (Oldroyd, 2013). Nowadays different bioformulated products have been prepared for biofertilizer, biocontrol, biostimulant, and enhancer of biological products and sold under the different trade names. For example, Novozyme (Denmark) incorporate flavonoids and the LCO promoter technology to improve the product yields of legume and nonlegume crops (http://www.bioag.novozymes.com). Tag team uses a combination of rhizobia strains with Penicillium bilaii and used for supplementing the phosphate and nitrogen uptake. Nitragin Gold contains rhizobia strains with patented slow-drying system, assures a high number of bacteria on the seed, and results in higher levels of nitrogen fixation and maximum yield in leguminous crops. Exopolysaccharides (EPS) include the group of important metabolites having a crucial role in root nodulation, root colonization, biofilm formation, carbon utilization, and toxin neutralization and secreted by PGPRs like pseudomonads and rhizobial bacteria (Tewari and Arora, 2014). In this way, EPS thus can be used as amendments in bioformulations or even as slow release bioprotectant carriers for the PGP microbe. Additionally, EPS supplementation in bioformulated products protects microbial cell from extreme pH, harmful radiations, osmotic shock, dessication, and protection from predators (Seneviratne et al., 2011). The supplementation of bioformulated products with the precursor of plant hormones such as L-tryptophan [precursor for indole 3-acetic acid (IAA) production] results into the enhancement of root hair formation,

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plant biomass, grain yield, and even capacity to fight pathogens (Zahir et al., 2010). Likewise, L-methionine (precursor of ethylene) which, when amended with PGP microbes resulted in enhanced growth. The use of phytohormones including gibberellic acid and cytokinins as additives in bioformulations came up with excellent performance under field condition. Bioinoculants prepared with macro or micronutrients (for plant) potash rocks, sulphur or insoluble zinc, and phosphates, along with solubilizing microbes or sulphur oxidizing bacteria can be much more effective. Apart from beneficial PGPRs bioformulated products prepared with endophytic and beneficial fungi also give good results. The Myc factors secreted by mycorrhizae activate signal transduction pathway or common symbiosis pathway in the host plants can be used in bioformulated derivatives for promoting the microbial association with plants. Biosurfactants are microbial products having antimicrobial, antiinsecticidal, and antiviral activities. These molecules are secreted by various PGPRs and function as dispersing agents have good emulsifying and wetting properties. Their role in bioformulated derivatives as carriers may come up with surprising results (Thavasi et al., 2015). Some of the commonly employed fungal bioformulations include RhizoMyco, Rhizoplex, and RhizoMyx. The multiinoculants RhizoMyco contains formultions of 18 endo and ecto mycorrhizae and growth-promoting substances and can be used for nutrient acquisition, root hair formation and plant growth promotion (Mehnaz, 2016). Some of the most commonly employed biostimulants that have been used in commercial bioformulations has been shown in Table 8.1

8.3  Production and marketing constraints The successful production of microbial bioformulation and delivery of the quality product in the market is a challenging task. Since bioformulated products employ living microbial species, it needs extreme care to maintain the microbial load and vigor without having any product contamination. Additionally, during manufacturing processes production technologies requires advanced level of sophisticated instruments so as to maintain high-quality products. The quality of microbial inoculants in bioformulated products plays a crucial role in supplying nutrients to the plants; product contamination should be avoided for achieving maximum benefits. In developing countries, the lack of knowledge, absence of sophisticated instruments and technologies, improper distribution, inexperienced manpower, technical difficulties, and importation laws for live inoculants are several factors that leads to the loss of viability, efficacy, and effectiveness of microbial products (Arora and Mishra, 2016). Apart from this, the major constraints that could be associated with effective bioformulation development may include high production cost, shelf life, and inconsistent performance. During manufacturing of bioformulated products, the potential hazards associated with microbial contaminants cannot be avoided as long as nonsterile carrier inoculants are widely used. The high cost production restricts the use of high tech advanced instruments with completely sterile condition during production of microbial bioformulation. The under performances of the microbial product could be

Type of microbial inoculants

Trade name

Microorganism employed

Assigned function

References

Inómix Biostimulant Inómix Biofertilisant

B. polymyxa (IAB/BP/01), B. subtilis (IAB/BS/F1) Bacillus megaterium, Saccharomyces cerevisiae, Azotobacter vinelandii, Rhizobium leguminosarum P. fluorescens, B. megaterium, S. cerevisiae

Promotion of plant growth, increase in root and shoot weight, more vigorous root system apparatus

IAB (Iabiotec), Spain

T. Stanes & Company Ltd, India ABiTEP GmbH, Germany http://www. tstanes.com/ products.html

Inómix Phosphore

Biofertilizer Symbion-N Symbion-P Symbion-K FZB24f Rhizovital 42 RhizoMyco RhizoPlex RhizoMyx

Phosphate availability

ABiTEP GmbH, Germany

Phosphate availability + root an shoot development Phosphate availability + N2 fixation

(Novozymes) www.novozymes. com (Mehnaz, 2016)

(Continued)

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Azospirillum, Rhizobium, Acetobacter, Azotobacter B. megaterium var. phosphaticum Frateuria aurantia B. amyloliquefaciens ssp.plantarum B. amyloliquefaciens B. amyloliquefaciens ssp. plantarum contains 18 species of endo- and ectomycorrhizae and growth-promoting substances. 18 species of endo- and ectomycorrhizal +  uniquely formulated blend of patented bacterial cultures+ stress reducing ingredients endomycorrhiza inoculant

Microbial bioformulation-based plant biostimulants

Table 8.1  List of commercialized bioformulated products under different trade name and used as biofertilizer, biocontrol products, and bioyield widely employed Europe, North America, and Asia.

Type of microbial inoculants

Trade name JumpStart JumpStart LCO Tag Team TagTeam LCO Cell-Tech (N Prove) Nitragin Gold Apron XL/ Allegiance FL compatible Amase Rhizosum N Liquid PSA Nitrofix Bioenraiz

Penicillium bilaii Penicillium bilaii + LCO Promoter Technology Penicillium bilaii + rhizobial strains (for legumes)

Assigned function N2 fixation

References Labiofam (www. labiofam.cu) Fertibio (www.fertibio. com)

Wheat growth promotion

Flozyme (http://www. flozyme.com/ agriculture)

Rhizobial strains Microbial inoculant + fungicide

Pseudomonas azotoformans N2-fixing bacteria (genera not described) Pseudomonas aurantiaca strain SR1 +  organic and inorganic nutrients Azospirillum brasilense Phytohormones extracted from Rhizobium Cocktail of more than 30 microbes Azotobacter chroococcum + Pseudomonas fluorescens

Phosphate availability, N2 fixation, plant growth promotion

Mapleton Agri Biotec Pty Limited (mabiotec.com) Phosphorus availability as substitute Biagro (http:// of Triple Super Phosphate (TSP) www.biagrosa. com.ar) N2 fixation, drought resistance, disease resistance +  bionematicide

Agrilife (www. agrilife.in) (Mehnaz (2016)

Microbial Endophytes

Inogro Bio Gold

Microorganism employed

202

Table 8.1  List of commercialized bioformulated products under different trade name and used as biofertilizer, biocontrol products, and bioyield widely employed Europe, North America, and Asia. (Cont.)

CataPult Nodulest 10 Agrilife Nitrofix

Si Sol B P Sol B Mn Sol B Micofert BuRIZE1 Mycobiol1 FOSFOSOL1 Phylazonit-M Zn Sol B Bio yield Enhancers

RhizoBio RhizoMyx

B. megaterium assisting phosphate solubilization Vesicular-arbuscular mycorrhizal (VAM) fungus Glomus intraradices +  Bacillus spp. Two different strains of Bradyrhizobium japonicum+ Sphagnum peat moss N2-fixing biofertilizers containing Azotobacter chroococcum, A. vinelandii, Acetobacter diazotrophicus, Azospirillum lipoferum and Rhizobium japonicum. Pseudomonas striata + Bacillus polymyxa, + B. megaterium Bacillus sp. Penicillium citrinum G. intraradices, G. etunicatum, Gigaspora sp. Glomus intraradices Glomus sp., Entrophospora colombiana, Acaulospora mellea Penicillium janthinellum

Phosphate-solubilizing biofertilizers Lantmannen Bioagri, Sweden

B. megaterium and Azotobacter chroococcum Thiobacillus thiooxidans 18 species of endo and ectomycorrhiza with a complex biostimulant Multi-inoculant formulation having multiinoculant species of endomycorrhiza fungi with biostimulant

Zinc oxidation and availability to plants Increased nutrient, water uptake, and Novozymes tolerance to abiotic stresses (www. Increased nutrient, water uptake, and novozymes. tolerance to abiotic stresses com)

Citric acid and oxalic acid producer for Mn solubilization Phosphate solubilization and mineral nutrition N2 fixation Silicate weathering

Cobos (2005) Lara (2008) MorenoSarmiento et al. (2007) Phylazonit Kft

Mineral absorption and translocation beyond the depletion zones of plant rhizosphere, induced changes in secondary metabolism leading to improved nutraceutical compounds

203

(Continued)

Microbial bioformulation-based plant biostimulants

Bio Phos+ Eppawala rock phosphate (ERP) mixture

Type of microbial inoculants Biocontrol Products

Trade name Met52

Microorganism employed Bioinsecticide, containing spores of the soil fungus Metarhizium anisopliae Bacterial-based biofungicide Trichoderma asperellum Bioinsecticide containing Beauveria bassiana

Cedomon Cerall

Bioinsecticide containing Metarhizium anisopliae

Cedress

P. chlororaphis + rapeseed oil

Bioscrop BT16

Biopesticide with P. chlororaphis and water.

MycoUp MycoUp Attack

Glomus iranicum var. tenuihypharum (a mycorrhizal fungus) (1.2 ×104 propagules/100 mL substrate)

Resid

Glomus iranicum var. tenuihypharum +  mineral clay substrates bentonite+ and smectite Biofungicide that contains Trichoderma viride

Bio Vaccine

References

Control of Cigarrinha (Mahanarva fimbriolata) infestations in sugarcane

Fertibio (www.fertibio. com) BioAgri (http://www. bioagri.se) Symborg (www. symborg.com)

Effective against phytopathogens and nematodes

Microbial Endophytes

Taegro TrichoderMax BoveMax MethaMax

Assigned function Insect control Soil borne and foliar diseases Soil borne crop diseases control of Broca (Hedypates betulinus) infestations

204

Table 8.1  List of commercialized bioformulated products under different trade name and used as biofertilizer, biocontrol products, and bioyield widely employed Europe, North America, and Asia. (Cont.)

Mycoinsecticides containing spores of Beauveria bassiana Mycoinsecticides with Gypsy moth (Lymantria dispar). Multicapsid nuclear polyhedrosis virus (LdMNPV)

Kills pathogenic fungi including Pythium, Rhizoctonia, and Fusarium spp. Applied using aerial or ground application equipment

BioKuprum

Containing spores of fungus Chaetomium cupreum (formulated as Wettable powder with 2 × 106 CFU/g) Spores and mycelial fragment of fungus F. proliferatum Powdery formulation contains spores of Ampelomyces quisqalis 2 × 106 CFU/g Bioformulation containing spores of T. harzianum (function as both biofungicide and bionematicide) T. harzianum

Used to protect agricultural, fruit, decorative, and flowering plants from beetle and moths Protects plants from rusts, blights, rots, and leaf spots Control over Downy mildew disease caused by several pathogens Controls several plant pathogens Controlling Botrytis, Fusarium, Pythium, Rhizoctonia, Gaeumannomyces, Sclerotinia, Sclerotium, Verticillium, and wood-rot fungi Postharvest decay of citrus fruits, apples, and pears caused by Penicillium sp. and Botrytis sp. Powdery mildew on greenhouse roses and cucumbers

Downycare Powderycare Ecosom TH

Binab (Fungicide)

Aspire Sporidex

Fungicide with Candida oleophila (using skimmed milk as carrier) Fungicide with Pseudozyma flocculosa (using skimmed milk as carrier)

Reardon and Podgwaite (1992) de Faria and Wraight (2007) Mehnaz (2016)

Microbial bioformulation-based plant biostimulants

Gypchek Boverin, Naturalis, Boverosil, Trichobass-P, and L BioGuard Rich Mycotrol ES MycotrolO

Punja and Utkhede (2003) Droby et al. (1998)

205

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attributed due to deficiencies in handling procedures and inexperienced manpower. Furthermore, “living microbial cells” employed in bioformulations have high sensitivity to temperature and other external conditions and requires enormous caution at the stage of manufacture/culture, transportation/distribution, and application (Arora and Mishra, 2016). All these processes requires huge amount of money invested in purchase of suitable carrier materials, packaging, and storage. Spurring the development of agricultural markets is the key factor for achieving targeted growth in bioformulation usage. Generally, firms with larger production facilities are associated with more expenditure on networks for grasping the market. Furthermore, one bigger obstacle is the registration of product (cost of registration), which is highly expensive and time consuming (Ehlers, 2006). The inconsistence performance and inadequate demand of the microbial product by the producers of bioformulations and investors necessitate their efficient storage, for providing prolonged shelf life. Furthermore, microbial shelf life is highly dependent on multiple factors, including production technologies employed, nature of carrier and packaging material used, mode, and distance of transport. However, the lack of technical knowledge, inappropriate handling experiences, and lack of subject knowledge makes most producers, shopkeepers, and farmers unable to provide an and efficient storage system for effective bioformulations. Furthermore, the other important factor that delimits the use of microbial products to the field for successful marketing is inconsistency observed in microbial performances while their transfer to the field/soil. The rapid decline in number of active cells from microbial population affects their potential in a new environment. The soil environment is a heterogeneous system with mixed biota under fluctuating local conditions, temporal, and spatial aspects, pertaining to the introduction should be critically evaluated for each release. Soil microbiostasis play a crucial role in growth/survival-inhibitory effect of the inoculant. The availability of nutrient resources and the hostility of the soil environment to incoming microbes under the presence of various abiotic and biotic factors influence the growth and survivorship of the inoculant and their potential for crop growth and productivity. Hence different species will show different performances as measured in terms of their activity and survival.

8.4  Bioformulation as biocontrol agents Biocontrol or biological control represents the reduction of inoculum density or diseases caused by pathogen or parasite in its active or dormant state, by one or more organisms accomplished naturally or through manipulation of environment, host or antagonist, or by mass introduction of one or more antagonists. Bioformulation based on biocontrol agent have been standardized, and mass multiplied in the form of biopesticides. The PGPR based bioformulations including Biofor PF-2 (Jaiva Kiran), Bioveer, Biozium, Biozin-PTB (Jaiva Kiran-2), Biozin-PTB, and talc-based biopesticide containing three aggressive strains of antagonists such as Pseudomonas aeruginosa, Trichoderma harzianum, and Bacillus brevis with standard adhesive and osmoticant have been developed by Department of Plant Pathology, Assam Agricultural University, Jorhat, Assam, India (http://dbtaau.ac.in/coordinator_pesticide.html).

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The biocontrol formulation has been tested and found efficient in biological control of wilt of ginger, wilt diseases of tomato, chilli, and controls rhizome rot, and also effective against damping off, foot root, root rot and wilt diseases of pepper, cabbage, and French bean. Similarly, BioPf-2 which contains beneficial microbes P. fluorescence and T. harzianum increases soil nutrients, rejuvenate soil, enhanced plant growth, and combat soil borne disease (Kumar et al., 2017a,b). Met52 is a bioinsecticide, containing spores of the soil fungus Metarhizium anisopliae. Taegro is a bacterial-based biofungicide/bactericide used for suppressing selected soil-borne and foliar diseases (Mehnaz, 2016). Beside this some of the most common biopesticides under registration process includes TrichoderMax, containing spores of T.asperellum. Similarly, BoveMax is a bioinsecticide, containing Beauveria bassiana for the control of Broca (Hedypates betulinus) infestations in Erva-mateplantation (Mehnaz, 2016). Some biopesticides such as Biollium, Biosona, and Biometa have been developed using microbial consortia, involving plant growth promoting microbe and entomopathogens, namely, B. thuringiensis, M. anisopliae, B. bassiana, and Verticillium. lecanii. However, the success of biostimulant or bioformulated product depends on the potency of microbial strains utilized and their mode and method of formulation used. In a recent study, it was demonstrated that bioformulations that consist of combination of PGPRs including P. flourescences Pf1, Bacillus subtilis Bs10 and biocontrol fungus T. viridae (Tv1) were found to be effective in reducing the incidence of peduncle blight under green house conditions (85.50%) (Durgadevi et al., 2018). Naraghi et al. (2018) suggested the biocontrol of important fungal pathogens including Rhizoctonia solani, Fusarium oxysporum, and Verticillium dahliae, using Nano formulations of antagonist fungus Talaromyces flavus. Among the fungal bioinoculants most of the registered bioformulated products employ species of T. harzianum, T. asperellum, T. gamsii, Coniothyrium minitans, Aspergillus flavus, and Chondrostereum purpureum (Auld, 2002).

8.5  Formulation and application methods 8.5.1  Solid Bioformulation Basically, the two most common types of bioformulations used are liquids and solids (Burges and Jones, 1998). However, modifications of these two types are frequently used nowadays all over the world. The most common type of solid bioformulations used includes granules (GR), micro granules (MG), wettable powders (WPs), waterdispersible granules (WDG), and dusts (Abadias et al., 2005; Guijarro et al., 2007a; Larena et al., 2003). Granular bioformulations include dry particles with active ingredient, carrier, and binder. Based on particle size coarse particles (100–100 µm) and MG (100–600 µm) bioformulated products are used. The concentration of active gradients in GR is 5%–20% (Brar et al., 2006). The most commonly used substrates for GR formation include cornmeal baits, wheat GR (Navon, 2000), GR formed with gelatinized cornstarch or flour (Tamez-Guerra et al., 1996), gluten (Behle et al., 1997), cottonseed flour and sugars (Ridgway et al., 1996), sodium alginate

208

Microbial Endophytes

(Guijarro et al., 2007b), gelatin or acacia gum (Maldonado et al., 2002) and diatomaceous earth (Batta, 2008). The wheat-based granular bioformulation of deleterious rhizobacterium Pseudomonas trivialis X33d (Mejri et al., 2013) and granular formulation of Serratia entomophila (sold under the trade name BioShield) has been reported (Young et al., 2010). Apart from GR-based formulation WPs have prolonged shelf life and generally contain 50%–80% technical powder, 15%–45% filler, 1%– 10% dispersant, and 3%–5% surfactant by weight (Brar et al., 2006). Some of the most common commercialized Trichoderma bioformulations are good example of WPs (Woo et al., 2014). Similarly, postharvest disease control was achieved with WP containing 60% B. cereus freeze dried powder, and 4% carrier as diatomite, 6% alkyl naphthalene sulfonate as wetting agent, 4% sodium lignin sulfonate as disperser, 1% K2HPO4 as stabilizer, and 0.1% β-cyclodextrin as ultraviolet protectant. In contrast, dust type bioformulated products have very finely ground mixture of the active ingredient (usually 10%) with particle size ranging from 50 to 100 µm. The bioformulated dust containing nonpathogenic F. oxysporum is sold under the trade name Biofox C and used in biocontrol of tomato, basil, carnation, and cyclamen (Kaur et al., 2011). The other types of bioformulated products are the modification of the types and includes WG/WDG that make WP more environmental friendly, nondusty, and quickly soluble in water but WG and WDG have higher concentration of dispersing agent. The WDG have excellent shelf life and have been widely employed against control of nematodes. Falk et al. (1995) reported the WDG bioformulated spores of antagonist fungus Ampelomyces quisqualis and used frequently for biocontrol of powdery mildew disease.

8.5.2  Liquid formulations Liquid formulations are aqueous suspensions made in oil, water, or combination of both oil and water (emulsion) (Schisler et al., 2004). Basically, a typical aqueous formulation contains 10%–40% microorganism, 35%–65% carrier liquid (oil or water), 3%–8% surfactant 1%–3% suspender, and 1%–5% dispersant, ingredient. The liquid formulations are available in the form of suspension concentrates (SCs), Oil miscible flowable concentrate (OF), ultralow volume (ULV) suspension (SU) and oil dispersion (OD). Among all these SCs are formulated by adding solid active ingredient with least water solubility and stability to hydrolysis (Tadros, 2013). OF is stable suspension of active ingredient(s) in a fluid intended for dilution in an organic liquid before use (Singh and Merchant, 2012). ULV SU is prepared formulations delivered through ULV equipment in the form of extremely fine spray (Singh and Merchant, 2012). In contrast, OD is a stable suspension of active ingredient(s) in water-immiscible solvent or oil (Michereff et al., 2009). Recently, spores of T. asperellum bioformulated on soybean oil-based carrier as OD was used to control cacao black pod disease. Many other Trichoderma-based liquid formulations are currently available and used in biocontrol, including Trichojet, Enpro-Derma, and Trichorich-L (Woo et al., 2014). It has been reported that oil-based bioformulations delivered in the form of foliar spray are useful and effective in enhancing the activity of entomopathogens (Feng et al., 2004).

Microbial bioformulation-based plant biostimulants

209

8.6  Microbial bioformulation-based plant biostimulants Microbial bioformulation-based biostimulants like various species of PGPRs (Azotobacter, Azospirillum, and Rhizobium spp.) and fungal biostimulants like Trichoderma spp. and various endophytic fungi including mycorrhizal fungi have been considered as promising means not only to secure crop productivity under nutrient challenged conditions (i.e., N and/or P deficiency) but also to avoid other environmental concerns (both abiotic and biotic stresses) (Rouphael et al., 2015; Ruzzi and Aroca, 2015). In fact, several studies done so far have demonstrated that PGPR and endophytic communities occupy several niches in the rhizospheric soil, and therefore modulate the microbial diversity in multidimensional manner both qualitatively and quantitatively, which results in positive impact on soil ecosystem (Fiorentino et al., 2018; Lace et al. 2015; Ruzzi and Aroca, 2015).

8.6.1  Bacterial-based plant biostimulants Interaction of bacteria with plants occurs by various means and ways possible. In general, most common way of association extends from soil to cell interior either transiently of predominantly within the rhizosphere (Colla et al., 2015). These associations of certain bacteria have been shown to influence several of the plant metabolic processes, ion homeostasis, promote water holding capacity, strengthening antioxidant defense system, and prevention of nucleic acid damage thus improving plant growth and productivity (Halpern et al., 2015). In agriculture, these bacterial biostimulants are now increasingly being exploited for enhancing plants abiotic stress tolerance and yield under adverse climatic condition (Calvo et al., 2014). Bacterial-based plant biostimulants are the combinations and formulation of compounds and several PGPRs that facilitate nutrition acquisitioning ability thus promoting better germination, root development, overall crop vigor, and stress tolerance against various biotic and abiotic factors (Seiber et al., 2014). Biostimulants are multifunctional ecofriendly formulations by restricting the use of chemical fertilizer and playing important role in the improvement of fruit color, quality, seed setting, enhancing soil fertility, enhancing nutrient assimilation, and translocation (Katiyar et al., 2015). From agricultural perspectives, these bacterial biostimulants are also termed as “beneficial bacteria,” which has been taxonomically, ecologically, and functionally characterized into two types: (1) Rhizobium like mutualistic endosymbionts and (2) mutualistic PGPRs, which are also generally known as biofertilizers (Calvo et al., 2014). These PGPRs are now intensely used as “plant probiotics” that contributes to plant morphogenesis, development, nutrition, as well as stimulate innate immunity of plants thriving under different agroecosystems (Halpern et al., 2015). Furthermore, PGPRs-based biostimulants have been considered to be easy-to-use agroecological tools for the stimulation of plant growth, nutrient uptake, and amelioration of abiotic stresses (Walker et al., 2012). Some PGPR-based biostimulants have strong biocontrol activities and have been reported to show beneficial association with host (Bhattacharyya and Jha, 2012). The bacterial genera that has the potential to act as biostimulants are Bacillus, Pseudomonas, Azospirillum, Azotobacter, Bradyrhizobium, and Rhizobium (Table 8.2),

Name of microorganism/strain

Plants/vegetable crops Effects on crop plant/vegetables

Rhizobium, Mesorhizobium, Bradyrhizobium Tomato, carrot, lettuce Azospirillum lipoferum, Azotobacter chroocccum, and A. brasilense Rhizobium tropici CIAT899

Cucumber lettuce, maize Bean

Pseudomonas sp. P. aeruginosa, fluorescens, P. putida, Pseudomonas sp. Pseudomonas aeruginosa

Broccoli, lettuce, cucumber Wheat

Burkholderia vietnamiensis AR112

Rice

Wheat, maize, pepper Bacillus cereus, B. amyloliquefaciens, Bacillus subtilis, B. megaterium Bacillus sp. Wheat Arthrobacter sp. and B.subtilis

Tomato

P. jessenii, P. synxantha, and a local AM fungi

Wheat

Increase in P and N amounts up to 40% and 42%, respectively, in soil. Nodule number enhanced by 70% and nodule mass by 43%. Plant shoot dry weight increased by up to 24% and root growth by up to 48% Enhanced nutrient acquisition, tolerance to high temperature stress and increase productivity Improved N and P uptake. Increase in leaf chlorophyll amounts and plant biomass under Zn stress (enhancement of antioxidative enzymes, ascorbic acid and total phenolics) Increased or equivalent weight and yield of traditional rice compared with 100% N chemical fertilization Enhanced drought tolerance, improving root length, and crop yield Increased plant tolerance to salinity. Plant dry weight increased up to 26% and 40% under 2 dS.m-1 and 6 dS.m-1 salinity level, respectively Inoculation of PGPR and AM together reduced fertilizer use by 25%. Combination was equivalent to 100% fertilizer application for plant growth, yield and nutrient uptake PGPR or AMF alone increased yield by up to 29% and 31%, respectively. Combining PGPR and AMF increased the yield by up to 41%

References Ghosh et al. (2015) Mangmang et al. (2015) Tajini et al. (2012)

Tanwar et al. (2014) Islam et al. (2014)

Araújo et al. (2013) Lim and Kim (2013) Upadhyay and Singh (2015) Adesemoye (2009) Mäder et al. (2011)

Microbial Endophytes

Bacillus amyloliquefaciens IN937a, Bacillus pumilus T4, Glomus intraradices

Increase tolerance to salinity stress, enhances nutrient uptake, and crop yield Increase germination, root length, nutrient assimilation

210

Table 8.2  List of promising plant growth-promoting rhizobacteria and other beneficial microorganisms as biostimulants with their potential benefits and effect on crop plants/vegetables for the reduction of chemical fertilizers.

Microbial bioformulation-based plant biostimulants

211

as members of these genera are genetically evolved to thrive under climate extremes by altering their metabolism, solute concentrations and accumulating high level of solutes thus increasing their tolerance (Philippot et al., 2013). These PGPRs when equipped high level of IAA and inoculated in soils can ameliorate plant’ abiotic stress induces oxidative damages (Calvo et al., 2014) by stimulating EPS production, which strengthen osmotic potential around the roots (Ferri et al., 2014). The role of PGPRs in the mitigation of plant's abiotic stress tolerance has been reported for salinity stress, temperature extremes, and moisture deficit for, for example, treating maize plants with Azotobacter strains has been shown increase K+ uptake and decrease Na+ levels thus improving plants growth against salinity stress (Olivares et al., 2015). Similarly, the inoculation of Azotobacter strains improves the ability of wheat plants to tolerate salinity stress by increasing membrane integrity, solute concentrations, and grain yield (Seiber et al., 2014). Inoculating soil with Rhizobium strain, namely, GRA 19, and GRL19, to two legumes plants resulted in the formation of larger nodules thus increasing nitrogen fixation process under salt stress. Similarly, Azospirillum brasilense have also been observed to increase the nodulation of chickpea plants thus improving the process of nitrogen fixation providing greater tolerance against salinity stress (Gozzo and Faoro, 2013). Tomato and sweet pepper plants treated with A. brasilense and P. dispersa showed enhance accumulation photosynthetic pigment content, secondary metabolites, and antioxidative enzymes under NaCl stress (Seiber et al., 2014). Despite of their positive and beneficiary effect on stimulating plants growth and productivity under adverse condition by improving primary or secondary metabolism, till date none of the bacterial biostimulants have been reported to improve physiological responses of plants under fluctuating environment (Corte et al., 2014). Therefore, detailed structural and functional characterization of these bacterial biostimulants is required to develop a comprehensive and systematic approach to understand their response on plant physiological processes under changing environmental condition. Furthermore, several bacterial biostimulants have also been reported to increase nutrient acquisition by formulating them with biofertilizers or some other inoculants need to be optimized as successful exploitation of bacteria as biostimulants require equal coordination of plant, biostimulants, and environment (Rose et al., 2014), which also need to properly addressed before use (Olivares et al., 2015). Meta genomics study of can be employed to answer above question by deciphering synergistic/antagonistic properties of bacterial biostimulants for developing plants specific bioformulations targeting desired trait for improving plants growth and productivity. Bacterial biostimulants have become indispensable tools for ensuring food and nutritional security leading to the development of sustainable agriculture by restricting the use of chemical fertilizers, which are also one of the causes of environmental pollution. Nonetheless, better understanding of mode of application, use and type of biostimulants for specific plants are required for their largescale formulation, production and application in vegetable production.

8.6.2  Fungal-based Biostimulants Several beneficial fungi have also been reported to have mutualistic association with plant roots in distinct ways, that is, from mutually beneficial relationship to

212

Microbial Endophytes

parasitism (Behie and Bidochka, 2014). Several studies have reported that both plant and fungi have coevolved with the evolution of terrestrial plants thus establishing the notion that parasitism-mutualism continuum is beneficial to improve ground and surface water contamination thus increasing crop growth and productivity (Johnson and Graham, 2013). The major fungal biostimulants comprised of mainly Trichoderma spp. and mycorrhizal fungi that have been known to enhance nutrient uptake and improve plant's growth in sustainable and environmentally manner, leading to enhance productivity of crop plants (Johnson and Gilbert, 2015). Apart from their growth promoting role, fungal biostimulants specially Trichoderma spp. have gained much importance as plant microbial biostimulant in agriculture owing to its multifunctional role in controlling adverse effect of biotic and abiotic stresses and their responses in vegetable crops (Simard et al., 2012). Trichoderma spp. have been reported to counteract the adverse effect of various plant pathogens, which include phytopathogenic fungi and certain bacterial diseases and apart from these Trichoderma strains have also proven their biostimulatory function by plant promoting growth and developmental processes under extreme environments leading improved nutritional quality and yield (Candido et al., 2013, 2015). Plethora of research have reported that Trichoderma spp. exert its biostimulatory function by improving the mechanism of root to shoot communication. They have also been known to stimulate plant growth and productivity by stimulating the biosynthesis of several phytocompounds such as indole-3-acetaldehyde, indole-3-ethanol, as well as other volatile compounds such as indole-3-carboxaldehyde. All these phytochemicals have been known to possess auxin like activity that enhances nutrient acquisition, assimilation, and solubilization thus improving overall plant architecture, that is root, shoot, and branching hence booming agricultural productivity (Colla et al., 2015). Mycorrhizal fungi belong to heterogenous group of taxa also known to have symbiotic association with more than 90% of the plants species (Sarkar et al., 2015). Among these heterogenous group, Arbuscule Forming Mycorrhiza (AMF) are the most common type of endomycorrhiza associated with most of economically important agricultural crops, which form the mutualistic association by penetrating their fungal hyphae in the root cortical cells of plants thus forming highly branched structure known as arbuscules (Candido et al., 2013). Increasing evidences have reported about the widespread benefit of AMF in promotion and development of sustainable agriculture by improving plant nutrient uptake ability, maintaining ion homeostasis, and improving plant growth and development under various biotic and abiotic stress condition (Colla et al., 2014). In the past recent years, several researchers have pinpointed beneficial role of AMF in strengthening agricultural productivity around the world and also reported that it is the AMF hyphal network which is responsible to stimulate their biostimulatory function as well as facilitating the interconnection of individual plants with other plants with in plant community (Behie and Bidochka, 2014). Beneficial fungi and their products are being widely used to promote plant growth and productivity by improving their nutrient assimilation rate, increasing their tolerance to several biotic and abiotic stresses. However, major limitation that makes their exploitation bit difficult on large scale is their biotrophic nature and lack of proper understanding of

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213

factors determining the true nature of population dynamics and host specificities for specific agroecosystems (Colla et al., 2015). Nonetheless, apart from AMF, other fungal biostimulants such as Trichoderma spp. (Ascomycota) and Sebacinales (Basidiomycota) have also been known to colonize the roots of several plants and they exert this function without coming in close contact with plants thus improving plant nutrient uptake and modulating their antioxidant defense mechanism with currently unknown mechanism (Mukherjee et al., 2012). Both Trichoderma spp. and Sebacinales are receiving due attention both as fungal formulations enhancing plant growth and productivity and as model organism for deciphering mechanism by which they exert these functions (Nicolás et al., 2014). Among all the sources of fungal biostimulants Trichoderma spp. are well characterized and have been extensively used for their biopesticidal and biocontrol abilities for enhancing innate immunity of plants against various biotic and abiotic stresses. The Trichoderma spp. have also been commercially exploited by various biotechnological industries as valuable sources of enzymes (Nicolás et al., 2014). Therefore, to stimulate the interaction of beneficial fungi with diverse plants and to exploit them to their full potential more crop plants should be treated with microorganism by practising better crop management practises. Metagenomics have emerged as a promising tool, which can help reap all the benefits of beneficial fungi by employing this technique to develop novel strategies for sustainable agriculture by reducing the use of chemical fertilizer, thereby maximize crop growth and productivity.

8.7  Mechanisms implicated in plant biostimulatory effects on crop productivity With the advent of modern biotechnological tools, it has become much easier to understand plant physiological changes; however, most these achievements are mainly restricted to model plants and under control environments (Przybysz et al., 2014). A newer challenge is now to strategically used these knowledges and understanding for detailed structural and functional annotation of biostimulatory effect of these beneficial bacteria and fungi (Fig. 8.1) on different agronomically important plants using modern meta and functional genomics approaches (AAPFCO, 2012). Among various techniques, the upper hand high-throughput plant phenomics techniques have developed for characterizing mutants produced in plants via interaction with fungal or bacterial biostimulants and have increased our understanding their mode of action on different plant genotypes thriving under substandard environmental conditions (Bahadur et al., 2017). Therefore, to bridge the gap between the biostimulatory effect of beneficial microorganism and their mode of interaction with plants implication of combining laboratory data with field data is an important prerequisite for understanding their role growth-promoting role (Fig. 8.2). In the past recent years, the biostimulants have been developed using traditional pharmacological approach, which involve the screening of potential microorganism following a systematic procedure involving both laboratory and field assessments

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Microbial Endophytes

Figure 8.1  Research and development strategies for bioformulation technology.

Figure 8.2  Key biostimulatory mechanisms targeted by beneficial bacteria and fungi upon interaction with plants.

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(Dotaniya et al., 2016). This method of development of biostimulants is much more efficient but this rigorous mode of selection results in high cost value which become difficult to sell in the market (Ghosh et al., 2015). Another way of developing biostimulants involves the collection of field data by observing them and their biostimulatory effect on different plants and then performing their systematic characterization under laboratory condition. As for an example, several soil scientists, and ecologists have first recorded the detailed field observation of different beneficial microorganism about their mode of interaction and they regulated growth and development of maize plants along with growth and compositions of bacterial or fungal populations upon interaction (Jaiswal et al., 2016). Such observations can provide the first step toward successful understanding of interaction of PGPRs with plants. Furthermore, it may also facilitate our understanding the effect of inter or intra specific variation of plants and the effect they produce upon interacting with different PGPRs. Several economical approaches are being developed and are commercialized that will facilitate the standardization and use of local microbiota instead of inoculating plants with synthetic microbial products. This mechanism will bridge the gap when the capacity of microbial biostimulants become limiting to maintain sufficient activity of beneficial microorganism with in the rhizosphere (Kumar et al., 2016a, 2016b). In the present era, the large-scale application of beneficial biostimulants will require local and temporal adapted tools for monitoring efficiency of biostimulants in terms of interaction, long term effects, effect on environment, and biological cycles should be taken into consideration in the decision-making process on the field and landscape levels. In the developing countries like India, several other factors such as availability of the products at proper time also influences successful adaptation of this technology, Moreover, emphasis should be made on the demonstration of beneficial effect of biostimulants by organizing agricultural extension programs to convince several low-level farmers by ensuring them good returns and crop productivity.

8.8  Current Scenario/Market Trends For successful marketing of bioformulated products that leads into commercialization, it is important to consider the type of bioformulated product as the market demands the product that has easy application with maximum efficacy. Microbe-based formulations for plant growth promotion, disease suppression are widely used across the globe (Gašicand Tanovic,́ 2013; Leggett et al., 2011; Naderifar and Daneshian, 2012). ́ However, one important factor that restricts user to use bioformulated products is discrepancy used in their naming. Both “biofertilizer” and “bioinoculants” products use living organisms (microbes) and their derived active compounds for plant growth promotion, nutrient uptake, and enhancing the crop productivity. However, most of the developed countries employ biofertilizer and biopesticides for enhancing the crop productivity for different crops. According to one report, it was found that the market of biostimulant was mainly located in Europe and was projected to have an annual increase of 12% and reaching $2241 million by 2018 (Calvo et al., 2014). European

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biofertilizer market is the most developed and widespread among all the regions and is expected to grow from around $2566.4 million in 2012 to $4582.2 million by 2017, at a calculated annual growth rate of 12.3%, from 2012 to 2017. It was reported that in India, about 151 biofertilizer production units are operated by government and nongovernment agencies (Mahajan and Gupta, 2009). The only nitrogen fixing biofertilizers represented 78% of the global demand in 2012 (Agro news, 2014). Furthermore, Bacillus thuringiensis (Bt)-based biopesticides covered 95% of the total microorganism used and were reported to be most successful biopesticides Bravo et al., 2011). It was estimated that about annual revenue of $210 million was generated from selling from 322 Bt products sold across the globe. Overall, among all the bioformulated products particularly, biopesticides, Europe has the fastest growing regional market for biopesticides showing annual average growth rate of 15.0% (Industrial Equipment News, 2011) and the United States represents the largest region of biopesticides worldwide.

8.9  Regulatory framework Today, the regulatory situation of biostimulants is very complex. The most common reason behind this is the lack of public awareness, use of technical terms and/or lack of proper definition for biostimulants, and most likely lack of acceptance of the concept of the biostimulants by regulatory bodies. From a regulatory point of view, there is no agreement globally over the definition of PBs and many EU and nonEU countries lack a specific legal framework (Caradonia et al., 2018; Rouphael et al., 2018; Yakhin et al., 2017). The two major routes through which biostimulants are marketed in Europe is either the national regulation on fertilizers or the other route including European pesticide law, which follows both supranational and national provisions for introducing plant protection products in the market. However, the regulatory status of biostimulants is indeed diverse, depending on whether or not they have been registered under the REACH regulation (EC No. 1907/2006 concerning the registration, evaluation, authorization, and restriction of chemicals). The regulatory status of biostimulants is indeed diverse, depending on whether or not they are registered under the REACH regulation, as fertilizing materials under national laws, as pesticides under European legislations, authorized or not in organic productions, etc. However, REACH regulation mainly covers chemicals and excludes microorganisms, which represent important group of biostimulants. In Europe, however, EC regulation No. 1107/2009 on plant protection products (‘PPPs’) has been used for plant biostimulants of multiple categories, which defines them more broadly. Several beneficial microorganisms including Trichoderma spp. have been registered as PPPs and classified as microbial biological control agents (Woo et al., 2014). The supplementation of host plants with Trichoderma inoculation have added advantages like assisting host plants in nutrient uptake and release, inducing systemic resistance in hosts, promotion of root and shoot growth (promotion of plant growth), resistance against various abiotic and biotic stresses, and other beneficial effects (Lorito and Woo, 2015), Although,

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many strains of this beneficial fungi have been employed in commercial bioformulation for plant disease control as biocontrol and have been registered as biopesticides still many Trichoderma spp. typically have not been indicated in the registered product disclaimer. Conversely, plant biostimulants like arbuscular mycorrhizal fungi (AMF; Rouphael et al., 2015), which are also capable of inducing systemic resistance conferring crop protection to disease and pest attack (Cameron et al., 2013). This concludes the fact that we should develop a new registration track for beneficial microbes, having multiple plant beneficial functions that are utilized either in single treatment or in microbial consortia in order to regulate the use of effective agricultural products that are “all inclusive” (e.g., biostimulant, biofertilizer, and biopesticide). Recently, the EU has decided to reformulate the previously established Fertilizers regulations to facilitate the internal marketing operations for products classified as fertilizers and also has provided a common legal framework for PBs currently fragmented across member states (Caradonia et al., 2018; Rouphael et al., 2018). Accordingly, under the newly developed regulation, “plant biostimulants will be CE marked as fertilizing products that stimulates the plant nutritional aspects and processes independently of the products’ nutrient content with the sole aim of improving one or more of the following characteristics of the plant and the plant rhizosphere or phyllosphere: amelioration of abiotic stresses, nutrient use efficiency, improving crop quality, and product yield, availability of confined nutrients in the soil and rhizosphere, humification and degradation of organic compounds in the soil.” PBs are thus to be defined-based on their proposed effects, or more correctly, “by the plant response they elicit rather than by their makeup,” since the group includes diverse inorganic and organic substances and/ or microorganisms, including protein hydrolysates, humic acids, seaweed extracts, PGPRs, endophytic fungi, N-fixing bacteria, and mycorrhizal fungi (du Jardin, 2015; Rouphael et al., 2018)

8.10 Conclusion From the point of successful marketing, the bioformulated product is not effective until it does not have good impact in field condition, reliability, and cost effectiveness. The entire bioformulation process is not only dependent on microbes utilized, their potential applications, functional diversity, physiological attributes, and response mechanism but also other factors determine the success of the formulated products including formulation type, fermentation processes, microbial population ad delivery system. It has been found that during fermentation processes several factors including media employed in production processes, oxygen transfer, concentration of constituents, incubation temperature, harvesting time and treatment done after harvesting all determine and affect the formulation development. Apart from fermentation processes, microbial delivery is an important factor that greatly influences the bioformulation market as microbial delivery system defines the utility of the bioformulated products, as sustainability of the product is highly dependent on delivery system. Although significant progresses have been made in this field, still we have not succeeded in producing

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such an elite formulation, which has broad spectrum activity and also counteracts the economic challenges. At present, for successful development of bioformulation we need to exploit the microbial machinery at its full potential and molecular mechanism involved in regulating their genetic flexibility in diverse environment with the collective effort of both microbiological and biotechnological aspects. Besides this we need to investigate and reevaluate the entire bioformulation processes at each and every step to make it more convenient, reliable, productive, and plugging of the loop holes.

Acknowledgments M.A. is thankful to the Indian Council of Medical Research (ICMR), New Delhi for research facilities in the form ICMR-Junior Research Fellowship and ICMR-SRF. We finally acknowledge to the Head, Department of Botany, Institute of Science, Banaras Hindu University for providing infrastructure facilities for this work. Author Contributions Statement M.A. conceived the idea and drafted the outline of the chapter. M.A. wrote the entire chapter KKR assisted in writing some sections of the chapter. A.Z., M.K.D., S.K., and V.S. contributed in writing some sections and also helped in reviewing the finally prepared chapter. R.S.U. provided necessary supervision and guidance during chapter preparation supervised the whole work. All authors finally approved the chapter for publication. Conflict of Interest None of the authors involved in this manuscript has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of this report. Weblinks http://www.biagrosa.com.ar http://www.bioagri.se http://www.flozyme.com/agriculture http://mabiotec.com www.agribioticproducts.com www.agrilife.in www.aurigagroup.com www.fertibio.com www.labiofam.cu www.novozymes.com www.symborg.com

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Genomic insights of plant endophyte interaction: prospective and impact on plant fitness

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Tejas C. Bosamiaa, Kalyani M. Barbadikarb, Arpan Modic a Biotechnology Lab, ICAR-Directorate of Groundnut Research, Junagadh, Gujarat, India; b ICAR-ICAR-Indian Institute of Rice Research, Hyderabad, Telangana, India; cAgricultural Research Organization, Volcani Center, Israel Chapter outline head 9.1 Introduction  227 9.2 Microbial diversity  229 9.3 Omics–unraveling plant–endophytic interaction  230 9.4 What makes microbes an endophyte?  234 9.5 Plant fitness: Plant–endophyte interaction  234 9.5.1 Genes involved in nutrients acquisition  234 9.5.2 Genomics aspects of mitigation of abiotic stress tolerance by endophytes  237 9.5.3 Genomics of alleviating biotic stress in host plants by endophytes  239

9.6 Perspectives: a way ahead  241 References  241

9.1 Introduction The plant is a habitat for diverse microorganism, commonly referred to as microbiota and these microbiota comprises of all the beneficial, harmful, and neutral microbes. Majority of the microbial community are living on nourishment provided by the plant without causing any harm to the host, such microorganisms are known as commensal microorganisms (Brader et al., 2017). The microbes are found either at outer surface, namely, rhizosphere or phyllosphere or in the inner surface of the plant and on the basis of inner and outer localization, microbe could be defined as endophyte and epiphyte, respectively. Endophytes are the microorganisms which can be isolated from surface sterilized plant tissue without causing any symptom of the disease (Hardoim et al., 2008). In this chapter, we have considered endophytes as microbes that reside in the plant and remain asymptomatically at least a part of their life cycle. Microbes are the major driving force for fundamental metabolic processes of soil and plant (Nannipieri et al., 2003). The fate of the microbes whether colonized as endophytes, epiphyte, or become pathogen depends on many factors such as genomic constitMicrobial Endophytes. http://dx.doi.org/10.1016/B978-0-12-819654-0.00009-0 Copyright © 2020 Elsevier Inc. All rights reserved.

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uent of microbes, host genotype, environmental conditions, and microbial interaction (Brader et al., 2017). Plant species without microbial endophytes are difficult to find in nature and plants with endophytes have shown more sustainability under environmental stress (Partida-Martinez and Heil, 2011; Timmusk et al., 2011). However, there is still a gray area in terms of identification and classification of microbial species in several ecological niches. The endophytes play a major role in the growth, development, and health of the plant by promoting the production of phytohormones, nutrition uptake, and the ability to cope up with biotic and abiotic stresses (Fig. 9.1). Microbes gain entry into plants using different ways including tissue wounds, stomata, lenticels, root cracks, and germinating radicles (Ali et al., 2014a). Microbial endophytes have a position of advantage over rhizospheric microbes since they colonize inside the plant and interact with a host more efficiently. It is an interesting area of research to determine the key difference in endophytic and rhizospheric microbes. Ali et al. (2014a) have identified several genes responsible for the endophytic lifestyle of bacteria by comparing the genomes of nine endophytic proteobacteria. The functional capabilities, genomic potential of microbial endophytes, and its relationship with plant has been addressed using the high throughput omics techniques based on next generation sequencing platforms (Muller et al., 2016). The bacterial endophytic mutant strains have been employed for studying the various processes of host-endophyte interaction. Alquéres et al. (2013) have shown that reactive oxygen species (ROS)-deactivating genes (superoxide dismutase and glutathione reductase) are important during the initial stages of colonization in G. diazotrophicus– rice interaction by deploying the transposon insertion mutants of the SOD and GR genes of

Figure 9.1  Schematic representation of briefing the role of plant–endophytes interaction in plant fitness. In the figure arrows denote interactions. Abbreviations: IAA, indoleacetic acid; GAs, gibberellins; CKs, cytokinins; ACC, 1 aminocyclopropane-1-carboxylic acid; ET, ethylene; KMBA, 2-keto-4-methylthiobutyric acid; ROS, reactive oxygen species.

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G. diazotrophicus strain PAL5. Molecular mechanisms of functional traits governed by microbial endophytes imparting beneficial attributes to plant health are still not clear. Nevertheless, the omics approaches along with mutational studies have shed light in understanding some of the complex endophyte-host interactions (Pinski et al., 2019). It is interesting to ask how the microbial community acts on the determination of plant phenotypes in a continuous changing environment. How plant microbiota alleviates stresses? How plant–endophyte symbiotic relationship enhances nutrient uptake and promotes growth? In this chapter, we have highlighted the role of endophytes in providing overall fitness to the host plant and genomic insight of plant–endophytic association in a multi-variant environment. We have also focused on the selection of defined microbial community as an eco-friendly and green solution for developing climate resilient agricultural technologies.

9.2  Microbial diversity Plants are surrounded and hosted by taxonomically diverse microbes. Bacteria come first in term of abundance followed by fungi, oomycetes, algae, protozoa, nematodes, and viruses (Muller et al., 2016). The microbial diversity present in root endosphere is less than in the rhizosphere and bulk soil (Liu et al., 2017). The number of bacterial population in the endosphere per gram tissue is also less as compared to the bacterial cell present in the rhizosphere (Bulgarelli et al., 2013). Host plants stringently select the specific community of microbes from soil to colonize as endophytes. Proteobacteria (∼50% relative abundance) is the most abundant phyla followed by Actinobacteria (∼10%), Firmicutes (∼10%), and Bacteroidetes (∼10%) (Liu et al., 2017). Among the three classes of proteobacteria, γ-Proteobacteria is most diverse and dominant as compared to α- and β-Proteobacteria. Among phyla actinobacteria, the most abundant species is Streptomyces sp. and Microbispora, Micromonospora, Nocardioides, Nocardia, and Streptosporangium are common genera. On the other hand, Chloroflexi, Armatimonadetes, Acidobacteria, Nitrospirae, Planctomycetes, and Verrucomicrobia are common phyla of bacteria but represent the small fraction of endosphere (Santoyo et al., 2016; Liu et al., 2017). However, the diversity and the predominance of endosphere microbes may depend on host plant species (Bodenhausen et al., 2013; Ding and Melcher, 2016). Mycorrhizal fungi colonization is limited to the plant's root and form a symbiotic association called mycorrhiza; in contrast fungal endophytes colonize and grow in all or any part of the plant. Fungal endophytes are broadly classified into four distinct groups, clavicipitaceous endophytes (class I), non-clavicipitaceous endophytes (class II), class III, and class IV. The class I endophytes are defensive mutualism of host and more common in grasses comprising genera, namely, Balansia sp., Epichloe sp., and Claviceps sp. (Vijayabharathi et al., 2016). Class II fungal endophytes are predominant with vascular and nonvascular plant species (Rodriguez et al., 2009). All class-II endophytes are members of the Dikarya (Ascomycota or Basidiomycota). The association of class-II endophytes has confirmed fitness benefits to plant such as stress tolerance, nutrient acquisition and increased growth and yields (Rodriguez et al., 2009). Class III endophytes are found exclusively in above-ground

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tissues, tropical trees, nonvascular plants, seedless vascular plants, and conifers. They are members of Dikaryomycota (Ascomycota or Basidiomycota), a brown to a blackish-pigmented fungus called as “mycelium radices atrovirens;” and dark septate endophytes are classified as class IV endophytes. Class IV endophytes are mostly asexual filamentous ascomycetes including Helotiales, Xylariales, and Pleosporales. Although class IV endophytes are more frequent in harsh, nutrient-limited arid, and semiarid areas (Knapp et al., 2018).

9.3  Omics–unraveling plant–endophytic interaction With the growing evidence of beneficial plant–endophytic interaction in host plant growth promoting activity (PGPR), alleviating certain abiotic/biotic stress, production of secondary metabolites, it becomes increasingly essential to unravel the molecular mechanism of the interaction. The identification, characterization, and taxonomic classification of endophytes are critical for its unique identity, and potential beneficial use in plant growth and health. The techniques used to study the plant-endophytes interaction has been summarized in Fig. 9.2. Classical molecular characterization relies on the deployment of molecular markers, namely, inter transcribed spacer (ITS), 23S rRNA, 18S rRNA for endophytic fungi, and 16S rRNA for endophytic bacteria. The deployment of omics techniques such as high throughput genomics, transcriptomics, proteomics, and metabolomics can unravel the molecular insights operating under plant–endophytic interaction. Genomics involves the high throughput sequencing of DNA or RNA of the microbial endophyte. Whole genome sequencing involves

Figure 9.2  Methods to access plant–endophytes interactions.

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the sequencing of DNA isolated from a target endophyte on an NGS platform for studying its genes, taxonomy, phylogeny, etc. Many bacterial and fungal endophytes isolates have been sequenced and the information is freely available in the NCBI. The comparative genomic analyses of endophytic bacteria identified conserved and unique genetic determinants of interaction during recognition of root exudates, surface adhesion, biofilm formation, penetration, colonization, etc (Pinski et al., 2019). Metagenomics refers to the DNA sequence-based information of genes and genomes of the endophyte/microbiome. Metatranscriptomics refers to the sequencing total RNA of microbiome, which gives functional or gene expression information. Transcriptomics refers to total RNA sequencing of a specific tissue of the host plant, at a specific given time and conditions. The interaction between colonizing endophyte in host plant can be studied through dual transcriptomics, which involves transcripts of both host plant and endophyte (Gómez-Godínez et al., 2019; Hara et al., 2019). Such dual transcriptomics shed light on the key transcripts involved in symbiosis, colonization, signaling molecules, effector molecules, etc. These techniques have been used in various plant–endophyte interactions to decipher different functions (Table 9.1). A type of transcriptome sequencing (RNA-Seq) is microRNA (miRNA) which is employed to know the role of specific miRNA in host plant–endophytic interaction at a given time and tissue. The miRNAs are small noncoding RNA that functions in posttranscriptional regulation of plant gene expression (epigenetic regulation). Specific miRNAs are reported to play a key role in the compatibility of the host plant with endophytic interaction. The miRNAs have regulatory response to the host plant growth promotion and the miRNA targets genes have been shown to be involved in the recognition of endophyte, perception of molecular signals, colonization, etc. (Thiebaut et al., 2014; Pentimone et al., 2018). Thiebaut et al. (2014) showed the regulatory role of small RNA (by upregulation) during association with endophytic nitrogenfixing bacteria in maize. Microarray is another technique for gene expression analysis, which is chip-based and gives large-scale information on many genes at a single time. It provides a comprehensive picture of the transcriptional activities at a given time facilitating the functional roles of the genes (Lahrmann et al., 2013). Customized microarray chips can be designed and developed to monitor the gene expression at different time points like colonization, interaction, etc., for host as well as endophytic microorganism. The high throughput sequenced data needs to be carefully processed further for appending context-based biological information. Several software, servers, algorithms, programs, platforms, and pipelines are available for accelerating the genomic, functional information like MG RAST, Qiime, etc. (Niu et al., 2018; Almeida and De Martinis, 2018). Proteomics includes the study for identification of the total proteins present in the tissue at a specific time. High throughput proteomic techniques, namely, Mass spectrometry (MS), LC-MS-MS, and MALDI-TOF/TOF are employed for studying the proteins involved in the interaction of host plant and endophyte (Kaul et al., 2016). Such information is useful for the identification of specific protein molecules expressed at that given time. A combined approach of omics tools like genomics with transcriptomics and proteomics is always better than a single approach for unraveling the molecular mechanism of host plant–endophyte relationship.

Target microbiota

Source/Tissue

Omics analysis

Chilli (Capsicum Genome sequencing and annuum), comparative genomics

Epiphytic microbiota

Apple

Metagenomics

Bradyrhizobium species

Sorghum Roots

Metagenomics and Proteomics

Whole plant microbiota

Aloe vera

Metagenomics

Burkholderia phytofirmans PsJN

Potato (Solanum Transcriptomics tuberosum L.) Plantlets

Key finding

References

A rich repertoire of pathogenicity genes encoding secreted proteins, effectors, plant cell wall-degrading enzymes, secondary metabolism-associated proteins, with potential roles in the hostspecific infection strategy, placing it next only to the Fusarium species. Understanding the biology and lifestyle Genome sequence required for designing efficient disease control regimens. Biocontrol strains belonging to genera: Filobasidiella spp., Talaromyces spp., Candida spp., Saccharomyces spp., Bacillus spp., and Enterobacter spp. were found Abundance (2.9%–3.6%) of Bradyrhizobium in the roots. Proteome analysis indicated that three NifHDK proteins of Bradyrhizobium species were consistently detected. Functional N2-fixing bacteria in sorghum roots are unique bradyrhizobia that resemble photosynthetic B. oligotrophicum S58T and non-nodulating Bradyrhizobium sp. S23321. The analyses revealed proteobacteria, firmicutes, actinobacteria, and bacteroidetes as the predominant genera which have been shown to produce beneficial bioactive compounds Transcripts upregulated in response to plant drought stress were mainly involved in transcriptional regulation, cellular homeostasis, and the detoxification of reactive oxygen species, indicating an oxidative stress response in PsJN. The activity of strain PsJN is affected by plant drought stress; it senses plant stress signals and adjusts its gene expression

Rao and Nandineni (2017)

Angeli et al. (2019) Hara et al. (2019)

Akinsanya et al. (2015) Sheibani-Tezerji et al. (2015)

Microbial Endophytes

Colletotrichum truncatum

232

Table 9.1  “Omics” based analysis to revealed plant–endophytes interaction.

Source/Tissue Omics analysis Tomato Metagenomics (Solanum lycopersicum)

Herbaspirillum seropedicae

Wheat (Triticum Transcriptome aestivum)

Azospirillum brasilense

Wheat (Triticum Transcriptome aestivum)

Phyllosphere associated fungi

Norway spruce (Piceaabies)

Root microbiomes

Salix purpurea cv Metatranscriptomics Roots petroleum hydrocarbon contaminated

Phyllosphereassociated bacteria

Echinacea purpurea (L.) Moench

MetaTranscriptomics

Metabolomics

Key finding Root-associated microbiomes in healthy and nematode-infected tomatoes indicated that nematode infections were associated with variation and differentiation of endophyte and rhizosphere bacterial populations in plant roots. The community of resident endophytes in tomato root was significantly affected by nematopathogenesis. Transcriptome comparison of root attached and planktonic bacteria revealed specific adaptations of bacterium such as expression of specific adhesins and cell wall remodeling to endophytic and epiphytic lifestyle. RNA-Seq transcriptional profiling of wheat roots colonized by A. brasilense strain FP2 revealed up-regulation of genes encoding proteins related to bacterial chemotaxi, biofilm formation and nitrogen fixation. The endophytes also enhanced the expression of plant genes related to nutrient uptake, nitrogen assimilation, DNA replication, and regulation of cell division. Transcripts originated from Dothideomycetes and Leotiomycetes species. Functional annotation of gene families indicating active growth and metabolism, with particular regards to glucose intake and processing and gene regulation. In response to contamination, 1745 Basidiomycota transcripts increased in abundance white rot Ascomycota genera (dominated by Pyronema), ectomycorrhizal (ECM) Ascomycota (Tuber), and ECM Basidiomycota (Hebeloma) by a poorly characterized putative ECM Basidiomycota due to contamination. Alkamide biosynthesis may be modulated by the bacterial infection. Plant–endophyte interaction influenced plant secondary metabolism affecting host therapeutic properties.

References Tian et al. (2015)

Pankievicz et al. (2016)

Camilios-Neto et al. (2014)

Delhomme et al. (2015)

Gonzalez et al. (2018)

Maggini et al. (2017)

Genomic insights of plant endophyte interaction: prospective and impact on plant fitness

Target microbiota Root microbiomes

233

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9.4  What makes microbes an endophyte? The establishment of plant–microbial association is a multifactorial mechanism. The changes in microbial endophytes diversity highly depend on the plant's geographic location, host microbes interactions, and environmental interaction (Edwards et al., 2015). The analysis of different plants–endophytes associations has revealed the factors responsible for the prompting of endophytic lifestyle includes host species, genotype, tissues, developmental stage, growing season, geographical location, soil type, host nutrient status, cultivation practice, and fertilization (Liu et al., 2017). The mechanisms of how endophytes penetrate and survive inside plant tissue in order to colonize within are still not fully understood. However, there are two types of lifestyle share by the microorganism, for instance, root microbial endophytes are profoundly influenced by microbial community of soil, only some of the rhizospheric microbes are able to colonize into the plant. Ali et al. (2014a) determined the gene involved in endophytic behavior in Burkholderia spp. In this study, the genomes of Burkholderia spp. (both endophytic and rhizospheric bacteria) were compared and identified the unique gene responsible for the endophytic lifestyle. These genes are further compared with the genome of eight endophytic bacteria to confirm the potential gene involved an endophytic lifestyle. The analysis revealed ∼40 potential genes such as encoding proteins for detoxification, secretion and delivery systems, plant metabolites modification, transcriptional regulation, transporter proteins, redox potential maintenance. All these genes were suggested to have potential involvement in endophytic behavior of bacteria. Similarly, two common dark septate endophytes (DSE) Cadophora sp. and Periconia macrospinosa from the same environment with different host preferences were sequenced and compared with another 32 ascomycetes of different lifestyles to gain insight into their lifestyle (Knapp et al., 2018). The genomic analysis of DSEs revealed higher copies of the genes encoding proteins related to carbohydrateactive enzymes (CAZymes), plant cell wall-degrading enzymes (PCWDE), secreted proteases and lipases, aquaporins, and melanin synthesis (Knapp et al., 2018). The higher copy number of CAZymes and PCWDE domains suggest that fungi are able to break down complex plant polysaccharides. This characteristic of fungi was found to be responsible for the endophytic behavior of different DSE fungi.

9.5  Plant fitness: Plant–endophyte interaction 9.5.1  Genes involved in nutrients acquisition The role of endophytes that facilitate the host plant growth is well understood (Gamalero and Glick, 2011; Glick, 1995, 2012). The plant growth promoting bacteria directly promote the growth either by assisting in the nutrient uptake or by synthesis or modulating the plant hormones (Santoyo et al., 2016). It also facilitates the growth indirectly by checking the colonization of harmful microbes in the host plant (Glick, 2015). The rhizospheric bacteria and endophytic bacteria both have been found to have growth-promoting actions. The rhizosphere is considered as a major

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source of endophytic population in the host plant (Germida et al., 1998; MarquezSantacruz et al., 2010). The mechanism of endophytes to promote plant growth is almost similar to that of microbes in the rhizosphere, the only difference is after colonization, the endophytic bacteria are not able to change the soil condition such as soil pH, temperature, and water content (Glick, 2012; Santoyo et al., 2016). Bender et al., (2016) reported the rich soils with diverse microbes and high organic matter to have less fertilizer requirements than conventionally managed soils. The direct growth promoting attributes of microbial endophytes includes the biological nitrogen fixation (BNF), phosphate solubilization, production of phytohormones, siderophores secretion, ACC deaminase activity, and biocontrol activity (Berg et al., 2015; Card et al., 2016). The BNF in a mutualistic relationship between Leguminosae plant and Rhizobiaceae bacteria have been well studied (Postgate, 1998). This symbiosis has been extensively studied to establish similar associations that might be developed with nonleguminous plants (Oldroyd et al., 2011). Cavalcante and Döbereiner (1988) postulated for the first time that the endophytic diazotroph (Gluconacetobacter diazotrophicus) might be involved in fixing a considerable amount of nitrogen. Since, the discovery of diazotrophic endophytes in sugarcane, many diazotrophs has been found to have association with nonleguminous plant that fix high amount of nitrogen biologically (Puri et al., 2018). The role of diazotrophic endophytes have also been reported in rice (Baldani et al., 2000; Gyaneshwar et al., 2001; Hurek et al., 2002; Araújo et al., 2013; Rangjaroen et al., 2015; Gururani and Chun, 2014), corn (Roesch et al., 2008; Szilagyi-Zecchin et al., 2014), sugarcane (Schultz et al., 2014), and wheat (Sabry et al., 1997; Gupta et al., 2013). The metagenome analysis of rice roots has shown that a vast number of microbial genes are involved in nitrification and ammonia oxidation processes (Sessitsch et al., 2012). The endophytic N2 fixing bacteria have both, nitrogen fixation (nifH) and denitrification genes in their genome (Straub et al., 2013). Similarly, foliar endophytic bacteria with genes involved in nitrogen cycle have confirmed N2 fixation in many sub alpine conifer species (Moyes et al., 2016). The genome and transcriptome analysis of root symbiont Piriformospora indica revealed genes responsible for the nitrogen uptake and transfer. The genome of P. indica contains “N” transporters including urea permease (DUR3), high-affinity ammonium transporters and amino acid-transporters (Zuccaro et al., 2011). The BNF by diazotrophic endophytes may provide low cost, stable N2 fixing strategy for the plant growth promotion in both native and non-native crops. Apart from “N,” phosphorus (P) and iron (Fe) are also essential nutrients required by plants. Phosphorus is found in the soils in both organic as well as inorganic form and iron is found in ferric oxide form. Though the bio-availability of both the nutrients is very low due to formation of insolubilized complexes, several reports indicated that microbes have the ability to liberate organic phosphate or by inorganic phosphates solubilization (Khan et al., 2010). The organic phosphorus is made available by endophytes through secretion of “P” mineralizing enzymes such as phytases, C-P lyases, and phosphatases. On the other hand, microbes solubilize inorganic phosphate by exudation of proton or carboxylate ions such as citrate, malate, or oxalate (Illmer et al., 1995; Chhabra and

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Dowling, 2017). In vitro activity of bacterial and fungal endophytes have been involved in phosphate solubilization by production of organic acid like gluconic acid (GA), malic acid, citric acid, salicylic acid, and benzene acetic acid (Jog et al., 2014; Otieno et al., 2015; Chen et al., 2014). Li et al. (2012) studied perennial grass-fungi (Achnatherum sibiricum-Neotyphodium sp.) association under the “N” and “P” limiting and nonlimiting conditions. It was emphasized that the association between plant and fungi were conditional and depends upon the availability of “N” and “P” in the environment. The fungal endophyte significantly increased the acid phosphatase activity and had greater “P” concentration in the root. Similarly, growth-promoting activity of endophytic fungi P. indica was examined in the maize plant. The endophyte inoculated plant increased biomass 2.5 folds than the non-inoculated plant under “P” limiting environment (Kumar et al., 2011). The transcriptome sequencing of soybean—P. indica association revealed the upregulation of genes related to lignin biosynthesis, regulation of transporter other nutrient acquisition pathway. The genes related to purple acid phosphatase pathway were significantly upregulated in colonized soybean (Bajaj et al., 2018). The potential mechanisms of uptake and transfer of “P” involved solubilize inorganic “P” by secretion of organic acids and transfer through a high-affinity phosphate transporter (PiPT). Ngwene et al., (2016) emphasized the role of four different PiPTs and two different acid phosphatases genes of P. indica in the regulation and uptake of “P” at different concentration of inorganic “P.” The study indicated that the uptake and transfer of “P” could involve either plant or fungi or both genes. In the case of iron acquisition, microorganisms producing siderophores play a major role. The siderophores are the compounds that chelate iron present in soil and make soluble complexes, which are directly absorbed by the plant. Around 500 different types of siderophores produced by various microorganisms have been reported (Ahmed and Holmström, 2014). The metagenomic analysis of the endophytic community of rice root revealed that a large number of genes are involved in siderophore biosynthesis, siderophore reception, and iron storage (Sessitsch et al., 2012). The endophytic and epiphytic microbes could change morphological and/or physiological responses of dicot plant species toward “Fe” deficiency (Romera et al., 2019). For instance, some endophytes and rhizobacteria induced the iron acquisition machinery in Arabidopsis by volatile organic compounds, independently of iron availability, and require a photosynthesis-related signal (Romera et al., 2019). Direct promotion of plant growth occurs when microbes facilitate the modulation of hormone levels by synthesizing one or more of the phytohormones auxin, cytokinin, and gibberellin. Endophytes belonging to genera Acinetobacter, Enterobacter, Pantoea, Pseudomonas, and Ralstonia could promote the growth by one or more mechanism such as phosphate solubilization activity, phytohormone, and siderophore production (Sobral et al., 2004; Loaces et al., 2011). Studies in plant endophyte interaction have identified genes responsible for biosynthesis pathway of phytohormones, namely, indole acetic acid (IAA) (Zúñiga et al., 2013), cytokinins (CKs) (Bhore et al., 2010), and gibberellins (GAs) (Shahzad et al., 2016). Sorty et al. (2016) reported the enhanced seed germination of wheat by IAA producing strain Enterobacter sp. NIASMVII from halo-tolerant weed. Several IAA producing bacteria have improved plant growth under nutrient-poor soil condition. The Serratia sp. from chickpea

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increased the grain yield of chickpea in nutrient deficits soil (Zaheer et al., 2016). The endophytic fungi Aspergillus fumigates influenced the endogenous phytohormones and also produced several exogenous gibberellins, which increased photosynthetic pigments and shoot biomass of soybean under salt stress (Khan et al., 2011). The GAs and IAA producing endophytic bacterium Sphingomonas sp. LK11 enhanced tomato growth (Khan et al., 2014). The production of ethylene (ET) is a common response of the plant to stress which may retard the growth of plant at higher concentration (Glick, 2014). 1-Aminocyclopropane-1-Carboxylate (ACC) is an immediate precursor of ET production in the plant. Several endophytes have the ability to use ACC as a carbon and nitrogen source by producing ACC deaminase (Karthikeyan et al., 2012; Ali et al., 2014b; Glick, 2014). Endophytes with ACC deaminase activity may protect the plant from the deleterious effects of stress condition by reducing the endogenous ACC level in the plant (Glick, 2014). A recent study found that ACC deaminase expressing endophyte Pseudomonas spp. reduced the stress-related ET and promoted the plant growth, leaf water contents, photosynthetic performance, and ionic balance of tomato plants (Win et al., 2018). A strain of Bacillus subtilis LK14 isolated from medicinal plant Moringa peregrine possessed phosphate solubilization, ACC deaminase and acid phosphatase activity that increased the shoot and root biomass and chlorophyll (a and b) contents of tomato plant upon inoculation (Khan et al., 2016). Such phytohormone producing endophytes can be isolated, characterized, and standardized for its dose of application in various crops for increasing crop productivity. Such endophytes can be commercialized as liquid formulation or in the form of capsules for application to the standing crop at a specific stage of life cycle.

9.5.2  Genomics aspects of mitigation of abiotic stress tolerance by endophytes The role of microbial endophytes to alleviate abiotic stresses in plants has been the area of great concern (Kasotia and Choudhary, 2014; Meena et al., 2017). Microbes had metabolic and genetic intrinsic potential to provide tolerance against abiotic stresses in the plants. Abiotic stresses such as drought, salinity, oxidative stress, temperature's stress, and heavy metal stress are a major constraint of agro ecosystem (Khare et al., 2018). The stress tolerance mechanisms employed by microbial endophytes in the plant include induction and regulation of stress-responsive genes, generation of ROS scavengers and production of anti-stress metabolites (Lata et al., 2018). Recently, Pandey et al. (2016) have reported Trichoderma harzianum colonization in rice significantly alleviated drought tolerance by modulating proline, SOD, lipid peroxidation product, and upregulation of aquaporin, dehydrin, and malondialdehyde genes. Similarly, Trichoderma harzianum application in NaCl affected Indian mustard has improved the accumulation of ROS-scavengers and osmolytes to nullify the adverse effect of salinity and decreased the NaCl uptake (Ahmad et al., 2015). Sheibani-Tezerji et al. (2015) performed a transcriptome analysis of B. phytofirmans PsJN colonizing potato in response to drought stress. The tolerance response to drought stress included upregulation of transcripts related to transcriptional regulation, cellular homeostasis, and the detoxification of ROS. The study also emphasized the expression

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of cell surface signaling element of PsJN was modulated to sense of changing environmental conditions and regulated stress-related metabolites accordingly. Similarly, PsJN colonizing grapevine showed earlier, faster, and better low-temperature stress responses than non-colonizing grapevine (Fernandez et al., 2012). The psychrotolerant endophytes, Pseudomonas vancouverensis OB155 and P. frederiksbergensis OS261 alleviated the chilling stress in tomato via reducing membrane damage and ROS levels. The colonized plants showed the higher activity of antioxidant in leave tissue and high expression of cold acclimation genes LeCBF1 and LeCBF3 compared with control plants (Subramanian et al., 2015). Similarly, endophytic bacteria B. phytofirmans strain PsJN increased Arabidopsis growth and strengthened cell wall under cold stress resistance (Su et al., 2015). The vesicular-arbuscular mycorrhizal is also important in mitigation of abiotic stress in the plant. These fungi develop very huge network of hyphae and increase the surface area of the root, thus increasing overall nutrient uptake by the roots. Several studies have indicated the arbuscular mycorrhizal fungi (AMF) associated salinity tolerance in host plant included Gossypium hirsutum (Liu et al., 2016), Oryza sativa (Porcel et al., 2015), Chrysanthemum morifolium (Wang et al., 2018), and Elaeagnus angustifolia (Chang et al., 2018). Similarly, AMF colonizing C3 (Leymus chinensis) and C4 (Hemarthria altissima) grasses have altered antioxidant enzyme and photosynthesis activity of plant in response to drought stress (Li et al., 2019) Phytohormones play a crucial role in the tolerance of abiotic stresses in plants (Wani et al., 2016). For instance, the endophytic halo-tolerant bacterium, B. Licheniformis HSW-16 enhanced the tolerance to salt stress and stimulated the growth of wheat through the production of IAA under saline soil conditions (Singh and Jha, 2016). In another study, a root-colonizing Pseudomonas spp. isolated from volcanic soil was able to mitigate under salt stress (500 mM NaCl) and high-temperature stress (40°C) by synthesizing IAA in maize (Mishra et al., 2017). The endophytic strain, B. subtilis NUU4 along with Mesorhizobium cicero IC53 enhanced the root and shoot biomass in chickpea (Cicer arietinum L.) under salt stress (Egamberdieva et al., 2017). Jaemsaeng et al., (2018) studied the effect of ACC deaminase producing endophyte Streptomyces sp. GMKU and its ACCD-deficient mutant on Thai jasmine rice under salt stress. The Streptomyces sp. GMKU 336 significantly promoted plant growth, chlorophyll content, osmolytes, and ion balance; but decreased ET, ROS when compared to plants not inoculated and inoculated with the ACCD-deficient mutant. The plant hormone abscisic acid (ABA) is a key regulator for the opening and closing of stomata and mitigates osmotic and other abiotic stresses in the plant. Several microbial endophytes modulate the ABA-mediated signaling pathway in the plant and may alleviate the abiotic stress tolerance. The expression of genes in the ABA signaling pathway of the wheat plant was modulated by the halo-tolerant Dietzia natronolimnaea, which is responsible for the salinity tolerance (Ilangumaran and Smith, 2017). Peskan-Berghöfer et al. (2015) emphasized the establishment of endophytic beneficial interaction between P. indica and A. thaliana roots required ABA. In a recent study, de Zélicourt et al. (2018) reported endophyte Enterobacter sp. SA187 isolated from the desert plant could alleviate the salt stress tolerance in Arabidopsis by the production of bacterial 2-keto-4-methylthiobutyric acid (KMBA) by regulating host ET signaling

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pathway. The Enterobacter sp. SA187 also improved the yield and related parameter of alfalfa plant under salt stress. Some microbial endophytes have the capacity to tolerate or resist high concentrations of pollutants and support the growth of plants under harsh soil condition. In a recent study, several bacterial endophytes were isolated from the Zn/Cd hyper accumulator plant Sedum plumbizincicola in which five isolates exhibit growth-promoting activities (Ma et al., 2015; Ullah et al., 2015). Similarly, endosymbiont bacteria Sinorhizobium meliloti strain CCNWSX0020 enhanced antioxidative defense response in Medicago lupulina under copper (Cu) stress (Kong et al., 2015). The fungi, Phialocephala fortinii, Rhizodermea veluwensis, and Rhizoscyphus sp. are the most dominant root endophytes significantly enhanced the growth of tree plant Clethra barbinervis under heavy metal stress condition. The endophytic fungi increased the potassium “K” uptake in shoots and reduced the concentrations of Cu, Ni, Zn, Cd, and Pb in roots to support the growth of the plant (Yamaji et al., 2016).

9.5.3  Genomics of alleviating biotic stress in host plants by endophytes Plant disease and insect pest have been the major constraints of agricultural products. Use of chemical pesticide and fungicide can inhibit the pest and pathogen but it adversely affects the environment. Endophytic microbes are found to be important in promoting plant growth indirectly by protecting them from biotic stress (Muller et al., 2016). The protection against plant pathogens by microbial endophytes include inhibition of pathogen by competition for niche and nutrients, production of antimicrobial compounds, lytic enzymes, and induction of plant immune system or induced systemic resistance (ISR) (Berendsen et al., 2012; Pieterse et al., 2014; Raaijmakers and Mazzola, 2012; Liu et al. 2017). Microbial endophytes control the growth of plant pathogen either directly through inhibition of pathogens or indirectly through enhancing plant immunity. The direct mechanism involves synthesis of antimicrobial compounds, production of chelators that reduce the availability of elements necessary for the growth of phytopathogen, production of toxic compound like hydrogen cyanide (HCN) and interruption of quorum sensing (QS) of plant pathogens (Liu et al. 2017; Pandey et al., 2019). The microbial endophytes associated defense mechanism is a complex phenomenon depending on various factors. The sphingomonads isolated from various plant species can give significant protection against the foliar pathogen in Arabidopsis rather isolated from air or water (Innerebner et al., 2011). In contrast, transcriptome analysis of Arabidopsis plant mutant revealed that plant responds differently to a member of its natural phyllosphere microbiota through the pattern-recognition receptor. Microbes were recognized by the plant-mediated component which triggers the expression of defense-related genes in the plant (Vogel et al., 2016). Furthermore, the mutation in Sphingomonas could affect the Arabidopsis plant protection against Pseudomonas syringae DC3000suggested that different mechanisms could contribute to plant protection (Vogel et al., 2012). The role of phyllosphere-colonizing microbes in plant protection against foliar plant pathogens are well documented (Innerebner et al., 2011; Vorholt, 2012; Ritpitakphong et al., 2016). Another mechanism

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to combat disease is microbial priming, which describes the preconditioning of immunity induced by microbial colonization after germination in the plant. The primed plant generates a faster and robust local and systemic response of plant upon pathogen attacks and restricts pathogen invasion (Conrath et al., 2015; Martinez-Medina et al., 2016). The microbe-free plant is more likely susceptible to pathogen infection since it generated a weaker immune response as compared to primed plant (PartidaMartinez and Heil, 2011). The priming is triggered by JA, SA, or ET-dependent signaling pathway, systemic acquired resistance (SAR) and ISR among which SAR is triggered by pathogenic microbes and regulated by SA-dependent manner whereas, ISR is conferred by beneficial microbes where ET and JA dependent pathways are a key player in the regulation of resistance response (Hacquard et al., 2017). Both types of induced resistance provide resistance against the vast range of plant pathogen (Pieterse et al., 2014). The downstream mechanism of priming state includes the accumulation of dormant MAPKs, elevated levels of PRRs, chromatin remodeling and activation of transcription factors (Hacquard et al., 2017). Recently, several studies have described the promising role of antimicrobial metabolites produced by endophytes against phytopathogens. The antagonistic properties of endophytic bacteria have been extensively studied in host plants included Nicotiana attenuata (Santhanam et al., 2014), Solanum trilobatum, and Solanum torvum (Bhuvaneswari et al., 2013; Achari and Ramesh, 2014). The lipoproteins compound of microbes like iturins, poaeamide induced the microbes associated molecular pattern (MAMP)- triggered immunity in plant and inhibited the growth of fungal pathogen (Han et al., 2015; Zachow et al., 2015). Similarly, volatile organic compounds are also conferring the resistance in the plant. Volatile organic compounds (VOC) from four strains of Hypoxylon anthochroum significantly inhibited the growth of F. oxysporum on cherry tomatoes (Macías-Rubalcava et al., 2018). Quorum sensing is an important mechanism for bacteria to survive in complex ecological niches by regulating the physiological activities of bacteria, reproduction, cell-to-cell communication and adaptation (Miller and Bassler, 2001). Yu et al. (2018) performed the comparative genomic analysis of twelve isolates to identify the genes required for the QS regulation in Pseudoalteromonas. The comparative genomic study and relative expression analysis revealed luxO and right origin-binding protein-encoding gene (robp) act as main regulatory genes in Pseudoalteromonas. Many bacterial species contain lux genes that produce the autoinducer N-acylated homoserine-lactone which is the key regulator to execute quorum sensing behaviors (Papenfort and Bassler, 2016). It has been reported that endophytes can interrupt the QS of phytopathogen and control the growth in ecological niche. For instance, Jose et al., (2019) suggested the endophytic bacterial strains of Bacillus and Variovorax species significantly inhibit the growth of Pseudomonas syringae pv. passiflorae by disrupting QS and associated virulence factors. The endophytic bacterial strains in Cannabis sativa L. disrupt cell-to-cell communication by intercepting in QS signals of biosensor strain Chromobacterium violaceum (Kusari et al., 2014). Despite these promising benefits of microbial endophytes in agriculture, it is practically difficult to deploy and establish a compatible interaction of microbes with the plant. Nation wise network-based approach should be made to bridge these gaps and to attempt commercialization of such products.

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9.6  Perspectives: a way ahead Microbial endophytes offer an opportunity by improving nutrient use efficiency, imparting stress tolerance to increase crop productivity. Nevertheless, characterization and identification of such production-related factors signify their existence and recognition among scientific community. Moreover, there is also a need to deploy modern cutting-edge technology for understanding the functional aspects of the beneficial plant–endophyte relationship. The standard protocols in terms of the biochemical, metabolome, proteome for identification, and characterization of endophytic spp. should be optimized and made reproducible for broad-spectrum range of endophytes classes. Such concentrated efforts will direct to availability of unique endophyte sequences. Exploring more and more environmental niches and ecosystems for identification of endophytes is essential. The molecular mechanism of cereal-endophyte vis-a-vis legume-endophyte should be unraveled. Endophytes from diverse ecosystems have potential in terms of imparting abiotic/biotic stress tolerance or special specific attribute. For example, there have been reports of colonization of rice roots by Gluconacetobacter diazotrophicus, an endophyte of sugarcane. In such cases, there is a need to understand the molecular mechanism of interaction in a plant–endophyte relationship on the genome, transcriptome, and proteome levels using a combined omics approach. Such information will shed light on the responses of host plants and aid in developing strategies for better tolerance or the deployment of endophyte as biofertilizer. Apart from the host plant–endophyte relationship, it is essential to understand the interaction between endophytes present in the plant tissue at a time. In such cases community microbiome analysis is essential. New strains of endophytes suitable for a particular combination of host, environment and microbial niche, such as generating new strain through mutagenesis, should be developed.

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Ullah, A., Heng, S., Munis, M.F.H., Fahad, S., Yang, X., 2015. Phytoremediation of heavy metals assisted by plant growth promoting (PGP) bacteria: a review. Environ. Exp. Bot. 117, 28–40. Vijayabharathi, R., Sathya, A., Gopalakrishnan, S., 2016. A Renaissance in plant growth- promoting and biocontrol agents by endophytes. In: Microbial Inoculants in Sustainable Agricultural Productivity, Springer, India. Vogel, C., Bodenhausen, N., Gruissem, W., Vorholt, J.A., 2016. The Arabidopsis leaf transcriptome reveals distinct but also overlapping responses to colonization by phyllosphere commensals and pathogen infection with impact on plant health. New Phytologist 212, 192–207. Vogel, C., Innerebner, G., Zingg, J., Guder, J., Vorholt, J.A., 2012. Forward genetic in planta screen for identification of plant-protective traits of Sphingomonas sp. strain Fr1 against Pseudomonas syringae DC3000. Appl. Environ. Microbiol. 78, 5529–5535. Vorholt, J.A., 2012. Microbial life in the phyllosphere. Nat. Rev. Microbiol. 10, 828–840. Wang, Y., Wang, M., Li, Y., Wu, A., Huang, J., 2018. Effects of arbuscular mycorrhizal fungi on growth and nitrogen uptake of Chrysanthemum morifolium under salt stress. PLoS One 13, e0196408. Wani, S.H., Kumar, V., Shriram, V., Sah, S.K., 2016. Phytohormones and their metabolic engineering for abiotic stress tolerance in crop plants. Crop J. 4, 162–176. Win, K.T., Tanaka, F., Okazaki, K., Ohwaki, Y., 2018. The ACC deaminase expressing endophyte Pseudomonas spp. enhances NaCl stress tolerance by reducing stress-related ethylene production, resulting in improved growth, photosynthetic performance, and ionic balance in tomato plants. Plant Physiol. Biochem. 127, 599–607. Yamaji, K., Watanabe, Y., Masuya, H., Shigeto, A., Yui, H., Haruma, T., 2016. Root fungal endophytes enhance heavy-metal stress tolerance of Clethra barbinervis growing naturally at mining sites via growth enhancement, promotion of nutrient uptake and decrease of heavymetal concentration. PLoS ONE 11 (12), e0169089. Yu, Z., Ding, Y., Yin, J., Yu, D., Zhang, J., Zhang, M., Ding, M., Zhong, W., Qiu, J., Li, J., 2018. Dissemination of genetic acquisition/loss provides a variety of quorum sensing regulatory properties in Pseudoalteromonas. Int. J. Mol. Sci. 19 (11), 3636. Zachow, C., Jahanshah, G., de Bruijn, I., Song, C., Ianni, F., Pataj, Z., Gerhardt, H., Pianet, I., Lämmerhofer, M., Berg, G., Gross, H., Raaijmakers, J.M., 2015. The novel lipopeptide poaeamide of the endophyte Pseudomonas poae RE*1-1-14 is involved in pathogen suppression and root colonization. Mol. Plant-Microbe Interact. 28, 800–810. Zaheer, A., Mirza, B.S., Mclean, J.E., Yasmin, S., Shah, S.M., Malik, K.A., et al., 2016. Association of plant growth-promoting Serratia spp. with the root nodules of chickpea. Res. Microbiol. 167, 510–520. Zuccaro, A., Lahrmann, U., Guldener, U., Langen, G., Pfiffi, S., et al., 2011. Endophytic life strategies decoded by genome and transcriptome analyses of the mutualistic root symbiont Piriformospora indica. PLoS Pathog. 7 (10), e1002290. Zúñiga, A., Poupin, M.J., Donoso, R., Ledger, T., Guiliani, N., Gutiérrez, R.A., González, B., 2013. Quorum sensing and indole-3-acetic acid degradation play a role in colonization and plant growth promotion of Arabidopsis thaliana by Burkholderia phytofirmans PsJN. Mol. Plant-Microbe Interact. 26, 546–553.

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Arpan Modia, Poonam Kananib, Ajay Kumara a Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, Rishon LeTsion, Israel; bDepartment of Agriculture Biotechnology, Anand Agriculture University, Gujarat, India Chapter outline head 10.1 Introduction  251 10.2 Endophytic fungi and their importance in agriculture  252 10.2.1 Historical evolution of endophytic fungi  252 10.2.2 Classification of endophytes  253 10.2.3 Entry of endophytic fungi into the host plant  253

10.3 Gene expression analysis  254 10.4 Gene expression analysis in plant–microbe interactions  259 10.4.1 Plant growth through fungal secondary metabolites  259 10.4.2 Biotic stresses  261 10.4.3 Abiotic stresses  262

10.5 Conclusion and future prospects  265 References  266

10.1 Introduction With recent and fast growing scientific breakthroughs, a nascent field of endophyte biology has come forward. Endophytic fungi residing in plants are exceedingly diverse group of fungi, typically classified in ascomycetes and anamorphic fungi (Arnold and Lutzoni, 2007), and it has been estimated that more than 1 million endophytic fungal strains residing nearby in the 300,000 plant species available worldwide (Strobel and Daisy, 2003). An imperative attribute of fungal endophyte is their incomparable diversity globally, as well as within individual, part of the plants and locations (Arnold and Lutzoni, 2007). Mainly, sustainable agricultural practices as well as increased production depend on strategies, which involve plant microbes interactions. Though there had been a lot of intended work program allocated for obtaining new and better understanding about how this all works out? But still we come up to many points where we are clueless, when various influential archetypes are involved regarding their specificity, identity, diversity, evolution, and their interaction with plants; various signaling Microbial Endophytes. http://dx.doi.org/10.1016/B978-0-12-819654-0.00010-7 Copyright © 2020 Elsevier Inc. All rights reserved.

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pathways which evolve are established in between and functionalize or channelize the interaction and move forward with the association in further period of their life cycle. Even though, there are few studies reported back on the isolation, diversity, and characterization of fungal endophytes colonized with different plant species (Fouda et al., 2015), but the role of gene expression intriguing these mechanisms is sparse, which can lead a new insights to acknowledge the breakthrough revelation in this area. With nascent advancement in technologies such as next generation sequencing (NGS) has added on to the basic knowledge and has led to deeper sense of understanding in relation to studies on plant host interaction (Wagner et al., 2016).

10.2  Endophytic fungi and their importance in agriculture Endophytes are termed as microorganisms (bacteria, fungi, and actinomycetes), indwelling inter- or intracellular in plant tissues for the entire or some part of their life cycle (Arnold, 2007). There can be a facultative or obligate complex relationship with the host and endophytes. They usually interact symbiotically with host plants principally via increasing host resistance against herbivores, thus they imitate like mycorhizae associated with mutual benefits to both host and fungi (Carroll, 1988; Gehring and Whitham, 1994). In general, plant confines endophytic growth conditions, so gradually to inhabit in that environment, endophytes employ various other mechanism to acclimatize in that surroundings via fabricating various plant growth-promoting active compounds (Dudeja et al., 2012).

10.2.1  Historical evolution of endophytic fungi For the first time, the word endophyte was termed by De Barry in 1866, and the reports are dated back 1900. Freeman (1904) reviewed on endophytic fungi in some publications and during 1930–90; numerous isolation of endophytic microorganism had been accounted from different plants and grasses. In terms of records obtained from fossils were linked with terrain plants for >400 million years ago (Krings et al., 2007). In the phase of evolution when there was transition from aquatic to terrestrial phases, the plants had faced various changes in extreme regimes such as temperature changeability, water level fluctuations, deprived nutrients in soil, and high carbon dioxide content. At that point of time fungi supported their own life and also helped in survival of plants to withstand unnatural circumstances. Also they have managed to become accustomed to milieu via some genetic variations together with entrapping few of the plants DNA (Selosse and Le Tacon, 1998; Bonfante and Selosse, 2010). Due to this course of evolution period and adaptability, there is wide range host system such as different ferns, shrubs, mosses, grasses, lichens, and coniferous and deciduous trees (Sun and Guo, 2012).

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10.2.2  Classification of endophytes There is a wide range of fungal endophytes known till date ever since from 19th century when endophytes came in light (Guerin, 1898). It has been estimated over a million of fungal endophyte species worldwide (Sun and Guo, 2012). In general endophytic fungi have been broadly classified in the two groups on the basis of phylogeny clavicipitaceous infecting some grasses and nonclavicipitaceous endophytes, which can be isolated from tissues of nonvascular plants, conifers, ferns, and angiosperm. Rodriguez et al. (2009) has categorized endophytes on the basis of colonization characteristics into four groups. Class 1 clavicipitaceous endophytes are found above and underneath plant tissues, such as aerial tissues, endorhiza, and in the rhizosphere; Class 2 are the one which disseminated vertically and horizontally in the plant system; and Class 3 are favorably accounted to basidiomycota and ascomycota in aerial tissues but disseminated horizontally in general. Class 4 consists of dark septate endophyte, which are mainly resides in the inter- or intracellular layers of cortical cells. There are various fungal endophytes have been isolated and reported, which mainly belong to Acremonium sp., Aspergillus sp., Cladosporium sp., Colletotrichum sp., Curvularia sp., Penicillium sp., Phyllosticta sp., Phomopsis sp., and Stemphylium globuliferum.

10.2.3  Entry of endophytic fungi into the host plant Basically the entry or infection of fungal endophytes inside the host tissue carried out through two manners, that is, horizontal and vertical transmission. Horizontal transmission is a prime mechanism of endophyte spreading from surrounding to the host tissue or within plant to plant transfer, whereas the seed descendants are infected through vertical transmission (Gallery et al., 2007). In account to horizontal transmission, the dispersal mainly occurs through endophytic inoculums but in case of asymptomatic host the transmission is debatable. In that scenario, it is proposed that inoculums are recovered from the dies off of host tissue that infects the fresh host (Sánchez Márquez et al., 2007). In some other cases, the diffusion of spores may be take place through phytophagous insects, as some fungal spores being resistant to gut digestion and present in fecal pellets that lead to transmission (Devarajan and Suryanarayanan, 2006). Also there are some unobtrusive ways of transmission, for instance in some grasses a microscopic layer of hyphae and conidia on the leaves surface causes infection of epichloë endophytes to new host (Tadych et al., 2007). The transfer and progression of endophytic fungi inside the host constitute developing hyphae or mycelia, through three possible ways (1) intercellular movements, (2) intracellular movement, and (3) both intercellular and intracellular movement (Fig. 10.1) (Guerin, 1898). There are scanty studies on vertical transmission, and it was discovered while investigating seed transmitting fungi (Gallery et al., 2007). Some of Epichloë and neotyphodium sp. of endophytes are transmitted vertically through seed infection, and the total of 100% infection rate is estimated (Schardl et al., 2004). The following table (Table 10.1) represents a list of fungal endophytes associated with their host organisms.

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Figure 10.1  Pathways for the physical entry of endophytes through roots. Intercellular entry represents the cell-to-cell transfer of endophytes probably thorough plasmodesmata. Another path involves the intracellular space for the movement of endophytes inside plant tissues. Inter- and intracellular entry comprises of both the movement mentioned earlier.

10.3  Gene expression analysis The functional characteristic of the organism is governed through one and only the specific entity called “gene,” and the expression analysis of genes gives peer knowledge for identification and interrelationship between various signaling, cellular, and molecular pathways (Casassola et al., 2013). The mechanism of gene expression is highly regulated and controlled both in eukaryotes and prokaryotes; therefore the study on gene expression provides an exact outlook associated with a particular given trait. Unraveling the mechanism associated to surmount diseases by fungal endophyte in cereal crops during the infection may unveil novel disease management strategies. The most significant way to address this is through the study during the interaction of host and endophyte-related differential gene expression. For a particular gene expression, it undergoes key steps, which lead to a functional gene. The most influential and determining step is RNA transcription from DNA, which forms a transcriptome (a complete set of RNA transcripts produced by genome), and in reference to plant transcriptome study, it ascribes about the differential gene and molecular mechanisms associated in plant–pathogen interaction (Polesani et al., 2008, Al-Taweel and Fernando, 2011). The gene expression studies are usually carried out through various approaches, such as Microarray, complementary DNA (cDNA)-amplified fragment length polymorphism, suppressive subtractive hybridization (SSH), complementary DNA (cDNA) libraries differential display reverse transcription PCR, RNA fingerprinting by arbitrary primed PCR, expressed sequence tag (EST) sequencing, representational difference analysis (RDA), serial analysis of gene expression (SAGE), and transcriptome sequencing [or

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Table 10.1  List of fungal endophytes in different classes with corresponding host organisms. Endophytes Acremonium sp. A. cucurbitacearum

A. zeae Acremonium sp. (ENF 31) Aspergillus sp. Aspergillus sp.

A. niger and A. carbonarius Cladosporium sp. C. oxysporum and C. sphaerospermum. C. herbarum Colletotrichum sp C. endomangiferae C. gloeosporiodes Curvularia sp. C. geniculata Curvularia sp. Penicillium sp. Penicillium simplicisssum (ENF22) Penicillium funiculosum LHL06 Phyllosticta sp. P. brazilianiae P. capitalensis

Phomopsis sp. Phomopsis sp. Phomopsis sp. Phomopsis castanea Stemphylium sp. Stemphylium globuliferum Stemphylium sedicola SBU-16

Host system

References

Cucumis melo and Citrullus lanatus Taxus chinensis Maize Oryza sativa L. and Zea mays L.

Armengol et al. (1998)

Datura stramonium, Moringa olifera, Prosopis chilensis Zea mays and Arachis hypogea

Mahdi et al. (2014)

Pine Trees

Paul and Yu (2008)

Cinnamomum camphora Triticum aestivum

He et al. (2011) Larran et al. (2002)

Mangifera indica L. Lycopersicum esculentumMill

Vieira et al. (2014) Larran et al. (2001)

Parthenium hysterophorus L.

Priyadharsini and Thangavelu (2017) Mahdi et al. (2014)

Datura stramonium and Moringa olifera

Liu et al. (2009) Wicklow et al. (2005) Potshangbam et al. (2017)

Palencia (2012)

Oryza sativa L. and Zea mays L. Glycine max L.

Potshangbam et al. (2017) Khan and Lee (2013)

Mangifera indica Punica granatum, Ficus benjamina Citrus sp. Mangifera indica

Wikee et al. (2013)

Oryza sativa Ginkgo biloba L. European chestnut

S.C. et al. (2017) Thongsandee et al. (2012) Washington et al. (1999)

Mentha pulegium. Taxus baccata

Debbab et al. (2009) Mirjalili et al. (2012)

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Figure 10.2  Widely used techniques for the analysis of gene expression. These techniques may require prior sequence information.

RNA sequencing (RNA-Seq)] (Fig. 10.2) (Casassola et al., 2013). Aided with these techniques there are also few reports on sequenced genome of fungal endophytes in plants as shown in Table 10.2. With the existing plenty technologies in market for the quantification of transcriptome are basically based on hybridization or sequencing approaches. Among this hybridization-based techniques engross the identification of differential gene expression based principally on nucleic acid hybridization (Clark et al., 2002). The experiment is initiated with total RNA or mRNA of given cells or tissue sample and is Table 10.2  List of fungal endophytes with sequenced genome. Fungal endophytes

References

Epichloe festucae E2368 Piriformospora indica Ascocoryne sarcoides Harpophora oryzae Penicillium aurantiogriseum NRRL 62431 Shiraia sp. slf14 Pestalotiopsis fici Rhodotorula graminis WP1 Microdochium bolleyi J235TASD1 Phialocephala scopiformis DAOMC 229536 Xylona heveae Aspergillus montevidensis ZYD4 Fusarium solani JS-169 Gaeumannomyces sp. Strain JS-464 Sphingomonas sp. LK11

Schardl et al. (2013) Zuccaro et al. (2011) Gianoulis et al. (2012) Xu et al. (2014) Yang Y. et al. (2014) Yang H. et al. (2014) Wang et al. (2015) Firrincieli et al. (2015) David et al. (2016) Walker et al. (2016) Gazis et al. (2016) Liu et al. (2017) Kim et al. (2017a) Kim et al. (2017b) Asaf et al. (2018)

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Figure 10.3  Timeline of sequencing technologies represented as first, second, and third generation of sequencing. After first generation, the sequencing methods were the flagships of different companies.

reverse transcribed to cDNA. They are preliminarily labeled with fluorescent dyes, being treated as “target,” and then they are hybridized onto a solid surface (microarray chip) loaded with immobilized huge known DNA sequences in an ordered array (Schena et al., 1995). The parallel information on genomic data and advancement in molecular technique like microarray had eased the performance of global analysis of enormous genes in solitary attempt (King and Sinha, 2001). The recent advancement in high throughput sequencing technologies has emerged a novel method called “RNA-Seq” for mapping and quantifying transcriptome, projected to transfigure the sequencing era of genomics (Marioni et al., 2008). RNA-Seq utilizes newly developed depth sequencing methodology. Basically the technique involves the use of RNA fragments or total RNA with poly A tail, and is converted to cDNA/EST library incorporated with adaptors at one or both the ends. All the molecules are sequenced to obtain short reading sequences from one or both the ends (single- end or paired-end sequencing). The sequencing reads ranges from 30 to 400 bp based on the technology being used. Nowadays various high throughput technologies are in demand such as Roche 454 Life Science, Ion torrent sequencing Life technologies, Illumina/Solexa sequencing, ABI/SOLiD sequencing, Pacific biosciences SMRT sequencing, Oxford nanopore sequencing (Kchouk et al., 2017) (Fig. 10.3). Basically, either sequencing by synthesis or chain termination technology, sequence information ends up with many short fragments, which are actually parts of coding region of the gene (EST). Sequencing data are processed, corrected, edited, and aligned to find the expressed genes qualitatively and quantitatively. Mostly, differentially expressed genes and identification of novel proteins associated with metabolism, stress, or physiological processes are identified using RNA-seq tools. After the complete processing of sequencing data through dry lab operations, various primers can be designed, which target specific genes involved in above-mentioned processes. Gene expression analysis with its absolute quantity can be done as one of the products of RNA-Seq, and with two different tissues or cell types, these quantities can be

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Figure 10.4  General outline of gene expression analysis in plants with an example of control and treated samples. 1, Total RNA is extracted from both the control and treated samples (particularly affected plant parts). 2, With the help of reverse transcriptase, these total RNA is subjected to form cDNA from mRNA population only, which represents expression populations to be studied. In the process itself, cDNA fragments are purified for downstream processing. 3, Purified fragments of cDNA from both the samples are subjected to sequencing. 4, Annotation of sequencing data from both the samples implies differentially expressed genes. 5, Using the sequence information, primers from individual gene (differentially expressed) can be synthesized which can be used to detect gene expression changes through real-time PCR (5a) in future experiments involving similar treatments. 6, Similar to primer synthesis of primers, a set of probes for differentially expressed genes can be made and used for microarray experiments (6a).

compared (Fig. 10.4). RNA-Seq is extensively used in nonmodel plants and many unknown enzymes and proteins of metabolic pathways, secondary metabolism, stressinduced pathways, and developmental processes are explored. Details of the technique and some of the examples are reviewed by Weber (2015). Quantitative real-time PCR-based method for the gene expression analysis is highly recommended, and budget-friendly technique yet equally reliable as others. Basically, differentially expressed genes within two or more different biologicalsamples (one of them being control or untreated sample) can be quantified using this technique. The quantification of gene expression has two pathways: absolute quantification and relative quantification. In absolute quantification, copy number of standard template is required and using serial dilution, standard curve of copy number versus threshold cycle (Ct) value can be plotted and ultimately absolute copy number of target gene can be calculated. In relative gene expression, Ct values of two samples with Ct values of respective samples of reference genes are plotted in 2−δδCt in order to find out relative values in terms of fold change (RQ) increase or decrease as compared to control. RQ

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value of control is always 1 as per the equation (Livak and Schmittgen, 2001). In terms of detection of fluorophores, two basic chemistries are used: SYBR green chemistries, where dye binds to DNA template (just like ethidium bromide) which may give nonspecific amplification and probe chemistry, where fluorophores attached probe also binds to the complementary sequence in the product so that nonspecific products can be avoided.

10.4  Gene expression analysis in plant–microbe interactions So far, numerous researchers have used endophytic fungi as a weapon for combating biotic and abiotic stresses, as well as plant growth promotion in numbers of species including field crops, medicinal plants, and horticultural plants (Card et al., 2016; Franken, 2012; Johnson et al., 2013; Khan et al., 2015; Lugtenberg et al., 2016; Waqas et al., 2015). The most common mode of action behind this internal strength of plants is the secondary metabolites secreted by endophytes and their fate in promotion of plants survival. However, mechanism at the transcriptome, proteome, and metabolome level can highlight many metabolic clues. In a study of plant–microbe interactions, many metabolic processes are involved, which can be revealed through transcriptome sequencing or less giant techniques like microarray.

10.4.1  Plant growth through fungal secondary metabolites Looking at the reduction in world wheat production and the major hindering factor responsible for poor germination, Banerjee et al. (2014) used fungal endophyte (endophytic Ascomycota mitosporic fungal isolate SMCD 2206) treatment of wheat seeds with direct and indirect contact. Here, indirect contact confirmed the influence of fungal hyphae on seed germination must progressed through fungal diffusible, or volatiles chemicals were secreted and reached to seeds during growth. Significantly, higher germination rate was observed in seeds placed on media with direct contact of endophyte culture. The energy of germination, which can be defined as number of days required to attain 50% germination, (Hubbard et al., 2012) was also higher in the same treatment, reaching the energy of germination (50%) within 2 days of seed inoculation. This led to the emergence of a new concept called as “mycovitalism,” which means to increase the vitality through fungal colonization (Vujanovic and Vujanovic, 2007); however, this kind of colonization does not need to be carried out with endophyte always. To evaluate the effects of endophyte treatments (direct and indirect along with control) on gene expression pattern of gibberellic acid biosynthesis and regulatory pathway, they selected two important genes, namely, GA3-oxidase 2 (GA3 biosynthesis) and 14-3-3 (negative regulator of GA3 biosynthesis). GA3ox2 expression was increased along with lower expression of its negative regulator. During seed germination, GA3 production is increased in order to break seed dormancy and higher expression of GA3ox2 (as compared to control) showed colonization has a positive impact on seed germination process.

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One of the preeminent features of fungal endophytes in its life cycle is their ability to induce the synthesis of secondary metabolites, while they are in the status of symbiosis. These secondary metabolites may act as biocides. A group of researchers (Soliman et al., 2013) explained this phenomenon with the help of Taxus, which served as host to the fungus Paraconiothyrium SSM001. This strain is actively involved in the production of taxol within the tree. Taxol is well known for its anticancer activity. First of all, when five different species of Taxol plant were evaluated for their taxol content and the genotype as well as density of its fungal endophyte Paraconiothyrium using 18s rDNA sequencing, it was observed that different plant species have different types of Paraconiothyrium but with only 1% nucleotide substitutions with SSM001 pathotype. However, when their level were measured using semiquantitative t-RFLP-based fluorescent fingerprinting, correlation was observed between the concentration of taxol and density of fungal endophytes. Similar correlation was also observed within same plant with different branches. Another set of experiment was conducted to study the effect of fungicide on taxol production. In vitro studies revealed that fungicide Maxim XL at the concentration of 100 nM inhibited the growth of these fungi completely. When 1-year-old mature trees were subjected with different concentrations of this fungicide (Maxim XL), the amount of taxol as well as fungal 18S rRNA expression level decreased with the increasing concentrations of fungicide applications. Thus, it was clear that fungal endophyte Paraconiothyrium has a correlation with the taxol production in various Taxus species. Now, the next question raised after this observation was whether the fungi themselves produce the taxol or they elicit the production within the plant. To study this, they targeted a key enzyme, DXR (deoxyxylulose-4-phosphate reductase) of MEP (Methyl-Erythrose-4Phosphate) pathway, leading to the synthesis of diterpenoid taxane and TS (taxadiene synthase), a rate limiting enzyme in taxol biosynthesis. Expression level of both the enzymes showed down-regulation in fungicide-treated plants as compared to control. The conclusion of this finding was clear that there is an elicitation of taxol biosynthesis due to endophytes as revealed by gene expression of analysis of two important enzymes of the plant. However, the authors also mentioned about the possibilities of fungal contribution toward taxol production directly during symbiosis. Rhodiola crenulata in interaction with fungal endophyte ZPRs-R11 (Trimmatostoma sp.) showed the accumulation of salidroside and tyrosol (Cui et al., 2017). The phenomenon was checked at signal transduction, enzyme gene expression, and metabolic pathway, leading to the production of these secondary metabolites. At 10 days of inoculation, 13.7 and 9.7 fold increment was found in salidroside and tyrosol accumulation, respectively in inoculated plants than control. As mentioned, the signal transduction process starts with the production of hydrogen peroxide (H2O2), nitric oxide, and salicylic acid (SA). Furthermore, among expressed genes, rate limiting enzyme, genes of salidroside biosynthesis pathways, were targeted to evaluate the expression pattern in endophyte-treated and untreated plants. These included, UDP-glucosyltransferase, tyrosine decarboxylase, monoamine oxidase, phenylalanine ammonia lyase (PAL), and cinnamic-4-hydroxylase (C4H). All these genes were up-regulated, and highest level of up-regulation was observed from UDP-glucosyltransferase to C4H.

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10.4.2  Biotic stresses Plant immunity or plant defense system comprises of many factors, starting from the direct recognition or detection of pathogenic molecules with the help of membrane receptors known as pattern recognition receptor, which detect pathogen-associated molecular patterns, whereas wall-associated kinases detect damage-associated molecular patterns as a result of cellular damage during infection. The major function of these receptors is to activate one of many signaling cascades. As a result of these cascades, pathogenesis-related proteins (PR proteins) are secreted, which helps to maintain cellular integrity and metabolism through hypersensitive response (HR), reactive oxygen species (ROS), cell wall modification, closure of stomata, and production of chitinase, proteinase inhibitor, defensins, and phytoalexins. Better findings with the molecular tools revealed that plant defense involves various organelles and classes of both proteins and nonproteins compounds. Defense-related functions are mediated through the production of PR proteins (Andersen et al., 2018). The expression of genes coding for PR proteins may provide clues for plants response. It is expected to be enhanced gene expression and subsequent PR proteins production during pathogen resistance. Mejía et al. (2014) studied the effect of foliar endophytic fungi Colletotrichum tropicale of Theobroma cacao on gene expression changes. Infected and uninfected leaves were designated as E+ and E−, respectively. Up-regulation of a gene coding for putative proline rich protein, aminly involved in hardening of cell wall was observed. Similarly, tubulins, highly expressed in wood forming tissues were also upregulated. This results suggested that in endophyte-infected leaves, host cells tried to strengthen the cell wall barrier, which ultimately helped them in pathogenesis. The difference between pathogenicity and endophytism has also been studied at molecular level. Xu et al. (2016) have shown that fungal pathogen Magnoporthe oryzae (causing organism for rice blast disease) and fungal endophyte Harpophora oryzae infected to rice seedling secreted many proteins and enzyme, which altered the host metabolism. They used RNA-Seq to check the difference in host transcriptome infected with both the fungi. The result revealed that 14 proteins were secreted exclusively in the endophytic infection, whereas 44 proteins were found to be uniquely secreted in pathogenesis. The Class of hydrolases proteins was found to be most abundant among other classes in both the cases. When comparative transcriptomic studies were carried out, genes coding for the enzymes such as peptidase, lipase, tyrosinase, cutinase, cellulase mainly involved in cell wall degradation process were down-regulated in endophyte-infected plants, which suggested its impact on lowering the rate of cell wall degradation, wherein the same genes were up-regulated in M. oryzaeinfected plants. Similar to this manner, genes involved in oxidation-reduction reaction were up-regulated during pathogenesis and down-regulated during mutualism. Gao et al. (2010) reviewed and proposed a pathway of mechanism, how endophytes help plant to combat in biotic stresses. The residential effects of endophyte may be direct, indirect, or ecological. In direct effects, pathogen growth is prevented by the synthesis of antibiotic and lytic enzymes. These antibiotics include alkaloids (altersetin against gram positive bacteria), volatile oils (tetrohydofuran, 2-methyl furan,

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2-butanone, and aciphyllene), and terpenoids (3, 11, 12-trihydroxy cadalene), which have antimicrobial properties. Indirect effect, on the other hand, is a plant’s response toward pathogen but partially or completely initiated by endophytes. There are morphological and biochemical changes like cellular necrosis, HR and production of phytoalexins. Systemic acquired resistance (SAR) and induced systemic resistance are two forms of plant’s response, which are mediated by SA and jasmonic acid, respectively. In SAR, PR proteins, such as chitinase and β-1, 3-glucanse, are produced by host, structural changes of cell wall and plasma membrane or localized cell necrosis occur to restrict entry, and spreading of pathogens. Besides these, the production of radical scavenging enzymes, such as superoxide dismutase (SOD) and peroxidase (POD) is also induced by endophytes. Phytoalexins include terpenoids and flavanoids mainly despite of the fact that these molecules are also synthesized by host plant upon stimulation by UV light and other environmental factors confirming abiotic stresses. One of the avoidance mechanisms imparted by endophyte is to increase the vigor of the plant itself by stimulating or producing plant growth hormones like auxins. Ultimately, combating with pathogen is also a race of growth and survival for both the organisms and growth of one organism is a limiting factor for the other during pathogenesis. After complete protocol of endophytism (host recognition, spore germination, penetration through epidermal tissues, and colonization), endophytes create their niche within intercellular spaces of host. This phenomenon is first and very important sign of prevention of ecological establishment of other pathogens (as they are also microbes like endophytes).

10.4.3  Abiotic stresses Preliminary study related to the effect of mutualism of Neotyphodium coenophialum with tall fescue in order to find differentially express genes through suppressive subtractive hybridization (SSH) by Johnson et al. (2003). In total, 29 genes were found to be up/down-regulated in endophyte-treated plants as compared to untreated controls. Within up-regulated, significant genes were RNA-binding protein, GDP dissociation inhibitor protein, metallothionein, herbicide safener-binding protein, polyadenylatebinding protein, ABC transporter, and omega-3 fatty acid desaturase, which conferred plasma membrane strengthening, RNA metabolism, reduction of heavy metal toxicity, and copper sequestration in roots (Scott-Craig et al., 1998; Tocher et al., 1998). Very few significant matches were found among down-regulated genes, which included Chlorophyll A-B-binding protein, enzyme aminopeptidase, and pathogenesis-related protein PR-10 and S-adenosylmethionine decarboxylase. Although, it was an identification study of differentially expressed genes, it opened the ways toward understanding the molecular mechanism of plants immunity mediated through endophytes. Further work was carried out on the same symbioants by Dinkins et al. (2010) with Affymatrix wheat genome array cheap and Barley1 genome array chip. Due to less similarities between tall fescue and wheat/rye transcripts only 14%–18% were detected with signal intensities. And also among them more numbers of down-regulated genes were found than up-regulated in infected seedlings and many of them showed contradictory results when semi-quantitative PCR was performed using primers generated from

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ESTs. However, one gene, involved in cellulose synthesis, was highly up-regulated in infected seedlings than uninfected seedlings. Although authors suggested further investigation for this gene, it was clear that strengthening the expression of this gene implied plant’s preparation toward resistance against biotic and/or abiotic stresses. An attempt was also done using molecular tool to characterize the plant endophyte interaction by Bailey et al. (2006) in cacao seedlings through gene expression analysis. Infected and uninfected plants were subjected to differential display, macroarray, and real-time PCR analysis. For endophyte Trichoderma, they used five different virulent strains and seedlings of Theobroma cacao were taken. In order to study colonization, they observed several plant ESTs comprising of genes, and found homology with other plants species, for ornithine decarboxylase, GST-like protein, zinc finger protein, wound-induced protein, carbohydrate oxidase, and EF-calcium-binding protein in the Trichoderma colonized plants. These proteins are known to have an involvement in biotic and abiotic stress resistance mechanism directly or indirectly (Capell et al., 2004; Carter and Thornburg, 2004; Dean et al., 2005; Dixon et al., 2002; Kim et al., 2004; McCormack et al., 2005; Walters, 2000). Apart from these over expressed genes, they also observed one down-regulated gene, aquaporin commonly known as major intrinsic protein. The down-regulation of this protein is also associated with drought resistance strategies, as this protein play important role in the transportation of water, small neutral molecules, and ions; during drought, the cell water conservation becomes a primary goal for plants (Smart et al., 2001); and thus the down-regulation of aquaporin plays important role in such preservation. A major breakthrough in the molecular insights of host–endophyte relation was observed, when Dupont et al. (2015) conducted an experiment comprised of perennial ryegrass infected with fungal endophyte Epichloe festucae. Expression pattern of almost one third of the host genes were affected (Fig. 10.5). Alterations in host gene expression were observed in three main areas: primary metabolism, secondary metabolism, and stress-related genes. And above these all genes involved in the transcription process, such as RNA polymerase, TATA-box-binding proteins, and transcription factors, were down-regulated, which, in turn, affected the down-regulation of 25% of genes. These included genes involved in the cell cycle, namely, cyclins and cyclin-dependent protein kinases. The infection of endophyte reduced the rate of cell cycle and cell growth, which was further demonstrated by reduction in the expression of genes involved in DNA synthesis, chromatin structure, and DNA repair. In this case, endophytes were imparting stress on host. Furthermore, the down-regulation of genes involved in carbohydrate metabolism, energy metabolism, lipid metabolism, nucleotide metabolism, and proteins of cell signaling was also observed. Among the physiological processes, photosynthesis rate was greatly affected, mainly Calvin cycle and the tetrapyrrole pathway which leads to chlorophyll biosynthesis. Further, chlorophyll contents were measured in infected and uninfected plants and concluded that the levels were at par, and photosynthesis rate had no correlation with the chlorophyll level. Coming to the second side of the coin, up-regulation was observed in expression of genes of cell wall biosynthesis, which included hemi-cellulose, cell wall modifiers and arabinogalactan proteins and leucine rich repeats. In case of phytohormones, genes involved in abscisic acid (ABA) biosynthesis were up-regulated. Now, these

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Figure 10.5  Up- and down-regulated genes during Epichloe festucae infection to ryegrass. Genes involved in cell wall biosynthesis, ABA biosynthesis, GA biosynthesis, lignin biosynthesis, and biosynthesis of secondary metabolites like anthocyanins (pigments) and flavonoids were found to be up-regulated, whereas genes involved in primary metabolism, physiological processes, and cell cycle metabolism were down-regulated (Dupont et al., 2015).

findings had slight controversy as the ABA level in infected plants did not elevated but, as an indirect indication, stomata closure, as affected by elevated ABA levels, was observed in infected plants to reduce the transpirational loss as a part of water conservation. Up-regulation of genes coded for enzymes involved in interconversion between inactive andactive forms of gibberellins was also found. A simple explanation could be given to this phenomenon with endophyte infection; number of trichomes on adaxial surface of leaf was observed in Arabidopsis (Perazza et al., 1998) seedlings treated with gibberellic acid. Secondary metabolism was greatly entertained by endophyte infection showing up-regulation of genes coded for various enzymes such as, arogenate dehydratase, PAL, C4H, p-coumaroyl-CoA synthase, cinnamoyl-CoA reductase, cinnmoyl alcohol dehydrogenase, caffeic acid O-methyl transferase, and hydroxycinnamoyl-CoA skimimate/quinate hydroxyl cinnamoyltransferase. However, up-regulation of various iso-forms of these enzymes were observed, which led to the biosynthesis of lignins. Similarly, under the battle of differential expression of genes involved in anthocyanin and flavonoids biosynthesis, where some iso-forms of the same enzymes were up-regulated and some of them showed down-regulation, production of both the chemicals were uplifted in infected plants. Finally, when it came to stress-related genes, interestingly, cold-induced genes were up-regulated and drought and heat responsive genes were found to be down-regulated. It was due to increased production of various solutes as a result of enhanced secondary metabolism, which acted as osmoprotectants and preventing host water loss.

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It is very well known that ABA plays vital role in drought stress by maintaining stomata closure and thereby reducing the water less. However, as a consequence of these mechanisms, many ROS such as H2O2, O2− (suoeroxide), and OH (hydroxyl), which are further taken care by the action of SOD, POD, catalase, etc. Up-regulation of these enzymes’ genes indicates plant’s ability to combat against such stresses. Nevertheless, many a times, plants require stimulation or help from the symbiosis (Abedi and Pakniyat, 2010). In their studies with maize seedlings, Kumar et al. (2009) inoculated P. indica and found that there was an increase in activity of catalase, glutathione reductase, glutathione S-transferase, and SOD. They also observed the decreased colonization of Fusarium verticillioides with increasing colonization of P. indica. However, they observed these changes through monitoring of enzyme activity.

10.5  Conclusion and future prospects Being an integral part of the plant’s life cycle, endophytes play, more or less, supportive role to help plant to cope up with adverse environmental conditions such as biotic and abiotic stresses. Fungal endophytes, in particular, have their own machinery to synthesize many chemicals, which directly or indirectly help for sustainability of their hosts. The first and most important factor for endophyte to reside in its host is to create the niche under which their existence can be secured. After successful establishment of intrinsic colonization, fungal endophytes start functioning. When plants are attacked by pathogen, these endophytes produce chemicals or chemical signaling molecules, which combat with pathogen. In case of signaling molecules, PR proteins are induced, synthesized, and transported to the site of action, upon which the process of pathogenesis slows down or stopped completely. There are several types of endophytes in these processes apply to all. In a combat with biotic or abiotic stresses, both endophytes and plants produce proteins, enzymes, or secondary metabolites (as a result of specific gene expression). Gene expression with this regard is a primary response to adverse conditions. Thus it is very important to know how these genes are expressed and what are the factors regulating them. Various methods for gene expression analysis are available, some of them need sequence information and some of them do not. RNA-Seq, SSH and microarray are the best methods for the identification of differentially expressed genes. RNA-Seq provides more information than microarray; still microarray technology is preferred one because of time saving and user-friendly protocols. However, real-time PCR has to be carried out in order to confirm the result of sequencing and microarray. When a single gene is expressed, it is a result of many metabolic processes each of which involves the synthesis of proteins or transcription factors serving as regulatory factors, and thus gene expression studies are important because it helps to identify quality and quantity of these factors through which molecular mechanism between plant–microbe interactions can be understood. Numerous works have been carried out based on NGS technologies for metagenomics, which has a limitation of identifying presence and absence of microbial communities associated with plant during plant–microbe interactions. Metatranscriptomic approach, on the other hand, highlights functional part of the plant–microbe interactions.

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Sun, X., Guo, L.D., 2012. Endophytic fungal diversity: review of traditional and molecular techniques. Mycology 3 (1), 65–76. Tadych, M., Bergen, M., Dugan, F.M., White, Jr., J.F., 2007. Evaluation of the potential role of water in spread of conidia of the Neotyphodium endophyte of Poa ampla. Mycol. Res. 111 (4), 466–472. Thongsandee, W., Matsuda, Y., Ito, S., 2012. Temporal variations in endophytic fungal assemblages of Ginkgo biloba L. J. Forest Res. 17 (2), 213–218. Tocher, D.R., Leaver, M.J., Hodgson, P.A., 1998. Recent advances in the biochemistry and molecular biology of fatty acyl desaturases. Prog. Lipid Res. 37 (2–3), 73–117. Vieira, W.A.S., Sami, J.M., de Morais, Jr., M.A., Hyde, K.D., Câmara, M.P.S., 2014. Endophytic species of Colletotrichum associated with mango in northeastern Brazil. Fungal Divers. 67 (1)doi: 10.1007/s13225-014-0293-6. Vujanovic, V., Vujanovic, J., 2007. Mycovitality and mycoheterotrophy: where lies dormancy in terrestrial orchid and plants with minute seeds? Symbiosis (Rehovot) 44 (1/3), 93–99. Wagner, M.R., Lundberg, D.S., Tijana, G., Tringe, S.G., Dangl, J.L., Mitchell-Olds, T., 2016. Host genotype and age shape the leaf and root microbiomes of a wild perennial plant. Nat. Commun. 7, 12151. Walker, A.K., Frasz, S.L., Seifert, K.A., Miller, J.D., Mondo, S.J., LaButti, K., Lipzen, A., Dockter, R.B., Kennedy, M.C., Grigoriev, I.V., Spatafora, J.W., 2016. Full genome of Phialocephala scopiformis DAOMC 229536, a fungal endophyte of spruce producing the potent anti-insectan compound rugulosin. Genome Announc. 4 (1), e01768-15. Walters, D.R., 2000. Polyamines in plant-microbe interactions. Physiol. Mol. Plant Pathol. 57 (4), 137–146. Wang, X., Zhang, X., Liu, L., Xiang, M., Wang, W., Sun, X., Che, Y., Guo, L., Liu, G., Guo, L., Wang, C., Yin, W., Stadler, M., Zhang, X., Liu, X., 2015. Genomic and transcriptomic analysis of the endophytic fungus Pestalotiopsis fici reveals its lifestyle and high potential for synthesis of natural products. BMC Genomics 16 (1), 28. doi: 10.1186/s12864-014-1190-9. Waqas, M., et al., 2015. Endophytic fungi promote plant growth and mitigate the adverse effects of stem rot: An example of Penicillium citrinum and Aspergillus terreus. J. Plant Interact. 10 (1), 280–287. Washington, W.S., Stewart-Wade, S., Hood, V., 1999. Phomopsis castanea, a seed-borne endophyte in chestnut trees. Aust. J. Bot. 47, 77–84. Weber, A.P., 2015. Discovering new biology through sequencing of RNA. Plant Physiol. 169 (3), 1524–1531. Wicklow, D.T., Roth, S., Deyrup, S.T., Gloer, J.B., 2005. A protective endophyte of maize: Acremonium zeae antibiotics inhibitory to Aspergillus flavus and Fusarium verticillioides. Mycol. Res. 109 (5), 610–618. Wikee, S., Lombard, L., Crous, P.W., Nakashima, C., Motohashi, K., Chukeatirote, E., Alias, S.A., Eric, H.C., McKenzie, E.H.C., Hyde, K.D., 2013. Phyllosticta capitalensis,a widespread endophyte of plants. Fungal Divers 60doi: 10.1007/s13225-013-0235-8. Xu, X.H., Su, Z.Z., Wang, C., Kubicek, C.P., Feng, X.X., Mao, L.J., Wang, J.Y., Chen, C., Lin, F.C., Zhang, C.L., 2014. The rice endophyte Harpophora oryzae genome reveals evolution from a pathogen to a mutualistic endophyte. Sci. Rep. 4, 5783. Xu, X., He, Q., Chen, C., Zhang, C., 2016. Differential communications between fungi and host plants revealed by secretome analysis of phylogenetically related endophytic and pathogenic fungi. PloS One 11 (9), e0163368. Yang, H., Wang, Y., Zhang Z, R., Zhu, D., 2014. Whole-genome shotgun assembly and analysis of the genome of Shiraia sp. strain Slf14, a novel endophytic fungus producing Huperzine A and Hypocrellin A. Genome Announc. 2 (1), e00011-14.

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Yang, Y., Zhao, H., Barrero, R.A., Zhang, B., Sun, G., Wilson, I.W., Xie, F., Walker, K.D., Parks, J.W., Bruce, R., Guo, G., Chen, L., Zhang, Y., Huang, X., Tang, Q., Liu, H., Bellgard, M.I., Qiu, D., Lai, J., Hoffman, A., 2014. Genome sequencing and analysis of the paclitaxelproducing endophytic fungus Penicillium aurantiogriseum NRRL 62431. BMC Genom. 15 (69)doi: 10.1186/1471-2164-15-69. Zuccaro, A., Lahrmann, U., Güldener, U., Langen, G., Pfiffi, S., Biedenkopf, D., Wong, P., Samans, B., Grimm, C., Basiewicz, M., Murat, C., Martin, F., Kogel, K.H., 2011. Endophytic life strategies decoded by genome and transcriptome analyses of the mutualistic root symbiont Piriformospora indica. PLoS Pathol 7 (10), e1002290.

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Endophytic fungi from medicinal plants: biodiversity and biotechnological applications

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Kusam Lata Ranaa, Divjot Koura, Tanvir Kaura, Rubee Devia, Chandranandani Negia, Ajar Nath Yadava, Neelam Yadavb, Karan Singhc, Anil Kumar Saxenad a Department of Biotechnology, Eternal University, Baru Sahib, Sirmour, Himachal Pradesh, India; bGopi Nath P.G. College, Veer Bahadur Singh Purvanchal University, Salamatpur, Uttar Pradesh, India; cDepartment of Chemistry, Eternal University, Baru Sahib, Sirmour, Himachal Pradesh, India; dICAR- National Bureau of Agriculturally Important Microorganisms, Mau, Uttar Pradesh, India Chapter outline head 11.1 Introduction  273 11.2 Endophytic fungi from medicinal plants  274 11.3 Biodiversity and distribution of fungal endophytes  275 11.4 Biotechnological applications  283 11.4.1 Production of novel anticancerous compounds  286 11.4.2 Cytotoxic secondary metabolites  291 11.4.3 Bioactive compounds for human health  291

11.5 Conclusion and future prospects  293 References  294

11.1 Introduction Microorganisms residing within the interior tissues of plant, spend the whole or part of their life cycle without causing any noticeable symptoms of infection to the host plants are referred to as endophytes (Petrini and Fisher, 1990; Strobel, 2012). Endophytic lifestyle of microbes plays an important part in maintaining the health of plants by providing nutrient and defense to the plants both against biotic and abiotic stress factors. Due to ubiquitous nature of endophytic fungi and their contribution in therapeutic use, in the present time they are the center of attraction for the scientists (Dias et al., 2012). Endophytic fungi are eukaryotic organisms, widely spread in nature. The word endophyte was first proposed in 1866 where “endo” meaning “within” and “phyte” meaning plant (Bary, 1866). There are about 1.5 million species of different fungi exist on our planet, out of which one million of them are endophytic in nature (Strobel and Daisy, 2003). They mostly reside in the internal tissues of roots, stems, leaves, flowers, and seeds (Bacon and White, 2000). The endophytes may be transferred either horizontally or vertically (Hartley and Gange, 2009). Microbial Endophytes. http://dx.doi.org/10.1016/B978-0-12-819654-0.00011-9 Copyright © 2020 Elsevier Inc. All rights reserved.

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Endophytic microbiomes play vital role in enhancing the growth of plants and also well known for their capability to produce bioactive compounds possessing application in biotechnology. In pharmaceutical and agrochemical industries the bioactive compounds known for their enormous applications. As in the present scenario, in the field of mycology enormous work has been performed for the production of bioactive compounds. In the developing countries about 80% of people use medicines derived from medicinal herbs. From the longtime medicinal herbs have been utilized as a vital resource of medicinal products. Medicinal plants are known for rich source of natural products and are extremely valued for the prevention of diseases and ailments (Pan et al., 2013; Yirga et al., 2011). From the medicinal plants different compounds of having applications in pharmaceutical industry used in anticancer agents, contraceptives, analgesics, antibiotics, diuretics, laxatives, etc. About 8000 different types of medicinal plants revealed for usage in dietary supplements, medicines, biocidal products, and other phytochemicals. The natural products or their byproducts are derived from microbes, plants, or animals (Strobel and Daisy, 2003). Endophytic fungi synthesizing bioactive compounds have diverse series of natural properties, for instance, antidiabetic, antibiotic, anticancerous, antimicrobial, antioxidant, and anti-inflammatory (Ruma et al., 2013). The endophytic fungi synthesizing different compounds are grouped into different categories such as, aliphatic metabolites, alkaloids, flavonoids, glycosides, lactones, phenyl propanoids, quinones, steroids, terpenoids, and xanthones (Zhang et al., 2006). The various studies have reported the Alternaria sp. (Host Catharanthus roseus) well-known to synthesize bioactive substances such as alkaloid, vinbrastin (Guo et al., 1998), Aspergillus niger and Alternaria alternata (Host Tabebula argentea) produces lapachol (Sadananda et al., 2011), Aspergillus niger (Host Taxus baccata) produces lovastatin (Raghunath et al., 2012), Colletotrichum gloeosporioides (Host Forsythia suspensa) produces phillyrin (Zhang et al., 2012), Dendryphion nanum (Host Ficus religiosa) produces antidiabetic compound (Mishra et al., 2013), Endophytic fungus Stachybotrys chartarum produces chartarlactams A-P and phenylspirodrimanes which exhibited potent antihyperlipidemic activity (Li et al., 2014b), Aspergillus flavus (Host Aegle marmelos) produces bioflavonoid (Patil et al., 2015), etc. Endophytic fungi residing inside the plants are natural synthesizers of chemicals (Owen and Hundley, 2004). These natural products or chemicals represent as the major storehouse for the discovery of novel substances with various activities for example antibiotic drug, antioxidants, anticancerous, etc., possessing an essential part in agronomy, pharmacy, and cosmetic sectors (Tan and Zou, 2001). Recently, more focus has been implemented for the isolation of fungal endophytes from medicinal plants with potential of synthesis of bioactive compounds.

11.2  Endophytic fungi from medicinal plants Endophytes have been isolated from all the different parts of plant, for example, roots, stem, leaves, fruits, flowers, bark, and scales (Pirttilä et al., 2008). After sampling, the plant samples assembled in an autoclaved bag should be processed within a proper

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time. To lessening the chances of contagion fresh plant material must be utilized for isolation of endophytes. The plant parts must be washed under the running tap water for the elimination of dust and debris (Radu and Kqueen, 2002). One of the crucial procedures for surface disinfection is the use of disinfectants by which external area of explants is sanitized to confirm that the isolates are endophytes (Schulz et al., 1993). After washing plant, with the sterilized cutting edge small piece of plant 2–4 mm should be cut and explant are sterilized by successive immersion in 70% ethanol for 1–2 min, 1%–6% sodium hypochlorite for 2–5 min or 15% hydrogen peroxide solution for 1 min, than rinsed in 70% ethanol solution for 1 min or 75% ethanol for 0.5 min, and washed in sterilized distilled water twice or thrice times (Rubini et al., 2005; Guo et al., 2000b; Raviraja et al., 1996). During fungal endophytes isolation from different parts of plant extremely sterilization should be maintained. Using a mortar and pestle the required samples must be immersed individually with 10 mL sterile 0.85% NaCl and further regulated for 60 s at high speed. Further using the standard serial dilution plating technique and enrichment method fungal endophytes may be isolated and incubated for 5, 15, and 30 days for the isolation of fast, medium, and slow growing fungal endophytes. Morphological and molecular techniques are important for the characterization of fungi, morphologically fungi were characterized on the basis of colony, color, size of spore, texture shape, growth rate, shape, and attachment of conidia (Domsch et al., 2007; Shan et al., 2012). For long-term preservation method, the spores and mycelia of fungal cultures collected in 20% glycerol in milli Q water (v/v) and preserved at −80̊C. Modern techniques of molecular biology including genomic DNA isolation, amplification of ITS gene, and sequencing of desired gene may be carried out for the identification of fungal isolate. For phylogeny and taxonomical relationship PCRamplified ITS gene should be sequenced and linked with already available database presented in the NCBI database with help of MEGA software.

11.3  Biodiversity and distribution of fungal endophytes The endophytic fungi have been isolated from the different medicinal plants. These endophytic fungi produced a wide range of industrially important bioactive compounds (Table 11.1). Older parts of endemic plant Cordemoya integrifolia such as leaves, petioles were colonized higher by fungal endophytes than comparatively younger leaves. The most dominant fungal endophytes are Pestalotiopsis sp. and Penicillium (Toofanee and Dulymamode, 2002). A total of 582 fungal endophytes were isolated from 81 Thai medicinal plant species (Wiyakrutta et al., 2004). By the microplate alamarBlue assay, extracts of 92 isolates fungal endophytes could inhibit Mycobacterium tuberculosis; Plasmodium falciparum inhibited by extracts of six fungal endophytic 40 isolates possessing antiviral activity against Herpes simplex virus type 1. The results suggested from Thai medicinal plants and different fungal endophytes synthesizes novel bioactive compounds. Medicinal plants capable of producing one or more bioactive compounds were studied for the association of endophytic fungi with these host plants. Puri et al. (2005) in their study reported that the endophytic fungus Entrophospora infrequens, isolated from the interior bark of medicinal

276

Table 11.1  Endophytic fungi with potential for producing bioactive compounds Host

Compound

References

Aspergillus flavus

Corchorus olitorius (jute)

Trichoderma atroviride

Lycoris radiate

Wulandari and Suryantini (2018) Zhou et al. (2017)

Rhizoctonia solani Microthyriaceae Dothiorella sp Stemphylium sp Penicillium sp Hypocreavirens Chaetomium globosum Aspergillus niger Aspergillus oryzae Trichoderma stromaticum Nodulisporium sp Fusarium subglutinans P. microspora Aspergillus terreus Fusarium sp. Colletotrichum gloeosporioides Cytonaema sp Xylaria, Phoma, and Hypoxylon spp. Pestalotiopsismicrospora Emericella sp Xylaria sp Hormonema sp Chloridium sp

Cyperusrotundus Paspalumconjugatum Avicennia marina Brguiera sp Cerberamanghas Premnaserratifolia Nymphaea nouchali Colpomeniasinuosa Hetero Cyperusrotundus Bontiadaphnoides Tripterygium wilfordii Timoniusmorobensis Artemisia annua Opuntia dillenii Vitex negundo

4-nitrobenzoic acid, 3-chlorophenyl ester (27.23%) and (+)-salsolidine (21.82%) 3-amino-5-hydroxy-5-vinyl-2-cyclopenten1-one dimer, atrichodermone A, atrichodermone B, and atrichodermone C Solanioic acid Sterigmatocystin Cytosporone B Infectopyrones A and B Pinazaphilones A and B Gliotoxin and Bisdethiobis gliotoxin Chaetoglobosin A and C Asperamide A, B

Torrya taxifolia Astragalus lentiginosus Anoectochilussetaceus Juniperuscommunis L Azadirachtaindica

Ethanolic extract Nodulisporic acid A Subglutinols A and B Pestacin and Isopestacin Acetyl choline esterase Equisetin Methanol Cytonic acid A and B Cytochalasins Torreyanic acid Secoemestrin D Helvolic acid Enfumafungin Naphthaquinone

Ratnaweera et al. (2015b) Almeida et al. (2014) Xu (2005) Zhou et al. (2014) Liu et al. (2015) Ratnaweera et al. (2016) Dissanayake et al. (2016) Zhang et al. (2007) Qiao et al. (2010) Ratnaweera et al. (2015b) Ondeyka et al. (1997) Lee et al. (1995) Harper et al. (2003) Ge et al. (2010) Ratnaweera et al. (2015a) Arivudainambi et al. (2011) Guo et al. (2000a) Wagenaar et al. (2000) Lee et al. (1996) Xu et al. (2013) Ratnaweera et al. (2014) Aly et al. (2011) Kharwar et al. (2009)

Microbial Endophytes

Fungal endophyte

Alternaria sp. Cryptosporiopsis quercina Cryptosporiopsis quercina Pestalotiopsis microspore Pestalotiopsis microspore Fusarium subglutinols Alternaria sp Fusarium oxysporum Chaetomium globosum Cephalosporium sp

Tropical plant

Compound Phomol Eupenicinicols A and B Volatile hydrocarbons Volatile hydrocarbons Volatile hydrocarbons demethylasterriquinone B-1, L-783,281 Ambuic acid

References Weber et al. (2004) Li et al. (2014a) Stinson et al. (2003) Tomsheck et al. (2010) Banerjee et al. (2010) Strobel et al. (2004) Li et al. (2001)

Nothapodytes foetida Cynodon dactylon Cantharanthus roseus Prumnopitys andina

Campothecin Naptha-y-pyrone Vincristine Peniprequinolone

Sabina vulgaris Tvipterigeum wilfordii Tvipterigeum wilfordii Torreya taxifolia Torreya taxifolia Taxus cuspidata Catharanthus roseus Catharanthus roseus Hypericum perforatum Paris polyphylla var. yunnanensis Azadirachta indica Nothapodytes foetida Apodytes dimidiate Sinopodophyllum hexandrum Sabina recurve Boswellia sacra

Podophyllotoxin Cryptocandin Cryptocin Pestaloside Torreyanic acid Subglutinols A & B Vinblastine Vincristine Hypericin Diosgenin

Puri et al. (2005) Zhang and Qi-Yong (2007) Kumar et al. (2013) Schmeda-Hirschmann et al. (2005) Eyberger et al. (2006) Strobel et al. (1999) Li and Strobel (2001) Lee et al. (1995) Lee et al. (1996) Kim et al. (2004) Li et al. (2004) Wang et al. (2006b) Kusari et al. (2008) Jin et al. (2004)

Azadirachtin Camptothecin Camptothecin Podophyllotoxin Podophyllotoxin Extracellular enzymes and Auxins (IAA)

Kusari et al. (2012) Puri et al. (2005) Shweta et al. (2010) Trivedi (1970) Kour et al. (2008) Khan et al. (2016)

277

Eupenicillium parvum Entrophospora infrequens Fusarium solani Alternaria sp Fusarium oxysporum Penicillumcitrinum, Preussia sp., Aureobasidium

Host Erythrina crista-galli Xanthium sibiricum Eucryphia cordifolia Perseaindica Ginkgo biloba

Endophytic fungi from medicinal plants: biodiversity and biotechnological applications

Fungal endophyte Phomopsis sp Eupenicillium sp Gliocladium roseum Hypoxylon sp Muscodoralbus Pseudomassari sp. Pestalotiopsis spp. and Monochaetia sp. Entrophospora infrequens Aspergillus niger Fusarium oxysporum Penicillium janezewskii

(Continued)

Fungal endophyte Fusarium oxysporum Penicillium chrysogenum Penicillium sp. Eupenicillium parvum by Fusarium proliferatum (MTCC 9690) Aspergillus niger Alternaria Fusarium oxysporum Curvularia sp., Choanephora Infundibuliphera Eupenicillium parvum Phomasp Diaporthephaseolorum, Trichoderma sp Fusarium solani

Compound Ginkgolide-B Huperzine A

References Cui et al. (2012) Zhou et al. (2009)

Azadirachtin A Rohitukine

Kusari et al. (2012) Kumara et al. (2012)

Resveratrol

Liu et al. (2016)

Vinblastine Vincristine Vindoline

Guo et al. (1998) Zhang et al. (2000) Pandey et al. (2016)

Azadirachtaindica Arisaemaerubescens Taxus wallichiana var. mairei

b-sitosterol Bacctatin III

Kusari et al. (2012) Wang et al. (2012) Li et al. (2015)

Camptotheca accuminata, Apodytes dimidiata Podophyllum peltatum Taxus brevifolia

Camptothecin

Shweta et al. (2010)

Podophyllotoxin Taxol (Paclitaxel)

Eyberger et al. (2006) Kusari et al. (2014)

Crocus sativus

(-)-(1R,4R)-1,4-(2,3)-indolmethane-1methyl-2,4-dihydro-1H-pyrazino-[2,1-b]quinazoline-3,6 dione Cytochalasin N, Cytochalasin H and Epoxycytochalasin H Epipolythiodioxopiperazine and Gliotoxin Phomenone, Phaseolinone

Zheng et al. (2012)

Phomopsis sp.

Gossypium hirsutum

Chaetomium globosum Xylaria sp

Ginkgo biloba Piper aduncum

Fu et al. (2011) Li et al. (2011) Silva et al. (2010)

Microbial Endophytes

Phialocephalafortinii Taxomycesandreanae and other several sp Penicillium vinaceum

Host Ginkgo biloba Lycopodium serratum Huperzaseretta Azadirachta indica A. Juss Dysoxylum binectariferum Hook.f Wine grape Carbernet Sauvignon Catharanthus roseus Catharanthus roseus Catharanthus roseus

278

Table 11.1  Endophytic fungi with potential for producing bioactive compounds (Cont.)

Host Polysiphoniaurceolata Nothapodytesfoetida Camptothecaacuminata Camptothecaacuminata Imperatacylindrica Cynodondactylon Mediterranean green alga Tripterygium wilfordii Ulva pertusa Adenophoraaxilliflora Artemisia annua Rhizophora mucronata Camellia sinensis Polysiphoniaviolacea Leaf, Hala-Bala Forest

Compound Chaetopyranin 1 Camptothecin 2 Camptothecin 2 Camptothecin 2 Chaetoglobosin U 6 9-Deacetoxyfumigaclavine 11 Emindole DA 12 Cytochalasin 1 13 Cytoglobosin C 17 Chaetominine 19 Daldinone C 20 Pestalotiopsone F 22 Pestaloficiol I 23 Epiepoxydon 27 Depsidone 1 28

Fusarium oxysporum PM0651480 Phomopsis longicolla ZSU44 Chaetomium globosum

Ephedra fasciculata Mimosopselengi Dicerandrafrutescens Mangrove plant Ephedra fasciculata

Beauvericin 29 Ergoflavin 30 Dicerandrol A 31 Secalonic acid D 34 Globosumone A 35

Aspergillus clavatus & Paecilomyces sp. Acremonium sp.

Taxus mairei & Torreya grandis Knemalaurina

Brefeldin A 37

Chaetomium chiversii Pestalotiopsisphotiniae Eutypella sp.

Ephedra fasciculata Roystonea regia Etlingeralittoralis

Radicicol 38 Photinides A–F 39–44 Eutypellin A 45

Brefeldin A 37

References Wang et al. (2006a) Puri et al. (2005) Rehman et al. (2008) Kusari et al. (2009b) Ding et al. (2006) Ge et al. (2009) Kralj et al. (2006) Lee et al. (1995) Cui et al. (2010) Jiao et al. (2006) Gu et al. (2007) Xu et al. (2009) Liu et al. (2009b) Klemke et al. (2004) Pittayakhajonwut et al. (2006) Zhan et al. (2007) Deshmukh et al. (2009) Wagenaar and Clardy (2001) Zhang et al. (2009) Bashyal et al. (2005); Wang et al. (2002) Wang et al. (2002) Chinworrungsee et al. (2008) Turbyville et al. (2006) Ding et al. (2009) Isaka et al. (2009)

279

(Continued)

Endophytic fungi from medicinal plants: biodiversity and biotechnological applications

Fungal endophyte Chaetomium globosum Entrophosporainfrequens Neurospora crassa Fusarium solani Chaetomium globosum Aspergillus fumigatus Emericellanidulans Rhinocladiella sp Chaetomium globosum Chaetomium sp IFB-E015 Hypoxylontruncatum Pestalotiopsis sp Pestalotiopsisfici Apiosporamontagnei BCC 8616

Host Podophyllum hexandrum Podophyllum peltatum Taxus baccata Polygonum senegalense Polygonum senegalense Aegicerascorniculatum Excoecariaagallocha

Emericellanidulans var. acristata

Mediterranean green alga

Fusarium oxysporum Aspergillus parasiticus Talaromyces sp Thielaviasubthermophila Thielaviasubthermophila Aspergillus niger Pestalotiopsismicrospora Stemphylium globuliferum Chaetomium sp Xylaria sp

Cylindropuntiaechinocarpus Sequoia sempervirens Seaweed Hypericum perforatum Hypericum perforatum Cynodondactylon Torreya taxifolia Mentha pulegium Salvia officinalis Sandoricumkoetjape

Halorosellinia sp &Guignardia sp Mycelia sterilia Preussia sp Taxomycesandreanae Pestalotiopsismicrospora Pestalotiopsisterminaliae

Mangrove plant Knightiaexcelsa Aquilaria sinensis Taxus brevifolia Taxus wallichiana Terminalia arjuna

Compound Podophyllotoxin 46 Podophyllotoxin Leucinostatin A 47 Alternariol 51 Altenusin 54 Leptosphaerone C 56 2-(70-Hydroxyoxooctyl)-3-hydroxy-5methoxybenzene Arugosin A 59 Arugosin B 60 Bikaverin 61 Sequoiamonascin B 65 Kasanosin A 66 Hypericin 68 Emodin 69 Rubrofusarin B 70 Torreyanic acid 71 Mixture of alterporriol G &alterporriol H 72 Cochliodinol 74 2-Chloro-5-methoxy-3-methylcyclohexa2,5-diene-1,4-dione 76 Anthracenedione 1 78 Spiromamakone A 84 Spiropreussione A 85 Paclitaxel 87 Paclitaxel 87 Paclitaxel 87

References Puri et al. (2006) Eyberger et al. (2006) Strobel and Hess (1997) Aly et al. (2008) Aly et al. (2008) Lin et al. (2008) Huang et al. (2009) Kralj et al. (2006) Zhan et al. (2007) Stierle et al. (2003) Kimura et al. (2008) Kusari et al. (2009a) Kusari et al. (2009a) Song et al. (2004) Lee et al. (1996) Debbab et al. (2009a) Debbab et al. (2009b) Tansuwan et al. (2007) Zhang et al. (2010a) van der Sar et al. (2006) Chen et al. (2009) Stierle et al. (1993) Stierle et al. (1993) Gangadevi and Muthumary (2008)

Microbial Endophytes

Fungal endophyte Trameteshirsuta Phialocephalafortinii Acremonium sp. Alternaria sp. Alternaria sp. Penicillium sp. Phomopsis sp.

280

Table 11.1  Endophytic fungi with potential for producing bioactive compounds (Cont.)

Host Aegle marmelos

Compound Paclitaxel 87

Eutypellasp KLAR 5

Etlingeralittoralis Knemalaurina

Phyllostictaspinarum XG8D (a basidiomycete)

Platycladusorientalis Xylocarpusgranatum

ent-4(15)-Eudesmen-11-ol-1-one 88 8-Deoxy-trichothecin 89 7α Hydroxytrichodermol Trichothecolone 90 7α-Hydroxyscirpene 92 Tauranin 93 Merulin A 94

Phomopsis sp

Tectona grandis

Phomoxanthone A 99

References Gangadevi and Muthumary (2009) Isaka et al. (2009) Chinworrungsee et al. (2008)

Wijeratne et al. (2008) Chokpaiboon et al. (2010) Isaka et al. (2001)

Endophytic fungi from medicinal plants: biodiversity and biotechnological applications

Fungal endophyte Bartaliniarobillardoides

281

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Microbial Endophytes

plant Nothapodytes foetida, produced anticancer drug compound Camptothecin. The fungal endophytes isolated from five different species of Garcinia plants by surface sterilization of leaves and branches. By agar diffusion method the antimicrobial activity of fungi were tested. Molecular identification of fungal isolates based on rRNA gene sequence analysis demonstrate that the isolates belong to Phomopsis sp., Botryosphaeria sp. displayed the strongest antimicrobial activity (Phongpaichit et al., 2006). Chen et al. (2007) demonstrated that from the roots of Bruguiera gymnorhiza, fungal endophyte Penicillium thomi was isolated. The isolation of fungal endophyte lead to the isolation of novel compound 4’,5 dihydroxy-2,3 dimethoxy 4(-hydroxy propyl)-biphenyl. Their cytotoxic effect was investigated against three human cell lines. From 29 Chinese medicinal plants 1160 endophytic fungi were isolated, Colletotrichum, Phoma, Phomopsis, and Xylariales were the main isolates and mostly synthesizes phenolic compounds (Huang et al., 2008). The fungal endophyte Bartalinia robillardoides isolated from an important medicinal plant Aegle marmelos and screened for the production of Taxol, an important anticancer drug (Gangadevi and Muthumary, 2008). Liu et al. (2009a) reported Acremonium, Phomopsis, and Pezicula were the dominant genera isolated from Taxus chinensis and screened for Taxol production. Ahmad et al. (2010) in their study reported the plant growth facilitating activity of fungal endophyte Penicillium sp. and Aspergillus sp. Both fungal endophytes were isolated from Monochoria vaginalis, one of the serious weed rice paddy in Korea. Both fungi significantly promoted the growth of plant by increasing root and shoot length during screening experiment and secreted higher amount of gibberellins. Withania somnifera (L.) Dunal is an important tropical medicinal plant belongs to family Solanaceae. For its wide range of therapeutic use in ayurveda it is known as Indian Ginseng. Turmeric (Curcuma longa L.) belongs to Ginger family, used in ayurveda as medicine to treat various diseases. 45 fungal endophytes have been isolated from turmeric plant and the activity of biotransforming curcumin into its derivative compounds using four different growth media potato dextrose broth (PDB), Czapek medium, synthetic low nutrient (SLN), and Sabouraud media. The conformation of biotransformed compound was confirmed by HPLC technique (Prana et al., 2010). Khan et al. (2010) studied the biodiversity of fungal endophytes and their ability to synthesize secondary metabolites from 20 different plants and also reported Alternaria alternata as one of the most dominant fungal endophyte. Xing et al. (2010) reported Phyllosticta sp. fungal endophyte residing in Panax quinquefolium. Aspergillus clavatus fungal endophyte isolated from Azadirachta indica and also biosynthesized silver nanoparticles (AgNPs). Using transmission-electron microscopy, atomic force microscopy, and X-ray diffraction spectrometry characterization of AgNPs carried out. Against Candida albicans, Pseudomonas fluorescens, and Escherichia coli antimicrobial activity was performed. AgNPs possess biomedical application by acting as antimicrobial agent (Verma et al., 2010). Tao et al. (2011) studied the chemical constituents of fungus Fimetariella rabenhorst by column chromatography. The five chemical compounds identified were 4-hydroxy-phenylethyl alcohol, nicotinic acid, d-galacitol, 2-anilino-1, 4-naphthoquinone, N-phenylacetamide. Goveas et al. (2011) for the first time identified 41 endophytic fungi from one of the endangered species of medicinal herb yellow vine. The dominant fungus Phomopsis jacquiniana colonization frequency was 4.6%. 53 fungal endophytes isolated from roots and stem of

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Dendrobium devonianum and D. thyrsiflorum. The dominant species of the two Dendrobium species is common was Fusarium. Phoma sp. displayed strong inhibitory activity against pathogens whereas, Epicoccum nigrum from D. thyrsiflorum also exhibited antibacterial activity. The results suggested Dendrobium species might be a potential source as of antibacterial or antifungal (Xing et al., 2011). Anitha et al. (2013) reported that about 14 fungal endophytes, namely, Aspergillus fumigatus, Aspergillus flavipes, Alternaria alternata, Aspergillus niger, Colletotrichum falcatum, Fusarium oxysporum, Gliocladium roseum, Leptosphaeria species, Nigrospora sphaerica, Pestalotiopsis species, Penicillium senticosum, Phomopsis jacquiniana, Phomopsis archeri, and sterile mycelia were isolated from endemic medicinal plants of Tirumala hills of India. Colletotrichum falcatum fungus found with a colonization frequency of 12.5%. From the medicinal plant lady's glove, grecian foxglove, blond plantain, and air potato a total of 132 microbial endophytes were isolated. The fungal endophytes were further characterized for extraction of bioactive compounds, namely, Cardiac glycosides Digoxin (C41H64O14), digitoxin (C41H64O13), steroidal saponin diosgenin (C27H42O3), glucoside (iridoid family), and aucubin (C13 H19 O8 H2O) (Ahmed et al., 2012). The different parts of plant such as roots, fruits, and bark of S. saponaria L. plant is used as blood depurative, tonics, and cough medicine. Different species of Alternaria, Curvularia, Cochliobolus, Diaporthe, and Phoma were isolated from S. saponaria L and colonized the host plant was confirmed by light and scanning electron microscopy (García et al., 2012). From the twigs of Buddleja asiatica different species of endophytic fungi Alternaria, Cladosporium, Epicoccum, Fusarium, and Phoma were reported (Chhetri et al., 2013). Thymus sp. belongs to family Lamiaceae. It is also used in pharmaceutical, cosmetic, and food industry. Thymus sp. were collected from its natural habitat in Iran. The most abundant genera were Alternaria, Phoma, and Fusarium (Masumi et al., 2015). Su et al. (2012) Xylocarpus granatum belongs to family Meliaceae. The most abundant fungal endophyte isolated is Rhizoctonia, Penicillium, Aspergillus, Trichoderma, and Mucor of Zygomycotina. With Oxford cup assay, the antimicrobial activity checked. Endophytic fungus Fimetariella rabenhorst isolated from Aquilaria sinensis. The Aquilaria sinensis is major source of medicine and perfume belonging to family Thymelaeaceae. Albizia is an important medicinal plant and many are cultivated as ornamental for their attractive flowers. Six fungal endophytes were isolated from Albizia plants were Aspergillus sp., Acremonium sp., Fusarium sp., Penicillium sp., Trichoderma sp., and Verticillium sp. Inoculation of Albizia sprouts with fungal endophytes resulted in increased growth parameters such as percentage of explant root and shoot. Statistical analysis revealed Trichoderma sp. and Fusarium sp. were potential as plant growth regulator under in vitro growth (Wulandari and Suryantini, 2018).

11.4  Biotechnological applications Throughout history, humans have utilized plants and plants derived products for the treatment of various diseases. Plant secondary metabolites or bioactive compounds are known to be synthesized by plants. Microbes inhabiting inside the tissues of host plant also known for their capability to synthesize the similar substances as synthesized by

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the host plant. The secondary metabolites, for example, alkaloids, flavonoids, terpenoids, steroids, etc., synthesized by microbes were well known for their vital role such as antimicrobial, antioxidant, anticancer, antihypercholesterolemic, and antidiabetic (Fig. 11.1). In the future, with increasing population the demand for pharmaceutical and agricultural products is increasing day by day and the future of endophytic fungi for the isolation of various useful compounds is bright (Fig. 11.1).

Figure 11.1  Secondary metabolites of endophytic fungi isolated from medicinal plants.

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Figure 11.1  (Cont.)

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11.4.1  Production of novel anticancerous compounds 11.4.1.1 Aldehyde Chaetopyranin is one of the benzaldehyde derivative isolated from fungal endophytes Chaetomium globosum (Wang et al., 2006a). Chaetopyranin revealed moderate or weak cytotoxic activities with IC50 values of 15.4, 28.5, and 39.1 mg/mL against three human tumor cell lines: HMEC, SMMC-7721, and A549. Using DPPH (1,1-diphenyl-2-picrylhydrazyl) the compound Chaetopyranin was also assessed for its radical scavenging abilities. The compound also revealed temperate activity with an IC50 value of 35 mg/mL, in comparison to an IC50 value of 18 mg/mL for the positive control BHT (butylated Hydroxytoluene) (Wang et al. 2006a).

11.4.1.2 Alkaloids Alkaloids are the low molecular weight, naturally occurring nitrogenous compounds. The structure of alkaloid consisting of carbon, hydrogen, and nitrogen whereas, some of the other alkaloids also contain oxygen, sulfur (McNaught and McNaught, 1997; Khalil, 2017). Plants containing alkaloids has been used by human being thousands of years ago for remedial and entertainment applications. Around 2000 BC, in the Mesopotamia medicinal plants having therapeutic use containing alkaloids were identified (Aniszewski, 2007). Alkaloids derived from plant revealed natural potency from an extent of lethal to therapeutic properties. In many pharmaceutical uses alkaloids are extremely essential bioactive compounds well-known to play an important part leading to improved commercial significance recently (Ahmad et al., 2013). Organisms ranging from microbes to animals and plants are known to produce alkaloids. Alkaloids have been reported to hold extensive series of pharmaceutical actions including malaria control, antiasthma, antitumor, parasympathomimetic, vasodilatory, almokalant, anesthetic, antimicrobial, and anti-hyperglycemic activities (Cushnie et al., 2014; Kittakoop et al., 2014; Russo et al., 2013; Shi et al., 2014; Sinatra et al., 2010). Irinotecan, sold under the brand name Camptosar (anticancer agents), made from the natural compound camptothecin mostly cure colorectal carcinomas, and small cell lung cancer (Shi et al., 2014). Vincristine marketed under the brandname Oncovin is a vinca alkaloid attained from the Madagascar periwinkle Catharanthus roseus utilized for the treatment of acute lymphocytic leukemia, Hodgkin's disease, neuroblastoma (Montgomery, 2017). Fungal endophyte Fusarium oxysporum and Talaromyces radicus isolated from Madagascar periwinkle plant reported to produces vinblastine and vincristine in appreciable amounts, which Induce apoptotic cell death (Kumar et al., 2013; Palem et al., 2015). Endophytic microbes have also been studied to synthesize the products as their host does. Camptothecin (CPT) is one of the pentacyclic quinoline alkaloid acts as an effective antineoplastic agent, during replication of DNA employs its cytotoxic effect by hindering the dissociation of the DNA–topoisomerase I enzyme (Ling-Hua et al., 2003; Pommier, 2006). These alkaloids mostly target the enzyme Topoisomerase I. CPT at a very low concentrations penetrates in the cells of vertebrate and target topo I and also binds to the complex developed by topoisomerase I when it

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cleaves DNA (Wall et al., 1966). A plant Camptotheca acuminata (Nyssaceae) native to mainland China,from the wood of plant primarily camptothecin was derived; it exhibited effective antitumor activities in animals (Wall et al., 1966). In 2005, endophytic fungi Entrophosphora infrequens isolated from Nothapodytes foetida plant, analyzed on the basis of molecular analysis reported to synthesize Camptothecin (Puri et al., 2005). Later on, Camptothecin was also isolated from Cyanea acuminate (Rehman et al., 2008). Camptothecin isolated from fungi were verified as a genuine and can be used as against different human cancer cell lines A549, HEP-2, and OVCAR-5 (Rehman et al., 2008). From Camptotheca acuminate, endophytic fungi Fusarium solani was reported known to synthesize two analogues of Camptothecin as 9-methoxycamptothecin 3 and 10-hydroxycamptothecin 4 and known to inhibit the DNA topoisomerase I (Kusari et al., 2009b). Chaetomium globosum fungal endophyte isolated from Artemisia annua reported for its ability to demonstrate cytotoxic activities against KB, K562, MCF-7, and HepG2 as human cancer cell lines with the production of alkaloids chaetoglobosins V (1) and W (2) (Zhang et al. 2010b). One of the new alkaloid named as chaetoglobosin U (1), reported to be synthesize by Chaetomium globosum fungal endophyte residing within the stem of healthy Imperata cylindrica (Ding et al., 2006). Cytoglobosins C 17 and D 18 two of the novel fungal alkaloids, isolated from fungal endophyte Chaetomium globosum, reported for their ability to possess cytotoxicity profiles against the tumor cell line A549 (Cui et al., 2010).

11.4.1.3 Chromones Chromone or 1, 4-benzopyrone is a derivative of benzopyran, on the pyran ring, keto group is substituted. Novel chromone Pestalotiopsone F 22 reported from the culture filtrate of fungal endophyte Pestalotiopsis sp. exhibited adequate cytotoxic effect against the murine cancer cell line L5178Y (Xu et al., 2009). One of the fungal endophyte Pestalotiopsis fici of Camellia sinensis reported to produce four different chromone derivatives compounds (Liu et al., 2009b).

11.4.1.4 Cyclohexanones The marine fungus Apiospora montagnei reported to produce new secondary metabolites diterpene myrocin A, apiosporic acid, 9-hydroxyhexylitaconic acid, and epiepoxydon. The structures of compounds were illustrated mostly by 1D and 2D NMR, MS, UV, and IR spectral data. The compound epiepoxydon displayed considerable cytotoxic effect against human cancer cell lines (Klemke et al., 2004).

11.4.1.5 Depsidones Depsidones are chemical compounds, sometimes found as secondary metabolites in lichens. They are esters that are both depsides and cyclic ethers, for example, norstictic acid (Hauck et al., 2010). Fungal endophyte belonging to the order Pleosporales was isolated from a leaf collected from the forest of Narathiwat Province, Thailand from which three new depsidones have been isolated, and their structures were analyzed spectroscopically. The compound depsidones revealed

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weak cytotoxic effect against breast and epidermoid carcinoma cell lines (Pittayakhajonwut et al., 2006)

11.4.1.6 Depsipeptides A depsipeptide is a cyclic peptide where one or more of its amide groups -C(O) NHR-, are substituted by the analogous ester, -C(O)OR (Buckingham et al., 2015). Depsipeptides are constructed artificially, for example, used as research tools and are also found in nature. The endophytic strains of Fusarium oxysporum isolated from Ephedra fasciculata and were deposited in the School of Life Sciences, Arizona State University. Beauvericin is a depsipeptide isolated from the fungal endophyte F. oxysporum repressed movement of the metastatic prostate cancer (PC-3M) and breast cancer (MDA-MB-231) cells (Zhan et al., 2007). Beauveria bassiana fungus earlier recognized to be an effective insect pathogen, synthesizes a compound, which is very lethal to brine shrimp. From the culture filtrate of fungus depsipeptide, also called as Beauvericin was isolated (Hamill et al., 1969).

11.4.1.7 Ergochromes Ergoflavin is a member of the class of compounds called ergochromes and has the molecular formula C30H26O14. These compounds were first isolated from the ergot fungus Claviceps purpurea, Phoma terrestris, Pyrenochaeta terrestris, Penicillium oxalicum, and Aspergillus species (Deshmukh et al., 2009). The human TNF-α and IL-6 considerably inhibited by ergoflavin in comparison to dexamethasone is one of the biological properties of ergoflavin (Hiragun et al., 2005). Fungal endophyte isolated from Mimosops elengi an Indian medicinal plant, from which ergoflavin, a pigment was isolated, reported to inhibit the production of TNF-α and IL-6. For evaluating the cytotoxicity of ergoflavin, flavopiridol, a known anticancer compound, was used as a standard (Deshmukh et al., 2009). An endophytic fungus Phomopsis longicola isolated from Dicerandra frutescens, reported to synthesize three different compounds designated as dicerandrols A 31, B 32, and C 33 classified as ergochromes. These compounds revealed considerable cytoxicity effect against the human cancer cell lines, A549 and HCT-116 (Wagenaar and Clardy, 2001). During 1970s for the first time secalonic acid was isolated from Penicillium oxalicum metabolite reported to be enormously lethal and teratogenic (Mayura et al., 1982; Steyn, 1970). From one of the mangrove endophytic fungus ZSU44 Secalonic acid D (SAD) was derived and in an experiment, secalonic acid D reported to exhibited effective cytotoxic effect and also led to cell cycle arrest of G1 phase related to downregulation of c-Myc (Zhang et al., 2009).

11.4.1.8 Esters Orsellinic acid and globosumones esters were isolated from the Chaetomium globosum endophytic fungi on Ephedra fasciculata (Mormon tea). By spectroscopically the structures of these compounds were studied. Against non-small-cell lung cancer, breast cancer, pancreatic carcinoma, and normal human fibroblast cells the orsellinic

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acid and globosumones esters were evaluated for their cytotoxic effect. Globosumones were found to be moderate active (Bashyal et al., 2005).

11.4.1.9 Lactones Brefeldin A is a lactone antiviral, different fungal genera has been reported such as Alternaria, Ascochyta, Penicillium, Curvularia, Cercospora, and Phyllosticta for the synthesis of Brefeldin A (Betina, 1992; Vurro et al., 1998). Brefeldin A has been reported to play various bioactive role, for instance, antifungal, antiviral, and anticancer activity (Demain et al., 1976). For the treatment of cancer, Brefeldin A is a very favorable compound as it shows cytotoxic effect against different cell lines such as Hela, MCF-7, HL-60. Two of the fungal endophyte Aspergillus clavatus and Paecilomyces sp. reported to produce brefeldin A (Wang et al., 2002). Cladosporium sp. isolated from Quercus variabilis also reported to produce secondary metabolite, brefeldin A, with a molecular formula of C16H24O4 (Wang et al., 2007). Eutypellin A was isolated as a yellow, amorphous solid with a molecular formula of C11H12O5 from endophyte Eutypella sp. BCC 13199 reported to exhibit cytotoxic activities (Isaka et al., 2009).

11.4.1.10 Lignans For considering the production of potential anticancer agent based upon natural products prototypes Podophyllotoxins are mostly instructive class of compounds. In 1880, the crystalline substance podophyllotoxin, was isolated first (Podwyssotzki, 1880) and the correct structure was assigned in 1951 (Hartwell and Schrecker, 1951). The name Podophyllotoxins derives from the North American plant Podophyllum peltatum Linnaeus and the Indian species Podophyllum emodi Wallich. Kaplan (1942) reported the topical application of podophyllin in oil. The plant Podophyllum hexandrum (Himalayan Mayapple; family: Berberidiaceae) selected as a source for isolation of the endophyte and reported the production of aryl tetralin lignans including podophyllotoxin by an endophytic fungus Trametes hirsuta and also held the probability of horizontal gene transfer among Podophyllum spp. and its resultant endophytic organism. Due to the potent application of aryl tetralin lignans, as an antioxidant, anticancer they are of high demand globally (Puri et al., 2006). Eyberger et al. (2006) also reported the production of lignan podophyllotoxin from Phialocephala fortinii. Podophyllotoxin is an aryl tetralin lignan, one of the most treasured natural product applied in the cure of a range of malignant conditions and moreover utilized as a strong antiviral agents and as antineoplastic drugs (Canel et al., 2000).

11.4.1.11 Peptides Leucinostatin antibiotic mainly composed of leucine and unidentified amino acids derived from the culture filtrate of fungi Penicillium lilacinum. It was first reported as cytotoxic to HeLa cell culture and also showed some inhibitory effect on Ehrlich subcutaneous solid tumor (Arai et al., 1973). From the Taxus baccata, an endophytic fungi Acremonium sp. was isolated comprising of symbiotic relationship. The culture filtrates of Acremonium sp. potentially consisting of biological properties as anticancer and antifungal (Strobel and Hess, 1997). In another study, in prostate stromal cells,

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Leucinostatin reported to considerably inhibit the expression as well as tumor growth (Kawada et al., 2010).

11.4.1.12 Polyketides Polyketides, leptosphaerone C, penicillenone, arugosin I, and 9-demethyl FR-901235 produced by fungal endophyte Penicillium sp. JP-1. Against A-549 cells, the compound leptosphaerone C displayed its cytotoxic effect while penicillenone showed cytotoxicity against P388 cells (Lin et al., 2008). Pestalotiopsis sp. also reported for synthesis of metabolites pestalotiopyrones, pestalotiopisorin, pestalotiollides, pestalotiopin, and four amides pestalotiopamides and the detailed chemical investigation of metabolites done with the NMR spectroscopy (Xu et al., 2011). The crude extract of fungal cultures Pestalotiopsis clavispora produces six new polyketide derivatives, such as pestalpolyol I, pestapyrones A and B, (R)-periplanetin D, pestaxanthone, norpestaphthalide A, and an isolation artifact pestapyrone. The compound pestalpolyol I exhibited strong cytotoxic effect against the mouse lymphoma cell line L5178Y (Hemphill et al., 2016). Alternaria sp. one of the fungal endophyte reported to synthesize several polyketides including alternariol 51, alternariol 5-O-sulfate 52, and alternariol 5-O-methyl ether 53. As these compounds possess the tendency of cytotoxic to L5178Y mouse lymphoma cells (Aly et al., 2008). From the extract of Phomopsis sp. BCC 9789 six new oblongolides, W1, W2, X, Y, Z, and 2-deoxy-4α-hydroxyoblongolid were isolated and the cytotoxic activity of oblongolides were detected against different cancer cells such as oral human epidermal carcinoma (KB) cells, NCI-H187 cells, and Vero cells (Bunyapaiboonsri et al., 2009).

11.4.1.13 Quinone Torreyanic acid is a dimeric quinone first discovered by (Lee et al., 1996) from an endophyte Pestalotiopsis microspora. The fungus Pestalotiopsis microspora was reported with cytotoxicity against human cancer cell lines. Torreyanic acid brings about cancer cell death by apoptosis (Li et al., 2003). From Mentha pulegium, Stemphylium globuliferum was isolated. Chemical investigation of the fungal extracts revealed the synthesis of different secondary metabolites. The compound alterporriol G and its atropisomer alterporriol H displayed the strong cytotoxicity (Debbab et al., 2009a). Cochliodinol is the main component of secondary metabolites synthesized by Chaetomium sp. endophytic fungus shows cytotoxicity against L5178Y mouse lymphoma cells (Tansuwan et al., 2007). Two novel benzoquinone metabolites, 2-chloro-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione and xylariaquinone A isolated from an endophytic fungus, Xylaria sp. The in vitro cytotoxic activities of both compounds were tested against Plasmodium falciparum and African green monkey kidney fibroblasts (Vero cells) (Tansuwan et al., 2007).

11.4.1.14 Spirobisnaphthalenes The Spirobisnaphthalenes (also called as bisnaphthospiroketals) are a group of naphthoquinone derivatives were first isolated 15 years ago (Weber et al., 1990). The

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compounds in this class are mainly divided into three subclasses: those with two oxygen bridges (Krohn et al., 1994), those with three oxygen bridges (Weber et al., 1990), and those with two oxygen bridges and one C–C bridge (McDonald et al., 1999). Spirobisnaphthalenes reported to exhibit biological activities, including nematicidal, antileishmanial, cytotoxic, antimicrobial, and antitumor whereas, in the treatment of cancer spirobisnaphthalenes compounds are of great interest (Chu et al., 1995; Sakemi et al., 1995; Wipf et al., 2001). In a study, van der Sar et al. (2006) reported spiro-mamakone, new relative of the spirobisnaphthalenes was found to show cytotoxicity (0.33 µM) under in vitro conditions toward the P388 murine leukemia cell line and also mostly effective against the Trichophyton mentagrophytes, Bacillus subtilis, and Cladosporium resinae. The culture extracts of Preussia sp. led to the isolation of three new spirobisnaphthalene analogues, such as spiropreussione, spiropreussione, and spiropreussomerin. The compound spiropreussione showed cytotoxicity toward A2780 and BEL-7404 cells and weak activity against Staphylococcus aureus (CMCC B26003) (Chen et al., 2009). Five spirobisnaphthalenes such as palmarumycin CP17, diepoxin k, diepoxin η, diepoxin ζ, and diepoxin γ were isolated from the acetone extract of the fungal endophyte Dzf12. The diepoxin k found to have antibacterial activity and diepoxin η and diepoxin ζ have both antibacterial and antifungal activities (Cai et al., 2009).

11.4.2  Cytotoxic secondary metabolites About 50 years ago the first chemotherapeutic agent was discovered and mustard gas was used as a chemical warfare agent during “World War-I” (Goodman, 1946). Recently, various anticancer chemotherapeutics, such as tubulin inhibitors, alkylating agents, topoisomerases-I and II targeting the DNA, were mostly prescribed. These compounds mostly target the dividing cancer cells than normal cells. The cytotoxicity assays widely consisting of the discovery of compounds like paclitaxel, camptothecin, and the vinca alkaloids that typically aim the cancer cells (Kharwar et al., 2011; Wu, 2006). The bioactive compounds synthesized by endophytic fungi could be utilized for the invention of novel drugs (Firáková et al., 2007). The fungal endophytes have received less consideration, as they are mostly inhabited inside the tissue of plant. The discovery of novel compounds is positively vital. The various studies reported the discovery of bioactive compounds by fungal endophyte is similar as isolated from higher plants (Stierle et al., 1993).

11.4.3  Bioactive compounds for human health Expansion of world population leads to increase in health problems of human, animal, and plants and increased resistance of pathogen toward drugs. Transmittable diseases are worldwide health challenge because of the drug resistance pathogens. Endophytic fungi have the capability to provide benefit to human by production of bioactive compounds application in pharmacy. Altomare et al. (2000) in his study reported fusapyrone and deoxyfusapyrone as antifungal compounds isolated from fungal

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endophyte Fusarium semitectum isolated from rice cultivar. Both the compounds showed antifungal activity against major pathogenic fungi Penicillium verrucosum, Cryptococcus neoformans Candida albicans, and Aspergillus fumigatus. Healthy leaves and stems of Thai medicinal plant were collected from Provinces of Thailand for isolation of fungal endophytes. The result suggested Thai medicinal plants provide a wide diversity of fungal endophyte having a potential of bioactive compound production (Wiyakrutta et al., 2004). Li et al. (2005) demonstrated fungal endophytes were tested by MTT assay on tumor cell line BGC-823. 9.2% and 30% of isolates described antitumor and antifungal activity. For discovery of natural products of human benefits fungal endophytes in association with medicinal plant are important source. Huang et al. (2007) investigated the correlation between total antioxidant capacity (TAC) and total phenolic content (TPC) for 292 distinct fungal endophytes. Some of the fungal endophytes showed good antioxidant activity. The investigation revealed that the produced metabolites can be a potential basis of novel natural antioxidant. Bark of the tree belonging to family Taxus is a source of Taxol. In the past three decades, Taxol is a compound consisting of treatment against different types cancer, such as, lung cancer, ovarian cancer, head and neck carcinoma, and other types of cancer. Taxol was first isolated from Taxus brevifolia from the fungal endophyte Taxomyces andreanae. The concentration of Taxol found in yew trees is very less, which leads to higher demand in market with high price rate. With the discovery of fungal endophytes producing Taxol leads to the possibility and wide availability of product at cheaper rate. Bartalinia robillardoides was isolated from the leaves of medicinal plant Aegle marmelos from Chennai city. Using HPLC the amount of Taxol production from fungal endophyte was quantified. By using apoptotic assay, result suggested natural product of fungal endophyte has strong cytotoxic activity against human cancer cells (Gangadevi and Muthumary, 2008). One of the most promising drugs of 21st century is Camptothecin (CPT). Both under in vivo and in vitro CPT exhibit antitumor activity (Uma et al., 2008). Secalonic acid D, isolated from a fungal endophyte from mangrove possess strong anticancer activity (Qi et al., 2009). Hazalin et al. (2009) in their study reported that endophytic microbes have potential of producing novel metabolites. A total of 300 fungal endophytes were isolated from National Park Pahang, Malaysia. Sporothrix sp. revealed tough cytotoxic effect against colorectal carcinoma (HCT116) and human breast adenocarcinoma (MCF7) cell lines. Fungal endophyte also shows potent cytotoxic effect against murine leukemia P388 cell line and human chronic myeloid leukemia cell line K562. Gordien et al. (2010) reported fungal endophyte isolated from Vaccinium myrtillus screened for Mycobacterium aurum and M. tuberculosis H37Rv. The fungal endophyte shows best activity against Mycobacterium aurum. The result indicates fungal cultures isolated from Scottish provenance are source of antimycobacterial agent for future. Vennila et al. (2010) carried research on evaluating the anticancer activity of Taxol on mammary tumor. Taxol derived from endophytic fungus Pestalotiopsis pauciseta isolated from Tabebuia pentaphylla medicinal plant. Injecting of fungal Taxol to animals during experiment brought back the blood urea and serum creatinine to normal level. The

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other biochemical parameters like hemoglobin, total cholesterol, protein, triglycerides, and phospholipids were in control. The endophytic fungus Lasiodiplodia theobromae from leaves of Morinda citrifolia was isolated by standard method. Taxol produced from fungal endophyte showed potential toxicity against human breast cancer. The endophytic fungus Lasiodiplodia theobromae serve as potential candidate for production of Taxol by genetic engineering (Pandi et al., 2011). The crude ethyl acetate extract of Phomopsis sp. GJJM07 was tested against the test pathogens. The antimicrobial activity was highest against the test pathogen Bacillus subtilis. By DPPH radical scavenging assay the Phomopsis sp. was also studied for in vitro antioxidant activity (Jayanthi et al., 2011).

11.5  Conclusion and future prospects In the present scenario, there is a huge requirement for the discovery of bioactive compounds from the natural sources, which can be utilized for the treatment of various diseases. Recently, more focus is laid on the production of bioactive compounds from endophytic fungi as they are excellent platforms for exploiting the biosynthetic route for bioactive compound synthesis. Various literatures reported the endophytic fungi isolated from medicinal plants provide a variety of bioactive metabolites which provide the opportunity for researchers for dealing with bioactive compounds of pharmaceutical significance such as alkaloids, peptides, flavonoids, phenolics, taxol, camptothecin, etc. Medicinal plants are a unique source of novel drugs and remedial compounds. About 100,000 different species of angiosperms were used for the medicinal purpose. Endophytic fungi isolated from plant have enlarged the attention of many investigators in basic and applied research fields due to their capability of synthesizing the same compound as originated from their host plant. Major challenge is the low yield of the active desirable compounds obtained from endophytic fungi. However, to meet the demand of pharmaceutical companies for increasing commercial production of medicines genetic engineering technology, proteomic, drug design techniques, microbial fermentation technology, and research consisting of identification of genes involved in the biosynthetic pathway will prove to be an advantage.

Acknowledgments The authors are grateful to the Department of Biotechnology, Akal College of Agriculture, Eternal University, Baru Sahib and Department of Environment, Science & Technology (DEST), Shimla, HP– funded project “Development of Microbial Consortium as Bio-inoculants for Drought and Low Temperature Growing Crops for Organic Farming in Himachal Pradesh” for providing the facilities and financial support, to undertake the investigations. There are no conflicts of interest.

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B. Shankar Naik Department of P.G. Studies and Research in Applied Botany, Kuvempu University, Shimoga, Karnataka, India; Department of Biology, Government Science College, Chikmagalur, Karnataka, India Chapter outline head 12.1 Introduction  307 12.2 Mechanism of mycosynthesis of silver nanoparticles  309 12.2.1 Intracellular synthesis of metal nanoparticles  309 12.2.2 Extracellular synthesis of silver metal nanoparticles  310

12.3 Silver nanoparticles from endophytic fungi and their efficacy in biocontrol  311 12.3.1 Effect of nanoparticles against plant pathogenic fungi  313 12.3.2 Antimicrobial activity of silver nanoparticles from fungal endophytes  314

12.4 Mechanism of antimicrobial action by silver nanoparticles  315 12.5 Factors affecting the mycosynthesis of metal nanoparticles  315 12.6 Nanoparticles in plant disease management  316 12.7 Conclusions  317 References  317

12.1 Introduction Nanotechnology is a branch of science, which deals with synthesis and application of nanosized particles (1–100 nm or 1.0 × 10−9) (Taniguchi, 1974). The term “nano” came from Greek word “nanos” meaning dwarf, which represents a measurement on the scale of 1 billion of a meter in size (Narayanan and Sakthivel, 2010; Pantidos and Horsfall, 2014; Thakkar et al., 2010). Nanoparticles are defined as clusters of atoms with a size range of 1–100 nm (Mohanraj and Chen, 2006; Thakkar et al., 2010). The important feature of these particles is their higher surface area to volume ratio, which results in increased catalytic activities with other particles (Pantidos and Horsfall, 2014). Nanotechnology provides a platform for application of these nanoparticles from different metals in areas such as electronics, catalysis, energy, textile, diagnostics, biomarkers, antiplatelets, cancer and cytotoxic studies Microbial Endophytes. http://dx.doi.org/10.1016/B978-0-12-819654-0.00012-0 Copyright © 2020 Elsevier Inc. All rights reserved.

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Figure 12.1  Methods of metal nanoparticle synthesis.

(Nanda and Majeed, 2014; Singh et al., 2016). Among several metal nanoparticles, silver nanoparticles (AgNPs) have gained increasing interest due to their unique characteristics, which can be tailored for a specific application by modifying size, shape, and morphology (Singh et al., 2017). Several physical and chemical approaches have been made for nanoparticle synthesis, including thermal decomposition, mechanical milling, laser ablations, polyol assisted, and chemical reduction methods (Chol et al., 2005; Simakin et al., 2004) (Fig. 12.1). In physical methods, the involvement of high temperature, radiations, and pressure associated with these methods may cause serious damage organisms and the environment (Alzahrani et al., 2015). Chemical methods are employed because large production of homogenous nanoparticles can be obtained in a relatively short time and control on size and distribution (He et al., 2015). However, chemical methods employ toxic chemicals, energy intensive, and produce hazardous wastes that are a major threat to the environment. Furthermore, physic-chemical methods of nanoparticle synthesis resulted in low production rate, high expenditure, time-consuming, and release toxic chemicals to the atmosphere (Mallick et al., 2004). Biological methods are simple rapid and cost-effective and involve the synthesis of nontoxic, clean and biocompatible nanoparticles. Moreover, biogenic reduction in aqueous medium resulted in stable, homogenous, polydispersed metal nanoparticles (Mallick et al., 2004). Algae, bacteria, actinomycetes, fungi, yeasts, and extracts of plant tissues are known to produce nanoparticles but, among these, fungi are considered as a better source for the stable metal nanoparticle synthesis (Muhsin and Hachim, 2014). Fungi are advantageous over other microbes for the synthesis of nanoparticles due to ease of scaling up downstream processing, economic feasibility, and nature of mycelia (Mukherjee et al., 2001; Pantidos and Horsfall, 2014). Fungi secrete a variety of enzymes/proteins when compared to other microbes such as bacteria, and it can produce metal nanoparticles in a large scale within a short time (El-Moslamy et al., 2017). AgNPs have great potential in a number of industries such as antimicrobials and electronics (Netala et al., 2016). Pure stable AgNPs are synthesized from Fusarium oxysporum with a size range of 5–15 nm extracellularly (Ahmad et al., 2003). AgNPs are considered as effective nanoparticles due to their excellent

Biosynthesis of silver nanoparticles from endophytic fungi and their role in plant disease management 309

electromagnetic, optical, catalytic, and antimicrobial properties against a wide range of disease-causing organisms (Siddiqi and Husen, 2016). Endophytic fungi are the microbes, which live inside healthy plant tissues without causing any overt symptoms. All plants in the natural ecosystems are known to inhabit by fungal endophytes. Endophytic fungi secrete a large number of enzymes, sec metabolites, alkaloids, and novel compounds having importance in medicine and industries (Netala et al., 2015; Yadav et al., 2015). These fungi are suitable for the synthesis of nanoparticles, as they could form large biomass, which can withstand high agitation and flow pressure in bioreactors (Singh et al., 2017).

12.2  Mechanism of mycosynthesis of silver nanoparticles The nanoparticles of a wide range of materials can be prepared by a number of methods, which are categorized into three main groups chemical, physical, and biological (Fig. 1). These methods follow either a top–down approach or bottom–up approach. In the top–down approach, the bulk materials are mechanically grinded and the resulting nanosized particles are stabilized by the addition of colloidal stabilizing agents, whereas in the bottom–up approach, the bulk metals are reduced by electrochemical methods (Amulyavichus et al., 1998). The physic-chemical methods are usually associated with certain disadvantages such as the requirement of expensive equipment and use of toxic reducing agents like sodium borohydride and N-N-dimethylformamide, which produce hazardous effects on the environment and health. Silver nanoparticle have great potential in biomedical applications. Biosynthetic methods come under the bottom–up approach, wherein the AgNPs are produced by reduction via enzymes and other metabolites secreted by biological agents. The use of AgNPs has emerged as a most promising approach for overcoming the antibiotic resistance of microorganisms. Silver is advantageous over other metals, as it exhibits higher toxicity to a broad spectrum of microorganisms and lower toxicity to mammalian cells (Sharma et al., 2014). Fungi can synthesize stable metal nanoparticles both intra/extracellularly in nanoscale with exquisite morphology (Gholami Shabani et al., 2013). However, the exact mechanism nanoparticle synthesis still not understood completely.

12.2.1  Intracellular synthesis of metal nanoparticles In intracellular synthesis, fungal biomass is treated with a metal salt solution and incubated for 2 h in the dark. The purification and downstream processing of metal nanoparticles from fungal biomass is tedious task and require long processing and analytical techniques, whereas in extracellular synthesis, the fungal filtrate is treated with metal salt solution and metal nanoparticles are recovered without the lysis of cell (Duran et al., 2011; Nanda and Majeed, 2014). During intracellular synthesis, the bioreduction of metal nanoparticles occurs below the cell surface due to electrostatic interactions of positively charged groups of enzymes present in the cell membrane (Golinska et al., 2015). Initially, the entrapment of metal ions takes place by lysine residues in the cell membrane (Riddin et al., 2006) Finally the formation of

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nanoparticles is achieved by enzymatic reduction of metal ions with the involvement of cell wall sugars (Mukherjee et al., 2001). However, intracellular synthesis of metal nanoparticles needs intense downstream processing, and particles are very smaller in size (Thakkar et al., 2010), whereas extracellular synthesis is cost-effective, faster, easier, and need simple downstream processing (Devi and Joshi, 2015). Few reports are available on intracellular synthesis of nanoparticles from fungi. Verticillium sp. is able to synthesis silver (Sastry et al., 2003) and gold (Mukherjee et al., 2001) intracellularly. Similarly, AgNPs were synthesized extracellularly from Phoma sp. (Rai et al., 2015), Aspergillus fumigatus (Ahmad et al., 2003; Bhainsa and D'Souza, 2006), and Fusarium oxysporum (Ahmad et al., 2003).

12.2.2  Extracellular synthesis of silver metal nanoparticles Fungi produce several extracellular metabolites when exposed to biotic/abiotic stresses naturally for their survival, which can reduce metal ions into nanoparticles (Mehra and Winge, 1991). During this process, toxic metal ions are reduced to nontoxic metallic nanoparticles through catalytic activities of enzymes/metabolites (Vahabi et al., 2011) (Fig. 12.2).

Figure 12.2  Green synthesis of metal nanoparticles.

Biosynthesis of silver nanoparticles from endophytic fungi and their role in plant disease management 311

Several mechanisms have been proposed for the extracellular synthesis of metal nanoparticles. (Duran et al., 2011). NADH-dependent reductase known to involve in the reduction of Ag+ ions and, subsequently, the formation of AgNPs by Fusarium oxysporum and Aspergillus terreus (Ahmad et al., 2003; Li et al., 2012). Anthraquinone and NADPH-nitrate reductase with excellent redox properties were responsible for the reduction of AgNPs, where quinone and NADPH acted as electron shuttle to fulfill the deficiency of aqueous silver ions (Ag) and convert it into Ag neutral (Ag0) (Duran et al., 2011). Many authors proposed that the synthesis of AgNPs involve the reduction of NADPH to NADP+ and the electrons generated during the reduction of nitrate to Ag ions and converting them to Ag0 were donated by hydroxyquinoline or quinines and NADPH (Kumar et al., 2007). Ingle et al. (2008) affirmed that the cofactor NADH and nitrate reductase enzymes were responsible for the synthesis of AgNPs by F. acuminatum. Mukherjee et al. (2008) suggested the Michaelis–Menten type synthesis of nanoparticles, where the reaction initially exhibits pseudo-zero-order kinetics with slow rate when the concentration of silver nitrate is higher and then follows high order kinetics when the concentration of silver nitrate lowers down significantly. Authors proposed that the bioreduction of metal nanoparticles was occurs through protein extract containing free amino groups or cysteine residues undergoes dehydrogenation with silver nitrate to produce AgNPs, while most likely free amino groups serve as a capping of AgNPs. Bioreduction of metal ions involving polypeptides/proteins was reported by Das et al., (2009), which was confirmed through FTIR spectra of fungal culture containing AuCl4. The presence of amide groups (I, II, and III) and disappearance of carbonyl groups present in the mycelia indicated the involvement of polypeptides in the bioreduction of metal nanoparticles. Similar results reported during the synthesis of AgNPs from Coriolus versicolor (Sanghi and Verma, 2009). Jain et al. (2011) reported that the involvement of 32 KDa protein secreted by A. flavus acted as a reductase in the reduction of bulk silver ions into AgNPs. Furthermore, the stability of AgNPs was achieved through capping by 32 KDa protein. Bansal et al. (2004) proposed that two extracellular proteins with molecular weight of 24 and 28 KDa were responsible for the synthesis of zirconian oxide nanoparticles by F. oxysporum. Chan and Mashitah (2002) reported the involvement of a diketone compound in the reduction of silver ions from three different species of macrofungi.

12.3  Silver nanoparticles from endophytic fungi and their efficacy in biocontrol Several species of fungal endophytes are used for the synthesis of AgNPs from different parts such as leaves, stems, roots, etc. The general from which nanoparticles are synthesized includes Aspergillus, Penicillium, Colletotrichum, Phomopsis, Alternaria, curvularia, Pestalotiopsis, etc (Golinska et al., 2015; Raheman et al., 2011) (Table 12.1). Singh et al. (2017) reported the synthesis of AgNPs from endophytic Alternaria sp.

Table 12.1  Metal nanoparticles obtained from endophytic fungi and their antimicrobial activity. Host plant

Metal

Range

Activity

Alternaria sp.

Raphanus sativus

AgNPs

4–30 nm

Aspergillus clavatus A. niger A. tamari, A. niger, P. ochrochloron A. terreus

Azadirachta indica Simarouba glauca Potentilla fulgens L.

AgNPs AgNPs AgNPs

41.9 nm 3.5–8.5 nm

Singh et al. (2017) Antibacterial (Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Serratia marcescens) Antimicrobial Verma et al. (2010) Antioxidant, antibacterial Hemashekar et al. (2017) Devi and Joshi (2012)

Calotropis procera

AgNPs

16.54 nm

A. versicolor Colletotrichum sp. Curvularia lunata Epicoccum nigrum E. nigrum Fusarium semitectum

Centella asiatica Pelargonium graveolens Catharanthus roseus Phellodendron amurense Solanum lycopersicum L. Withania somnifera

AgNPs Gold AgNPs AgNPs AgNPs AgNPs

3–40 nm 510–560 nm 10–50 nm 1–22 nm 37 nm 10–20 nm

F. solani Guignardia mangiferae Nemanta sp. P. oxalicum Papulaspora pallidula Penicillium sp. Penicillium sp.

Withania somnifera Citrus sp. Taxus baccata L. Phlogacanthus thyrsiflorus Mesembryanthemum sp. Curcuma longa C. longa

AgNPs AgNPs AgNPs AgNPs AgNPs AgNPs AgNPs

10–50 nm 5–30 nm

Penicillium spinulosum

AgNPs

25–30 nm

Pestalotia sp. Pestalotiopsis microspora Phomopsis liquidambaris

C. longa, Catharanthus roseus Syzygium cumuni Gymnema sylvestre Salacia chinensis

AgNPs AgNPs AgNPs

12.40 nm 2–10 nm 18.7 nm

Trichoderma harzianum

Tomato

AgNPs

12.7 nm

8–90 nm 25–30 nm 25 nm

Antibacterial (S. typhi, S. aureus, E. coli) Antimicrobial, cytotoxic Antibacterial Antifungal Antifungal Antibacterial (Porphyromonas gingivalis) Antibacterial, cytotoxic Antibacterial, antifungal, cytotoxic Antibacterial Antibacterial Antitumour, anti bacterial Antibacterial (E. coli, S. aureus) Antibacterial(P. aeruginosa. Klebsiella pneumoniae)

References

Rani et al. (2017) Netala et al. (2016) Shiv Shankar et al. (2003) Ramalingam et al. (2015) Qian et al. (2013) Abdel-Hafez et al. (2017) Halkai et al. (2017) VIjayan et al. (2016) Balakumaran et al. (2015) Farsi and Farokhi (2018) Bhattacharjee et al. (2017) Muhsin and Hachim (2016) Singh et al. (2013) Singh et al. (2013)

Raheman et al. (2011) Netala et al. (2016) Seetharaman et al. (2018) El-Moslamy et al. (2017)

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Singh et al. (2016) Antibacterial Antioxidant, anticancer Antimicrobial, larvicidal (Aedes aegypti, culex quinquefasciatus) Antifungal

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from healthy leaves of Raphanus sativus. These nanoparticles exhibited antibacterial activity against several human pathogenic bacteria. The synthesis of AgNPs from endophytic Pestalotiopsis microspora isolated from healthy leaves of Gymnema sylvestre has been reported (Netala et al., 2016). According to FTIR studies phenolic compounds and proteins were involved in the reduction and capping of AgNPs. The UV-VIS analysis confirmed the synthesis of spherical AgNPs with an average size range of 2–10 nm by showing an absorption peak at 435 nm. Selected area diffraction and X-ray diffraction studies determined the crystalline nature of AgNPs with face-centered cubic lattice phase. The AgNPs exhibited antioxidant activity against 2,2-diphenyl-picrylhydrozyland H2O2 radicals with IC50 values of 76.95 ± 2.96 and 94.95 ± 2.18 µg/mL, respectively. Synthesis of AgNPs from endophytic Penicillium sp. isolated from leaves of Curcuma longa was reported (Singh et al., 2013). The optimization displayed maximum absorbance of 420–425 nm at pH7, 25°C with 1 mm AgNO3 concentration and 15–20 g of fungal biomass. TEM analysis revealed the formation of spherical, welldispersed nanoparticles with size ranging from 25,030 nm. The FTIR showed bands at 1644 and 1538 cm−1 corresponding to the binding vibrations of amide I and II bands of proteins. The AgNPs exhibited strong antagonistic activity against multi drug resistant bacteria E. coli and S. aureus with a max zone of inhibition of 17 mm and 16 mm, respectively at 80 µL of AgNPs. Rani et al. (2017) reported the synthesis and in vitro antibacterial activity of AgNPs from endophytic A. terreus isolated from C. procera. The AgNPs showed strong antibacterial activity against S. aureus (15.67 ± 0.58 mm) and E. coli (15.67 ± 0.58 mm) MIC concentration was observed in the range of 11.43– 308  µg/mL. Nucleic acid degradation with protein leakage was observed after the treatment with nanoparticles. Muhsin and Hachim (2016) reported the biosynthesis of AgNPs from endophytic Papulaspora pallidula. The AgNPs exhibited a high growth inhibition rate (52.685%) against human larynx carcinoma cell line (Hep-2) at a concentration of 3.13  µg/µL. A significant antibacterial activity (27–35.5 mm dia) was observed when gentamycin was used with AgNPs. AgNPs from endophytic Fusarium semitectum isolated from Withania somnifera showed antibacterial activity against (17.33 and 18 mm dia zone of inhibition) against Porphyromonas gingivalis (Halkai et al. (2017).

12.3.1  Effect of nanoparticles against plant pathogenic fungi Several researchers reported the antifungal activity of AgNPs both in vitro and in vivo conditions (Singh et al., 2013). Abdel-Hafez et al. (2016) reported the synthesis of spherical monodispersed AgNPs from endophytic Alternaria solani F10(KT221914). FTIR studies indicated that Ag ions were reduced and capped by extracellular proteins and metabolites. The AgNPs showed antifungal activity against different isolates of A. solani, which causes tomato early blight disease. AgNPs synthesized from endophytic Trichoderma harzianum SYA F4 strain using Taguchi design conditions. The nanoparticles showed maximum inhibition zones at concentrations of 100 µg/ mL against A. alternata (43 mm), Helminthosporium (35 mm), Botrytis sp. (32 mm),

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and P. arenaria (28 mm). An antioxidant compound curvularin isolated from the endophytic Epicoccum nigrum (ASU1) was reported to be used in the synthesis of AgNPs (Abdel-Hafez et al., 2017). Authors suggested the reduction and stability of nanoparticles were due to both hydroxyl and carbonyl functional groups present in the curvularin. The AgNPs showed strong antifungal activity against A. solani. Similarly, stable AgNPs were synthesized from endophytic Epicoccum nigrum isolated from the cambium of Phellodendron amurense (Qian et al., 2013). Authors found that an alkaline pH and elevated temperature favored the reduction of silver ions and subsequently formed AgNPs.

12.3.2  Antimicrobial activity of silver nanoparticles from fungal endophytes The antimicrobial properties of AgNPs against a wide range of microbial pathogens might be due to damage and pore formation in cell membrane. The nanoparticles synthesized from endophytic Pestalotiopsis microspora VJ1/ VS1 from healthy leaves of Gymnema sylvestre exhibited strong cytotoxic activities against different cancerous cells (Netala et al., 2016). Furthermore, concentration-dependent apoptotic changes were observed in SKOV3 cells, which include cell membrane bebbling, cell shrinkage, pycnotic nuclei, and fragmented nuclei. Balakumaran et al. (2015) reported the synthesis of AgNPs from endophytic Guignardia mangiferae (BiosPTK4). The antibacterial study revealed that AgNPs formed pores, which affected the cell permeability results in cell death. The AgNPs showed strong cytotoxic activities against Vero, HeLa, and MCF-7 at the IC50 values 63.37, 27.54, and 23.84 g/mL, respectively. Seetharaman et al. (2018) studied synthesis AgNPs from endophytic Phomopsis liquidambaris strain SA1. The AgNPs arrested the growth of second- and fourth-instar larvae of Aedes aegyptii and Culex quinquefasciatus in dose-dependent method. Bhattacharjee et al. (2017) studied the effect of AgNPs with a combination of antibiotics. They reported the synthesis of stable AgNPs from endophytic Penicillium oxalicum isolated from Phlogacanthus thyrsiflorus. The AgNPs with the combination of streptomycin showed antibacterial activity against four pathogenic bacteria. Similarly, AgNPs synthesized from endophytic Curvularia lunata showed strong activity with chloranphenicol, erythromycin, kanamycin against E. coli, S. paratyphi with ampicillin, B. subtilis with erythromycin (Ramalingam et al., 2015). Netala et al. (2016) reported antibacterial activity of AgNPs synthesized by endophytic fungus Aspergillus versicolor ENT17 isolated from Centella asiatica. The AgNPs were highly stable due to their high negative zeta potential value of −38.2 mV. The AgNPs showed effective free radical scavenging activity with IC50 values of 60.641 g/mL. They also exhibited activity against Gram-positive and-negative bacteria, and pathogenic fungi. Raheman et al. (2011) reported the antibacterial activity of AgNPs synthesized by Pestalotia sp. isolated from healthy leaves of Syzygium cumuni in combination with antibiotics. The AgNPs showed significant antibacterial activity with gentamycin against S. aureus.

Biosynthesis of silver nanoparticles from endophytic fungi and their role in plant disease management 315

12.4  Mechanism of antimicrobial action by silver nanoparticles The mechanism of antimicrobial activity of nanoparticles has been suggested by several studies. The antibacterial activity of nanoparticles is depended on cell wall composition of Gram-positive and-negative bacteria. According to studies, AgNPs accumulates in cell cytoplasm or binds with sulfur components of proteins present in the bacterial cell membrane forms pores, which leads to damage/disruption of normal function of the cell and finally results in cellular death (Jung et al., 2008). Similarly inside the cell proteins/enzymes are the potential sites for binding with AgNPs. This binding is said to be cause for the destabilization of protein/enzyme machinery involved in cellular metabolic pathways (Jung et al., 2008). AgNPs are known to target protein synthetic pathways, nucleic acids, and cell wall membranes (MarambioJones and Hoek, 2010). The disruption of bacterial cell membrane affects respiratory pathways, cell division, and DNA replication result in cellular death (Morones et al., 2005). When silver ions dispersed in an aqueous medium, it may contribute to the antimicrobial activity of metal nanoparticles (Morones et al., 2005). It has also been suggested that AgNPs bind with sulfur, nitrogen, and oxygen atoms of electron donor groups present in thiols or phosphates on amino acids and nucleic acids (MarambioJones and Hoek, 2010). The ATP synthesis is affected as the 30S subunit of ribosomes get denatured, which are required for the synthesis of enzymes and proteins in cellular metabolism (Marambio-Jones and Hoek, 2010). This creates oxidative stress in the cell cytoplasm leads to the formation of reactive oxygen species (ROS) such as oxidative radicals, H2O2, and toxic hyperoxide intermediates, which causes oxidative damage to DNA, lipids, and proteins responsible for cell death (Chauhan et al., 2013). The increased accumulation of ROS and stress responsive hormones in the cell cytoplasm results cellular dysfunction (Chauhan et al., 2013). However, microbial cells express several stress responsive genes during oxidative stress such as superoxide dismutase, catalase, glutathione transferase, and ascorbic peroxidize are a few examples of enzyme, which repair the damage caused by ROS (Chauhan et al., 2013; Torres et al., 2009).

12.5  Factors affecting the mycosynthesis of metal nanoparticles The enzyme production by fungi is affected by several factors in the growth parameters (Alghuthaymi et al., 2015; Singh et al., 2013). Optimization conditions of fungal culture medium affect and improve product yield (Alghuthaymi et al., 2015). Nanoparticle synthesis is directly affected by the time period of incubation, temperature, pH, and nature of the metal (Jain et al., 2011) and biomass concentration of fungal species (Li et al., 2012) and colloidal system, which controls the size, shape, dispersity of the nanoparticles formed.

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Figure 12.3  Applications of silver nanoparticles in plant protection.

12.6  Nanoparticles in plant disease management Nanotechnology has potential application in plant disease management in different ways (Fig. 12.3). Direct application of nanoparticles in the soil on seeds or leaves prevents the pathogen invasion both in plants and rhizosphere. The chemicals such as pheromones, SAR-inducing chemicals and the pesticide can be applied with a controlled release through carbon tubes, cups, etc (Khan et al., 2014). Slow and timely release of nanoformulations in the region of plant roots prevents the invasion microbial pathogens (Khan et al., 2014). These reduce the amount of pesticide significantly required for disease control (Khan and Rizvi, 2014). Nanoemulsions are one of the classical formulations, which can be used as pesticides to control crop diseases. These are effective in very low quantities and least hazardous to the environment (Khan and Rizvi, 2014). Nanoemulsions are very effective due to their kinetic stability, smaller size, low viscosity, and optical transparency. Nanopesticides contain very small-sized particles, which increase the dispersion and wettability of agricultural formulations (Bergeson, 2010). They offer larger surface area and exhibit increased affinity to target organisms. Nanoemulsions, naoencapsulators nanocontainers, and nanocages are proven to be effective delivery techniques in plant disease control techniques (Bergeson, 2010). Corradini et al. (2010) effectively delivered chitosan nanoparticles (antibacterial) for slow release of NPK fertilizer. Kaolin clay-based nanolayers can also be used for controlled release of fertilizers (Liu et al., 2006). Nanoclay materials will provide encapsulating agrochemicals with high aspect ratio (Ghormade et al., 2011). Generally there are three kinds of controlled release systems, namely, (1) zero-order release, first-order release, and square root time release (Khan and Rizvi, 2014). In crop protection programs, The nanopesticide formulations can be applied in the form of biodegradable microbial polymers like polyhydroxy alkanates, macro sugars, proteins (Pepperman et al., 1991), synthetic polymers, and inorganic materials (Chuan et al., 2013). Nanoparticles can be used as biomarkers or diagnostic

Biosynthesis of silver nanoparticles from endophytic fungi and their role in plant disease management 317

tools for detection of bacteria, virus, and fungi (Yao et al., 2009). The early detection of microbial diseases helps the scientists to develop necessary formulations for disease management (Khan and Rizvi, 2014). Nucleotide changes in bacteria and viruses can be detected through nanochips (Lopez et al., 2009). Yao et al. (2009) utilized a fluorescence silica nanoparticles in combination with antibodies to detect Xanthomonas axonopodis pv. vesicatora causal agent of bacterial spot disease in plants. Similarly Singh et al. (2010) used nanogold-based immunosensors that could detect karnal bunt diseases in wheat (Tilletia indica), using surface plasmon resonance. (Gc/Lc-mS) can be replaced to detect pesticide residues by nanomaterial-based nanosensors. These offer high sensitivity, low concentration detection limits, super selectivity, fast responses, and small sizes (Liu et al., 2008). The nanoformulations are considered to be safe and eco-friendly options for plant disease management programs. However, the increased release of AgNPs in the environment will adversely affect both the organisms and the environment (Banik and Sharma, 2011).

12.7 Conclusions Mycosynthesis of nanoparticles from fungal endophytes is a key area of research in nanobiotechnology. The nanoparticles produced from endophytic fungi are stable safe, nontoxic, effective, and eco-friendly. There is a need for the elucidation of exact mechanism of silver nanoparticle synthesis. Improvement in optimization conditions of media will help to produce nanoparticles in large scale within a short period of time. Antimicrobial activity of AgNPs against a wide range of microbial pathogens, gaining more interest among the researchers in synthesis and application of nanoparticles in plant protection.

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Morones, J.R., Elechiguerra, J.L., Camacho, A., et al., 2005. The bactericidal effect of silver nanoparticles. Nanotechnology 16, 2346–2353. Muhsin, T., Hachim, A., 2014. Mycosynthesis and characterization of silver nanoparticles and their activity against some human pathogenic bacteria. World J. Microbial. Biotechnol. 30, 2081–2090. Muhsin, T., Hachim, A., 2016. Antitumor and antibacterial efficacy of mycofabricated silver nanoparticles by the endophytic fungus Papulospora pallidula. Am. J. Bioeng. Biotechnol. 2 (1), 24–38. Mukherjee, P., Ahmad, A., Mandal, D., Senapathi, S., et al., 2001. Fungus mediated synthesis of silver nanoparticles and their immobilization in the mycelia matrix- A novel biological approach to nanoparticle synthesis. Nano Lett. 1, 515–519. Mukherjee, P., Roy, M., Mandal, B.P., et al., 2008. Green synthesis of highly stabilized nanocrystalline silver particles by a non-pathogenic and agriculturally important fungus T. asperellum. Nanotechnology 19, 103–110. Nanda, A., Majeed, S., 2014. Enhanced antibacterial efficacy of biosynthesized AgNPs from Penicillium glabrum (MTCC1985) pooled with different drugs. Int. J. Pharm. Tech. Res. 6, 217–223. Narayanan, K.B., Sakthivel, N., 2010. Biological synthesis of metal nanoparticles by microbes. Adv. Coll. Inter. Sci. 156, 1–13. Netala, V.R., Bethu, M.S., Pushpalatha, B., Baki, V.B., Aishwarya, S., Rao, J.V., Tartte, V., 2016. Biogenesis of silver nanoparticles using endophytic fungus Pestalotiopsis microspora and evaluation of their antioxidant and anticancer activities. Int. J. Nanomed. 11, 5683–5696. Netala, V.R., Bobbu, P., Ghosh, S.B., et al., 2015. Endophytic fungal assisted synthesis of silver nanoparticles, characterization, and antimicrobial activity. Asian J. Pharm. Clin. Res. 8, 113–116. Pantidos, N., Horsfall, L.E., 2014. Biological synthesis of metallic nanoparticles by bacteria fungi and plants. J. Nanomed. Nanotechnol. 5, 233. doi: 10.4172/2157-7439.1000233. Pepperman, A.B., Kuan, C.W., McCombs, C., 1991. Alginate controlled release formulations of metribuzin. J. Controlled Release 17, 105–112. Qian, Y., Yu, H., He, D., Yang, H., Wang, W., Wan, X., Wang, L., 2013. Biosynthesis of silver nanoparticles by the endophytic fungus Epicoccum nigrum and their activity against pathogenic fungi. Bioprocess Biosyst. Eng. 36, 1613–1619. Raheman, F., Deshmukh, S., Ingle, A., et al., 2011. Silver nanoparticles:novel antimicrobial agent synthesized froman endophytic fungus Pestalotia sp. isolated from leaves of Syzygium cumini (L). Nano Biomed. Eng. 3, 174–178. Rai, M., Ingle, A.P., Gade, A.K., Gade, M.C.T., Duran, N., 2015. There Phoma spp. synthesized novel silver particles that posess excellent antimicrobial efficacy. IE Nanotechnol. 9, 280–287. Ramalingam, P., Muthukrishnan, S., Thangaraj, P., 2015. Biosynthesis of silver nanoparticles using an endophytic fungus, Curvularia lunata and its antimicrobial potential. J. Nanosci. Nanoeng. 1, 241–247. Rani, R., Sharma, D., Chaturvedi, M., Yadav, J.P., 2017. Green synthesis characterization and antibacterial activity of silver nanoparticles of endophytic fungi Aspergillus terreus. J. Nanomed. Nanotechnol. 8, 4. Riddin, T.L., Gericke, M., Whiteley, C.G., 2006. Analysis of theinter-and extracellular formation of platinum nanoparticles by Fusarium oxysporum f sp. lycopersici using response surface methodology. Nanotechnology 17, 3482–3489. Sanghi, R., Verma, P.A., 2009. A facile green extracellular biosynthesis of CdS nanoparticles by immobilized fungus. Chem. Eng. J. 155, 886–891.

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H.C. Yashavantha Raoa, N. Chandra Mohanab, Sreedharamurthy Satishb a Department of Biochemistry, Indian Institute of Science, Bengaluru, Karnataka, India; b Microbial Drugs Laboratory, Department of Studies in Microbiology, Manasagangotri, University of Mysore, Mysuru, Karnataka, India Chapter outline head 13.1 Introduction  324 13.2 Incidence of microbial endophytes diversity  325 13.3 Comparison of native and alien endophyte inoculants  326 13.4 Growth promotional aspects due to symbiosis  327 13.4.1 Direct mechanisms of plant growth promotion  327 13.4.2 ACC deaminase activity  328 13.4.3 Micro and macro nutrient  328 13.4.4 Abiotic stress  328 13.4.5 Phytoremediation  329

13.5 Deciphering disease suppressive mechanisms  329 13.5.1 Indirect mechanisms of plant growth promotion  329 13.5.2 Competition for niche and nutrition  329 13.5.3 Antagonism  329 13.5.4 Induced systemic resistance  330 13.5.5 Endophytes as biocontrol agents against pests  331 13.5.6 Mode of action by entomopathogenic fungi  331 13.5.7 Host range and host specificity of entomopathogenic endophytes  332 13.5.8 Development of endophyte inoculants  332 13.5.9 Types of inoculation for delivery  333 13.5.10 Foliar inoculation  333 13.5.11 Stem inoculation  333 13.5.12 Seed dipping  334 13.5.13 Root dipping  334 13.5.14 Soil spray  334 13.5.15 TwinN  334

13.6 Commercialization of endophyte products for sustainable agriculture  334 13.6.1 Bio vaccine  335 13.6.2 Biofertilizers  335 13.6.3 Biocontrol products  336

13.7 Bio market  336 13.7.1 Registration of product  337 13.7.2 Quality control  337 Microbial Endophytes. http://dx.doi.org/10.1016/B978-0-12-819654-0.00013-2 Copyright © 2020 Elsevier Inc. All rights reserved.

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Efficacy of the products  337 Regulation of endophyte-based bioproducts market  337 Challenges in endophyte commercialization  337 Possible toxicity assessment  338

13.8 Recent developments and applications of microbial endophytes  338 13.8.1 Auto fluorescent protein (AFP) technique  338 13.8.2 Genome studies  338 13.8.3 Genetic engineering  338

13.9 Conclusion and future perspectives  339 References  339

13.1 Introduction Microbial endophytes have been evolved over the period of time from being just defined as the microorganisms living inside the host plants indicating their location and type of association with their host. They are ubiquitous in higher plant species which live asymptomatically in the internal tissues and exhibit various relationships with their host. Microbial endophytes enter into the plants through wounds, root hairs, epidermal conjunctions, and naturally occurs as a result of plant growth (Shah et al., 2019). Besides entering to the plants through wounds or natural openings, endophytic microorganisms actively penetrate the plant internal tissues using cellulase and pectinase hydrolytic enzymes (Paramanantham et al., 2019). They are well-known to increase plant growth and yield by nitrogen fixation, solubilization of potassium, zinc, and phosphorus; production of phytohormones like gibberellins, cytokinin, and auxin and having antagonistic activities as well as by reducing the stress ethylene in host plants (Patle et al., 2018; Rao et al., 2015a). Several studies have been documented for understanding the structure and composition of plant-associated endophytes, which indicates plant-microbe, microbe-microbe interactions as well as abiotic factors that lead to plant endobiome composition and structure (Rakshith et al., 2016; Rao et al., 2017a). The plant endobiome is well-known to increase plant defense ability against several invading pathogens and insect herbivores. Several reports on the plantmicrobe interactions involved in endobiome provide an alternative way for different biosynthetic metabolic pathways which are responsible for the biosynthesis of several bioactive and novel biomolecules of commercial significance (Rao and Satish, 2015; Rao et al., 2017b; Sheik and Chandrashekar, 2018). Indeed, plant endobiome is an important factor in global biogeochemical cycles. Hence, the use of plant endobiome is considered to bear the potential to promote production of plant secondary metabolites, plant disease protection, and chemical inputs, leads to more sustainable agricultural applications with enhanced productivity (Singh et al., 2017). Plant-microbe interactions have been extensively studied and explored since decades (Chisholm et al., 2006; Rao et al. 2015b; Strobel and Daisy, 2003; Zhang et al., 2006). However, cognizance of plant–microbe interactions and their relationship is highly complex (Rosenblueth and Martínez-Romero, 2006; Saikkonen et al., 1998; Saikkonen et al., 2004). But it has established certain microbial interactions which could exert

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a positive effect, with respect to endophytes applications. Endophytes have been previously defined as microbes inhabiting internal tissues of host plant without expending any negative effects (Breen, 1994; Faeth, 2002; Hardoim et al., 2008; Mack and Rudgers, 2008). Endophytes extend advantages to the host plant like plant growth promotion and defense against invading pathogens (Clay, 1988; Conn et al., 2008; Gao et al., 2010; Saikkonen et al., 2010). Endophytic behavior is often related to a set of genes, however, there is no definite understanding to specify the genes involved (Card et al., 2016; Sevilla and Kennedy, 2000). The direct benefits of endophytes to host plant include phytohormone production (Khan et al., 2011; Shi et al., 2009; Waqas et al., 2014), biocontrol against phytopathogens and pests due to antimicrobial secondary metabolites (Clay, 1989; Downing and Thomson, 2000; Mejía et al., 2008), iron chelators, due to siderophore production (Bartholdy et al., 2001; Lacava et al., 2008; Loaces et al., 2011), phosphate solubilizing compounds (Otieno et al., 2015; Taurian et al., 2010), nitrogen fixation (Cocking, 2003; Hurek and Reinhold-Hurek, 2003; James, 2000), induced systemic tolerance (Kloepper and Ryu, 2006; Vu et al., 2006), and antagonism (Clay, 1991; Coombs et al., 2004; Ramesh et al., 2009; Schulz et al., 1999). Plant growth promotion due to phytohormones produced by endophytes results in changes in the morphology and structure of host plant (Gaiero et al., 2013; Hardoim et al., 2008; Santoyo et al., 2016). The production of phytohormones such as indole acetic acid (De Battista et al., 1990), cytokinins (Frugier et al., 2008), gibberellic acid (Waqas et al., 2012), ethylene (Camehl et al., 2010), and auxins (Merzaeva and Shirokikh, 2010) would enable endophytes for their applications in sustainable agriculture. The ability of endophytes reducing the atmospheric nitrogen, which is limited for plants would be addressed biologically as an alternate to chemical fertilizers. The insoluble phosphate could reduce from endophytes by expulsion of organic acids converting them to soluble orthophosphate for plant uptake and utilization. Endophytes are well-known produced low-molecular weights called siderophore, which chelate iron for plant uptake and utilization. Some of them include catacholate, hydroxymate, and phenolate (Das et al., 2007). Indirect mechanism of benefits of endophytes confers tolerance to stresses like drought, cold, and hypersaline condition through mechanism, such as induced systemic resistance (ISR) and pathogenesis (Rodriguez and Redman, 2008; Waller et al., 2005).

13.2  Incidence of microbial endophytes diversity The term biodiversity is used to describe the variation in population associated with organisms or between their populations. The understanding of biodiversity could open many avenues at different scientific levels. The taxonomic studies with systematics along with population biology could give insights for evolution, their conservation and most importantly for their efficient utilization for various applications. Microbial endophytes association with the plant is ubiquitous; the existence of endophyte-free plant is a rare exception (Arnold, 2007; Porras-Alfaro and Bayman, 2011). Microorganisms in an ecosystem will directly influence the functioning and biodiversity of ecosystems.

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In this regard, the incidence of endophytes in plants can influence the potential factors to determine the diversity in ecosystems and also they could alter the structure and functioning of plants, which is highly complex and unpredictable. The changes in a shift of incidence in endophyte population could trigger changes in plant community composition due to various factors such as survivorship, competition (Rodriguez et al., 2009). Abiotic conditions could modify plants, which may have consequences in dynamics and diversity of the endophyte community (Bulgarelli et al., 2013). Hence, knowledge of different factors is imperative for environmental conservation and sustainable agriculture. Over the past decades, sufficient collective efforts from all over the globe have been concentrated for the diversity and role of microbial endophytes for sustainable agriculture. Endophytic assemblages with respect to endophytic associations have shed considerable knowledge in this regard (FROeHLICH and Petrini, 2000; Rao et al., 2015c). Several reports regarding diversity data have been published from actinobacteria, bacteria, and fungi (Arnold, 2007; Rao et al., 2015a). Most of the reports are based on culture-dependent studies and very fewer reports on culture-independent techniques are available. The major disadvantage of the culture-dependent technique is that it does not reflect the aspect and functionality of unculturable microbiota, which could cause hindrance in understanding the complex phenomenon of host-endophyte relationship. The diversity incidences of bacteria and fungi have been reported more when compared with actinobacteria due to their slower growth rate. The endophytic bacteria have been reported from the following phylum Firmicutes, Proteobacteria, and Bacteroidetes with a varying distribution where the highest incidence was reported from Proteobacteria whereas least from Bacteroides (Andreote et al., 2009). The incidences of fungal endophytes are reported in phylums Ascomycota and Basidiomycota where Ascomycota is the most dominant (Rodriguez et al., 2009). Tian et al. (2004) reported the population diversity of four rice cultivars. The results revealed that Fusarium and Streptomyces genera to be predominant. The study also revealed the incidence of endophytic fungi to be more diverse in leaf and actinomycetes in roots. The diversity study by Naik et al., (2009) in rice reported Streptomyces sp., Chaetomium globosum, Penicillium chrysogenum, Fusarium oxysporum, and Cladosporium cladosporioides as dominant species. Endophytes diversity was found to be lower during summer and high in winter suggesting the effect of climate on endophyte colonization.

13.3  Comparison of native and alien endophyte inoculants Introduction of alien microbes imposes the threat for existing native endophytes, in turn leading to unforeseen disturbance. The disturbance could be due to elaborate interactions exerted due to the presence of alien endophytes or absence of native endophytes (Bonnardeaux et al., 2007). The extent of invasion studies is more been focused on macroorganisms than microbes. The invasion risk associated with microbes is poorly

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understood. The general focus on pathogenic microbes found to be invasive is prioritized for control and elimination, but the invasion of nonpathogenic microbes should also be considered even though there is low risk as it is not been detected (Pringle et al., 2009). A recent comparison study on indigenous mycorrhizal fungi Rhizophagus irregularis from yam and Acaulospora from cassava with commercial Rhizophagus intraradices revealed a high rate of colonization by native flora. The overall growth and yield of the plant was also found to be increased. One significant observation from the study was that the Rhizophagus irregularis from yam induce highest colonization between two indigenous microflora indicating the selective importance of native microflora from target plant species would be advantageous (Kouadio et al., 2017).

13.4  Growth promotional aspects due to symbiosis Microbial endophytes colonization in host plant is considered as a symbiotic association. Microbial endophytes enhance their immune response and protection in the host plant and get benefited from secondary metabolites produced from these microbes aiding in plant growth (Berg, 2009). The symbiotic association is a coevolution of endophytes with host plant changes in microbial diversity that occurs depending on genotype, stage of growth, physiology, plant organs, and other ecological parameters. The evolution of endophytes brings in changes in cellular and molecular levels, which is highly complex. The intimate relationship occurring between endophyte and host plant is a positive selection process for evolution, which confers beneficial for successful propagation and survival. They accord plants with negative hinderance of biotic and abiotic factors. These interactions have not only beneficial in terms of plant health, growth, development, and production but also soil quality, which could stabilize adverse ecological conditions due to various anthropological activities (Choudhary, 2012). The positive effects of significantly increased plant biomass, dry matter yield, and grain yield would also produce higher income from agronomically with sustainability.

13.4.1  Direct mechanisms of plant growth promotion 13.4.1.1  Production of phytohormones Endophytes produce phytohormones like indole-3-acetic acid (IAA), cytokinins, and gibberellins, which can stimulate the growth, reproduction, and germination. It also has a major role in conferring to biotic and abiotic stress. Endophytes have a crucial role in physiological changes in plants. Reddy et al., (2014) demonstrated when wheat was treated with Metarhizium robertsii, M. brunneum, and Beauveria bassiana, there was a substantial increase in overall plant yield and stand counts. Cotton plants inoculated with B. bassiana and Purpureocillium lilacinum resulted in improved growth of plants along with biomass (Lopez and Sword, 2015). Artificial inoculation of the endophytic bacterium Pseudomonas spp. strains in cotton-improved plant height and number of nodes on the stem (Erdogan and Benlioglu, 2010).

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13.4.2  ACC deaminase activity One of the major aspects in this regard is enzymatic hydrolysis of ACC (1-Aminocyclopropane-1-Carboxylate). ACC has involved in the ethylene biosynthetic pathway, its intermediary between methionine to ethylene conversion. Endophytes utilize ACC exudate from plants before the oxidation occurs due to ACC oxidase in the plant. These endosymbionts cleave the ACC deaminase to α-ketobutyrate and ammonia, followed by utilization of ammonia decreasing ACC with simultaneous reduction of ethylene in the plant system. This phenomenon helps the host plants in stress tolerance along with the plant growth. Extensively studied phytohormone IAA which aid in cell division and elongation contributes indirectly in plant growth as well as plant defense response (Ali et al., 2014; Glick, 2014; Sun et al., 2009). Other than IAA, several plant hormones such as cytokinins and gibberellin can stimulate plant growth and modify plant morphology based on environmental conditions.

13.4.3  Micro and macro nutrient Micro and macronutrients are necessary for the entire microorganisms, as they act as a cofactor for numerous enzymatic reactions occurring in the biological system. For example, iron which exists in ferric state (Fe3+) under aerobic conditions forms hydroxides and oxyhydroxides, which are insoluble. Siderophores could also trigger IAA biosynthesis, which is a beneficial aspect as mentioned earlier. For growth and development of biological system phosphorous is very much required. In nature, soluble phosphorous exists in two forms, that is, monobasic and dibasic soluble forms. The available natural soluble phosphorous is limiting and in heavy metal concentration, P-uptake is highly affected leading to retardation in plant growth. Endophytes aid in conversion insoluble phosphorous to soluble forms by acidification, chelation, exchange reactions, and release of organic acids (Jog et al., 2014). Endophytes could also solve phytotoxicity imposed by high metal concentrations by biosorption and bioaccumulation mechanisms.

13.4.4  Abiotic stress Microbial-endophytes interaction with host plants has been proved to bestow abiotic stress tolerance and to minimize the obstructive ecological impacts on native as well-cultivated plant communities. The intrinsic associations help the host plant in the acquisition of nutrients during stress. Symbiosis with endophytes confer a variety of tolerance to hosts such as heat tolerance in high temperate regions; salt tolerance in plant communities present in coastal regions (Choudhary, 2012). Other mechanisms include biological nitrogen fixation and also the release of PGP factors facilitating the vegetation. When wheat was artificially inoculated with Azospirillum brasilense provided the host plant to mitigate water stress with better grain yield (Furlan et al., 2017). Induction of systemic tolerance against water and salt stress was observed in tomato and pepper plants by Achromobacter piechaudii (Paul et al., 2017).

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13.4.5 Phytoremediation Certain plants have the ability to strive on contaminated soil, this is associated with genetic as well as physiological changes employed to cope up with soil contaminants. One of the mechanisms includes harboring of endophytes especially bacteria for phytoremediation. These endophytes have evolved to tolerate high concentrations of pollutants and simultaneously exhibit plant growth promotion. Endophytes in such cases are involved in various activities like synthesis of siderophores, phosphate solubilization, ACC deaminase activity, production of IAA, cytokinins, and gibberellins (Kumar et al., 2015; 2016). Studies have revealed that endophyte resistance to heavy metals such as Cd, Zn, and Pb (Ma et al., 2015; Ullah et al., 2015). When Sedum plumbizincicola inoculated with endophytic bacterium Bacillus pumilus strain elevated the uptake of Cd, also increase in the root and shoot length of plants were observed. Thus, overall phyto-extraction capacity of the plant was observed along with plant growth promotion indicating the potential role of endophytes in phytoremediation.

13.5  Deciphering disease suppressive mechanisms 13.5.1  Indirect mechanisms of plant growth promotion The indirect mechanism of plant growth promotion is due to suppression of pathogens on plants by inhibitory substances or by ISR in the host. Biocontrol of phytopathogens using endophytes was first described by Timothy (Paulitz and Bélanger, 2001). Endophytes as biocontrol agents have been interesting, but have not been received considerable attention in this regard. Considering the present situation and challenges imposed by phytopathogens, employing endophytic microorganisms would surely create greater demand as well as the market. The isolation of native endophytes of particular species in their respective geographical location and assessment of its effects on phytopathogens as well implications on plant growth could provide dual benefits for sustainable agriculture.

13.5.2  Competition for niche and nutrition Endophytes could essentially deprive the space and nutrition against phytopathogens. Due to intrinsic properties and better adaptation to host as well as its environment, endophytes contend against invasive phytopathogens. The ability of native endophytes is relatively high on colonization in plant tissues when compared with invading pathogens (Backman and Sikora, 2008).

13.5.3 Antagonism Antagonism refers to hostility toward microorganism to another; it can be either by parasitism or antibiosis. Parasitism is inhibition of another organism, with respect to fungus it can be termed as mycoparasitism. Mycoparasitism involves penetration

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of the parasite into the host hyphae with specialized structures such as haustoria and release of secondary metabolites resulting degradation of fungal structure enabling nutrient uptake from host fungus (Clay, 1991). The widely studied Trichoderma spp. could be the best example while explaining mycoparsitism. Trichoderma spp. can secrete a wide range of cell wall-degrading enzymes which is crucial for mycoparasitism. Enzymes like cellulase, xylanase, pectinase, glucanase, lipase, amylase, protease, endochitinases, β-glucanases, and proteases can degrade fungal cell walls (Adams, 2004). The hyphae of Trichoderma spp. coils around pathogens forming specialized structures like hooks, appressorium, haustoria breaking the cell wall, and release of antimicrobial compounds leading to death (Markovich and Kononova, 2003; Verma et al., 2007). Four Pseudomonas strains isolated from the roots of Xanthium strumarium, Portulaca sp., Gossypium hirsitum, and Convolvulus arvensis showed suppression of disease incidence due to Verticillium wilt (Erdogan and Benlioglu, 2010). Antagonism against Ralstonia solanacearum, causative agent bacterial wilt was exhibited by two bacterial strains belonging to Streptomyces genera isolated from a tomato plant, mode of action was observed due to the production of siderophores and ACC deaminase activity Tan et al. (2011). The endophytic bacterium strain HA02 had significant inhibition against Verticillium dahlia which is the causative agent of Verticillium wilt of cotton (Li et al., 2012). A study carried out by Ramesh and Phadke, (2012) using bacterial endophytes from the grape, watermelon, and papaya against Ralstonia solanacearum revealed the antagonistic activity of isolates. The results showed maximum inhibition by Enterobacter cloacae from papaya followed by Bacillus subtilis (EB-06) and B. flexus from watermelon and least by B. pumilus from the grape. Antibiosis interaction by secretion of various secondary metabolites having antimicrobial properties or biostatic can suppress or reduce the growth of phytopathogens. Biocontrol of phytopathogens such as Phytoptera infestans and Phytoptera capsica by Purpureocillium lilacinum which are documented to produced antibiotic leucinostatins (Wang et al., 2016). The culture-based study carried out by Mohan et al. (2015) revealed antagonistic potential of eight ectomycorrhizal fungal isolates Alnicola sp., Laccariafraterna sp., Lycoperdonperlatum sp., Pisolithusalbus sp., Russulaparazurea sp. Scleroderma citrinum, Suillusbrevipes sp., and Suillussubluteus sp., against Alternaria solani, Botrytis sp., Fusarium oxysporum, Lasiodiplodia theobromae, Phytophthora sp., Pythium sp., Rhizoctonia solani, Sclerotium rolfsii, and Subramanio sporavesiculosa. The highest inhibition was shown against Suillusbre vipes while P. albus being least. The effective biocontrol of Capsicum bacterial wilt by Ralstonia solanacearum was observed by bacterial endophyte from Bacillus amyloliquefaciens due to production of antimicrobial protein LCI (Hu et al., 2010).

13.5.4  Induced systemic resistance Plants have enabled themselves with diverse defense mechanisms to intercept and counter adverse negative impacts posed by invading pathogens. These pathways are triggered when the invasion of pathogens and pests occur. They involve specific pattern-recognition receptors, which could be either pathogen or microbe-associated

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molecular patterns (PAMPs or MAMPs) based on signaling molecules from microbes or damage-associated molecular patterns (DAMPs), which are plant-based. Endophytes could trigger MAMPs prior to infection stage by phytopathogens rendering ISR to host plant. Disease by Verticillium dahlia in oilseed rape was found to be suppressed by Serratia plymuthica along with plant growth stimulation, the role of AHL-mediated signaling for disease suppression was found to be in crucial in this regard (Müller et al., 2009). Olive root endophyte of Pseudomonas fluorescens was found to be effective against Verticillium wilt of olive. The isolate induced a broad range of defense response along with induction of systemic defense responses (Cabanás et al., 2014).

13.5.5  Endophytes as biocontrol agents against pests Entomopathogenic fungi as a biocontrol agent against pests have been an emerging area of interest. Various genera of fungal endophytes have been reported to be entomopathogenic. The instigation of entomopathogenic fungi was studied using soil fungus Beauveria bassiana (Ascomycota) which was found to be effective against more than 700 insect pests, also being first commercial biopesticide. Many reports have been available in this regard from 1980s. Biocontrol using endophytes can also be employed for management of invertebrates’ pests (Jaber and Salem, 2014). Metarhizium, Beauveria, Lecanicillium, and Isaria are commercially available as biopesticides. Protection against both phytopathogens and arthropod pests render endophytes to be dual biocontrol agent. Biocontrol of Thripstabaci was found to be effective in onion with Fusarium sp., Hyprocrealixii, and Trichoderma sp. (Muvea et al., 2014). Artificial inoculation of B. bassiana for the control of H. armigera was successful in tomato plants (Qayyum et al., 2015a). Application of B. bassiana and M. brunneum in melon was effective in inducing a significant mortality rate of Bemisia tabaci for the pest management of Cucumis melo L (Garrido-Jurado et al., 2017). Even though endophytes have developed strong antagonism against phytopathogens, aiding in plant growth and induction of tolerance, there are several challenges need to be addressed for employing them as biocontrol agents. The artificial inoculation could essentially face strong competition from microbial diversity which is already established in host plant and can also be influenced by the effect of ecological changes. The effectiveness could vary from lab trials and field trials. This may be attributed to a variety of factors such as less viability during storage, toxicity to untargeted organisms or may have poor colonization rate.

13.5.6  Mode of action by entomopathogenic fungi The mechanism of infection in insects occurs through adhesion, penetration, proliferation, and death. Adhesion of entomopathogenic fungi to insects is due to adhesion genes or hydrophobicity exerted by conidia. The hydrophobic lipid layer aid in attachment of propagules. In specific adhesion by genes include adhesion proteins such as hydrophobins, some of the genes involved in this regard includes Mad1, ssgA, HYD1, HYD2, HYD3 (Moonjely et al., 2016). The conidia adhered to the cuticle

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germinate with aid of insect components such as lipids, chitins, and proteins leading degradation of the cuticle. The lipids in the cuticle provide the major carbon source for conidial germination. After germination, the hyphae protrudes into cuticle releasing various proteolytic enzymes such as proteases, esterases, N-acetylglucosaminidases, chitinases, and lipases. The specialized hyphal structures formed appressoria confirms the successful penetration. After successful penetration, yeast-like bodies is formed in hemolymph now called blastopores ultimately resulting in death. In addition to these mechanisms entomopathogenic fungi such as Beauveria and Metarhizium also produce insecticidal metabolites such as beauvericin and destruxins (Strasser et al., 2000). A study conducted by Kim et al., (2016) reported suppression of powdery mildew by Zucchini Yellow Mosaic Virus (ZYMV) and aphids in cucurbits.

13.5.7  Host range and host specificity of entomopathogenic endophytes The endophyte employed as controlling pests should be able to survive and maintain a symbiotic relationship with the host plant along with nontoxicity for nontarget organisms. Therefore, the target range and target specificity of entomopathogenic endophyte of interest should be determined to avoid detrimental changes. Metarhizium robertsii species is often considered as a potential candidate due to narrow host specificity and also it does not cause any time-lapse problems (Wang and Leger, 2005).

13.5.8  Development of endophyte inoculants The endophyte inoculants for sustainable agriculture have a potential market of interest. It also has several challenges associated with the solutions which can develop the various industry for large-scale agronomical practices. The high uneconomical use of chemical and its adverse negative impacts could essentially be addressed using endophyte technology. The native endophyte diversity with related crop wild relatives is more suitable for tangible benefits like improved colonization and adaptability, thus reducing the chances of the impact of alien species for the environment which could be detrimental. The establishment of endophyte diversity in particular genera from the diverse environment or more endemic to that region would provide significant correlations on diversity dominance. To evaluate the diversity dominance for selecting endophytes, the most crucial factors to focused are sampling sites, plants should be disease-free, ecological parameters of sampling site (temperature, pH, water conditions, and salinity), sites selected should be free from external influence of anthropological activities, that is, undisturbed natural habitat, should not consist of any invasive species, plant growth stages (Busby et al., 2017). After establishing dominant species, the second step is to screen for potential endophytes as inoculants could be assessed. The endophyte to be screened for application should not be pathogen for either human or plant, genetic variation between the host plant and endophyte should be minimum, the ability of potential isolate should grow on a range of substrates, purity of culture should be considered along with its ability to produce spore for artificial inoculation. Efficacy of single endophyte along with a mixture of endophyte inoculants in the host

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Figure 13.1  Different artificial inoculation methods for the colonization of microbial endophytes and their applications.

plant could also be considered for efficient plant improvement and yield. The tolerance assessment for various biotic and abiotic stresses should be validated for at least three generations with significant statistical results would be required (Faye et al., 2013). The successful translation from controlled environments to field trials would establish the successful commercialization. With global climate change and negative impacts by chemicals used in agriculture the employment of endophyte could provide a vision for sustainable agriculture.

13.5.9  Types of inoculation for delivery The effect of artificial inoculation for colonization of endophytes depends on various biotic and abiotic factors, along with inoculation methods, growth media, and the density of inoculum. There are several inoculation strategies described such as foliar (micro-slit method, spraying conidial suspension), stem (micro-slit method), seed dipping, root dipping, soil spray, and callus culture. Some of these techniques have been observed to show a high success rate which is briefly discussed in Fig. 13.1.

13.5.10  Foliar inoculation Foliar inoculation has been found to be successful in the majority of crops. Artificial foliar inoculation of Beauveria bassiana suppressed disease severity of downy mildew by Plasmopara viticola in grapevine (Jaber, 2015). Similarly, this method has been successful efficient in colonization in wheat, corn, bean, tomato, and pumpkin (Gurulingappa et al., 2010), soybean (Russo et al., 2015), tomato (ResquínRomero et al., 2016), bean (Jaber and Enkerli, 2017), and grapevine (Rondot and Reineke, 2018).

13.5.11  Stem inoculation Stem inoculation has also been found to be efficient in colonization of endophyte inoculants could be attributed to direct injection of conidia inside plant tissues by passing the external defense barriers. The stem inoculation has been successful in cacao (Rubini et al., 2005) and coffee (biocontrol of pest berry borer) (Posada et al., 2007).

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13.5.12  Seed dipping The presoaking of seeds in conidial suspension has been reported by Powell et al. (2009) in tomotoes (B. bassiana); bean (Akutse et al., 2013), and tobacco (Russo et al., 2015).

13.5.13  Root dipping The dipping young seedling roots in conidial suspensions have been found most effective in banana when compared with other techniques (Akello et al., 2007). Also, the method had in successful in the recovery of B. bassiana in tomato (Qayyum et al., 2015), tobacco, corn, wheat, and soybean (Russo et al., 2015).

13.5.14  Soil spray Application of conidial suspensions around root seedlings has also been found suitable for endophytes colonization. Reports of soil drenching in sorghum (Tefera and Vidal, 2009), tomato (Elena et al. 2011); common bean (Parsa et al., 2016), cassava (Greenfield et al., 2016) has been found to be successful. Soil drenching by the conidial suspension of B. bassiana and M. brunneum resulted in improved growth of sweet pepper plants.

13.5.15 TwinN TwinN is a dried microbial inoculum packed with vacuum which provides prospicient shelf life period. For usage, first dissolved in less water and later in large amount of water. TwinN contains a consortium of microorganisms including growth hormones, nitrogen fixation, and phosphate solubilizers. These microorganisms can inhabit in the root, shoot, rhizosphere, and leaf as endophytes. It can be used as an inoculant for crops as well as trees. This TwinN can be well applied in irrigation, sprinkler, and spray depends on crops.

13.6  Commercialization of endophyte products for sustainable agriculture Over the past 60 years, agriculture has been dependent on synthetic chemical pesticides and fertilizers resulting in the evolution of resistance. Endophytic microbes as an alternate to the traditional agricultural practices have been recently focused and have found wide interest globally by researcher communities (Fig. 13.2). These endophytes are desired as they can host a wide range of benefits from plant growth to plant protection. But due to the legislations imposed on biopesticides based on regulations for synthetic substances, it has brought barriers for biomarket of endophytes. The major bioinoculants employed and marketed globally includes rhizobium that helps in nitrogen fixation and phosphate solubilizers which is likely to be increased in demand.

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Figure 13.2  Biocommercial beneficial applications of microbial endophytes.

Most of the bioinoculants producers were from the United States and around 20–30 nations globally (Olson, 2015; Ravensberg, 2015). The use of general standards has hindered performance and viability of bioinoculants. Therefore, employment of native endophytes to region specific could solve wide issues such as performance, viability, endophyte adaptability, colonization efficacy, and storage efficiency. However, the need of strict regulations and quality control should not be neglected in this regard. The regulatory regimes and economic implications would be discussed below with product market aspects, product evaluation, and risk factors involved. As the endophyte application in agriculture is still in infancy the following evaluation would be based on available bioproducts in existence.

13.6.1  Bio vaccine Bio Vaccine is a fungicide which contains Trichoderma viride and provide protection to the plant against rot and wilt diseases. It destroys fungal pathogens like Fusarium spp., Pythium, and Rhizoctonia which causes various rot and wilt diseases. Trichoderma viride emerges as coils around pathogens which degrades cell wall synthesis of fungal pathogens by producing various enzymes like celluloses and chitinases. This process is also called as mycoparasitism where one fungus kills other fungus by reducing their growth and metabolic functionality. It also enhances systemic resistant to destroy plant pathogens. It helps to enhance nutrients and moisture in the root system and also increases the stress tolerance.

13.6.2 Biofertilizers The term “biofertilizer” is a product which are not chemically synthesized, biodegradable, and can be used as a fertilizer. However, biofertilizer entail as a fertilizer

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containing living organisms which is classified as phosphate-solubilizing and nitrogen-fixing biofertilizers which contains fungi or bacteria. They have been employed in many kinds of formulations. AgriLife has launched fifteen biofertilizer products based on nitrogen fixers, phosphate solubilizers, potassium, ferrous, sulfur, manganese solubilizers, and zinc-mobilizing microbes in the market. Each biofertilizers contains one bacterial strain and for each nutrient suppliment, specific biofertilizers are available (Mehnaz, 2016). JumpStart contains Penicillium bilaii, a fungus which provides phosphate availability to plants. P. bilaii colonizes the plant root and helps to release various organic compounds to the soil which breaks the bond between phosphate and elements. Plants get more access to phosphate whereas the fungus gets their nutrient supplements from the plant, establishing a symbiotic relationship. It is mainly suitable for canola, wheat as well as legume crops. TagTeam is a multi-action inoculant that is suitable for legume crops. It provides more usage of phosphate and helps to fix more nitrogen. It comes with a combination of a fungus Penicillium bilaii and rhizobia strains. This natural product is available in the form of granular and liquid formulations to use on soybean, pea, dry bean, lentil, and chickpea. RhizoMyco is a biofertilizer that contains eighteen species of endo as well as ectomycorrhizae and growth-promoting agents. RhizoMyco is available in the form of soluble or injectable form to give broad-spectrum benefits for enhanced nutrient supplements and increases root systems. RhizoMyx is a well-known endomycorrhiza inoculant which is designed to increase the plant performance by enhancing root nodules development and providing nutrient suppliments more available.

13.6.3  Biocontrol products Met52 is one of the potent bioinsecticides which contains spores of soil fungus Metarhizium anisopliae. The suspended spores of Metarhizium sp. attaches to the over surface of target insects which germinates, penetrates to the exoskeleton, and grows inside the pest. Later the death of target pest/insects will take place in a few days. Taegro is a bacteria-based biofungicides/bactericides which is used to suppress the selected soilborne and foliar diseases.

13.7  Bio market Bioproducts in sustainable agriculture include biopesticides and biofertilizers which are derived naturally from animals, plants, and microbes for the plant growth promotion and protection. The current bioproduct market includes around 3 billion US dollars. The major market is located in the United States where more than 200 products are available followed by the European Union (EU) around 60. The rate of demand has been increasing around 10% global market annually. The growing concerns related to

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synthetic analogues due to their negative effects on human health and environment, the bioproduct market would gradually over take in the future. Also, the unavailability of new chemical substitutes with new stringent regulations has reduced the chemical product market. The global attention in this regard has shifted to the inclination scientific community and general population toward ecofriendly and safer technologies like bioproducts. The revenue for the North American biofertilizer market has risen over 72.5% (Dunham and Trimmer, 2015). The market price in the EU of bioproducts is around 25% more than synthetic analogues (Czaja et al., 2015; Olson, 2015).

13.7.1  Registration of product The technical data required for bioproduct registration are quality, purity, and stability; efficacy; crop safety and maximum residue levels (Snyder, 2015).

13.7.2  Quality control The qualities of endophytes bioproduct are impeded due to natural microflora and varying their functions in a new host. There is no available standard for endophytes in this regard for their performance and availability (INVAM, 2008).

13.7.3  Efficacy of the products The overall yield gain through the application of endophyte bioinoculants could be ascertained as a success. But there is always the possibility of end results to be varied or inconsistent sometimes may become contradictory. The use of native endophytes would ascertain that the above complications to be minimized. Also, collective soil dynamics with native microflora could also affect the performance of bioproduct (Verbruggen et al., 2013).

13.7.4  Regulation of endophyte-based bioproducts market The bio market includes scientists, regulators, marketers, and end-users in commercialization. The scientists are involved in the earlier stages of product development followed by regulators then marketers with the final end-users. If there is disagreements between them, that needs to be resolved by mutual agreements. Assessment of risks needs to be evaluated which needs new regulation as there is no existing market in this regard to endophytes. Tailoring of these new regulations is most important. The elimination of tiresome lengthy process is one of the major concerns need to focus. Product development already involves a good amount of time, so it should not delay the new product commercialization.

13.7.5  Challenges in endophyte commercialization The limited companies in bioproduct-based industries are one of the major problems. The lack of initial investment is the main lacuna. The government and private funding

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for the development of these products are crucial. The government should provide improved regulations and should emphasize small and medium scale startups. The strategic investment in infrastructure is also a need for large-scale production. The regulatory frameworks for registration of bioproducts should be implemented with the view of faster commercialization for the products to enter the market.

13.7.6  Possible toxicity assessment The beneficial aspects of microbial endophytes are intriguing, but toxin production potential of these microbes should not be neglected. It should be regulated as the prohibition of toxin-producing endophytes for agricultural and industrial applications. The critical assessment and complete characterization of endophytes should be employed to carry out the assessment of genomic data with lab and field investigation.

13.8  Recent developments and applications of microbial endophytes 13.8.1  Auto fluorescent protein (AFP) technique Auto fluorescent protein technique has been employed to study the plant-microbe interaction and their colonization. AFP as a marker system, coding for green fluorescent protein has been successful in the monitoring of colonized endophytes in root tissues (Tombolini and Jansson, 1998). Green fluorescent protein has an advantage of fluorescing without additional requirement of substrate or cofactor. This method has been using poplar plants using different artificial inoculation techniques used in the b-glucuronidase (GUS) reporter system (Germaine et al., 2009).

13.8.2  Genome studies With the advancement of genome technologies whole genome of several endophytic bacteria like Enterobacter spp., Stenotrophomonas maltophilia, Pseudomonas putida are available (www.jgi.doe.gov). The valuable insights of mechanisms at molecular level would enable better understanding along with base for experimental design (Rao et al., 2016; Rao and Satish, 2015).

13.8.3  Genetic engineering The genetic modulation of Metarhizium robertsii was carried out by Wang and St Leger, (2007) for expressions neurotoxin from scorpion, reduced the survival of the tobacco hornworm by 28%. Four insect toxins were engineered in Metarhizium acridum, the synergistic effect of the combination of toxins reduced the incidence of acridids by 48% when compared with wild type (Fang et al., 2014). The whole genome analysis might enable the exploitation of gene clusters and depicting mechanisms

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of complex interactions associated with endophyte-plant host relationship (Mohana et al., 2018). The genetic engineering of GRAS endophytes can enable tailored benefits without affecting nontarget organisms should be more explored.

13.9  Conclusion and future perspectives The depth of scientific knowledge about microbial endophytes and their mechanism is highly in its infancy with published research literature is either scarce or not fully understood. No microbial technology can be considered a successful technique until its commercial availability is proved. The endophytes specificity with the host plants is a major hurdle for large-scale production. The host-specific studies of endophyte are much required before initiating the large-scale production, which involves microbial technology-based advance in research. More efforts are needed in the formulation of host plant specific inoculum doses of microbial endophytes. The optimized and enhanced host specific inoculants will certainly reduce the cost for production of bulk inoculum with their applications and thus might increase productivity. New strategies of exploration like the discovery of novel endophytic strains or endophyte gene alterations are on the apparent horizon of replacing the need for host-specific targets. Instead, new endophytic microbes can be screened for suitable traits from medicinal plants growing under unique niche and extreme environment conditions. The alternative strategy of genetic manipulation can fit host plants with new traits like disease resistance, phytoremediation and other applications could more suitably regulate the metabolism.

Acknowledgments Authors thank University Grants Commission (UGC), New Delhi for the award of Dr. D.S. Kothari Post-Doctoral Fellowship to the first author (No.F.4-2/2006 (BSR)/BL/17-18/0234). N. Chandra Mohan thanks ICMR for the award of Senior Research Fellowship (Award no. 45/69/2018-PHA/BMS/OL).

References Adams, D.J., 2004. Fungal cell wall chitinases and glucanases. Microbiology 150 (7), 2029– 2035. Akello, J., Dubois, T., Gold, C.S., Coyne, D., Nakavuma, J., Paparu, P., 2007. Beauveria bassiana (Balsamo) Vuillemin as an endophyte in tissue culture banana (Musa spp.). J. Invertebr. Pathol. 96 (1), 34–42. Akutse, K.S., Maniania, N.K., Fiaboe, K.K., Van den Berg, J., Ekesi, S., 2013. Endophytic colonization of Vicia faba and Phaseolus vulgaris (Fabaceae) by fungal pathogens and their effects on the life-history parameters of Liriomyza huidobrensis (Diptera: Agromyzidae). Fungal Ecol. 6 (4), 293–301.

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Sheik, S., Chandrashekar, K.R., 2018. Fungal endophytes of an endemic plant Humboldtia brunonis wall. of Western Ghats (India) and their antimicrobial and DPPH radical scavenging potentiality. Ori. Pharm. Exp. Med. 18 (2), 115–125. Shi, Y., Lou, K., Li, C., 2009. Promotion of plant growth by phytohormone-producing endophytic microbes of sugar beet. Biol. Fertil. Soils 45 (6), 645–653. Singh, M, Kumar, A, Singh, R. and Pandey, K.D., 2017. Endophytic bacteria: a new source of bioactive compounds, 3 Biotech, 7: 315. Snyder, S.W. (Ed.), 2015. Commercializing Biobased Products: Opportunities, Challenges, Benefits, and Risks. Royal Society Chem. Publisher: Royal Society of Chemistry. Strasser, H., Vey, A., Butt, T.M., 2000. Are there any risks in using entomopathogenic fungi for pest control, with particular reference to the bioactive metabolites of Metarhizium, Tolypocladium and Beauveria species? Biocontr. Sci. Technol. 10 (6), 717–735. Strobel, G., Daisy, B., 2003. Bioprospecting for microbial endophytes and their natural products. Microbiol. Mol. Biol. Rev. 67 (4), 491–502. Sun, Y., Cheng, Z., Glick, B.R., 2009. The presence of a 1-aminocyclopropane-1-carboxylate (ACC) deaminase deletion mutation alters the physiology of the endophytic plant growthpromoting bacterium Burkholderia phytofirmans PsJN. FEMS Microbiol. Lett. 296 (1), 131–136. Tan, H., Zhou, S., Deng, Z., He, M., Cao, L., 2011. Ribosomal-sequence-directed selection for endophytic streptomycete strains antagonistic to Ralstonia solanacearum to control tomato bacterial wilt. Biol. Control 59 (2), 245–254. Taurian, T., Anzuay, M.S., Angelini, J.G., Tonelli, M.L., Ludueña, L., Pena, D., Fabra, A., 2010. Phosphate-solubilizing peanut associated bacteria: screening for plant growth-promoting activities. Plant Soil 329 (1–2), 421–431. Tefera, T., Vidal, S., 2009. Effect of inoculation method and plant growth medium on endophytic colonization of sorghum by the entomopathogenic fungus Beauveria bassiana. BioControl 54 (5), 663–669. Tian, X.L., Cao, L.X., Tan, H.M., Zeng, Q.G., Jia, Y.Y., Han, W.Q., Zhou, S.N., 2004. Study on the communities of endophytic fungi and endophytic actinomycetes from rice and their antipathogenic activities in vitro. World J. Microbiol. Biotechnol. 20 (3), 303–309. Tombolini, R., Jansson, J.K., 1998. Monitoring of GFP-tagged bacterial cells. Bioluminescence Methods and Protocols. Humana Press, pp. 285–298. Ullah, A., Heng, S., Munis, M.F.H., Fahad, S., Yang, X., 2015. Phytoremediation of heavy metals assisted by plant growth promoting (PGP) bacteria: a review. Environ. Exp. Bot. 117, 28–40. Verbruggen, E., van der Heijden, M.G., Rillig, M.C., Kiers, E.T., 2013. Mycorrhizal fungal establishment in agricultural soils: factors determining inoculation success. New Phytol 197 (4), 1104–1109. Verma, M., Brar, S.K., Tyagi, R.D., Surampalli, R.Y., Valero, J.R., 2007. Antagonistic fungi, Trichoderma spp.: panoply of biological control. Biochem. Eng. J. 37 (1), 1–20. Vu, T., Sikora, R., Hauschild, R., 2006. Fusarium oxysporum endophytes induced systemic resistance against Radopholus similis on banana. Nematology 8 (6), 847–852. Waller, F., Achatz, B., Baltruschat, H., Fodor, J., Becker, K., Fischer, M., Franken, P., 2005. The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield. Proc. National Acad. Sci. 102 (38), 13386–13391. Wang, C., Leger, R.J.S., 2005. Developmental and transcriptional responses to host and nonhost cuticles by the specific locust pathogen Metarhizium anisopliae var. acridum. Eukaryotic Cell 4 (5), 937–947.

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Wang, C., St Leger, R.J., 2007. A scorpion neurotoxin increases the potency of a fungal insecticide. Nat. Biotechnol. 25 (12), 1455. Wang, G., Liu, Z., Lin, R., Li, E., Mao, Z., Ling, J., Xie, B., 2016. Biosynthesis of antibiotic leucinostatins in bio-control fungus Purpureocillium lilacinum and their inhibition on Phytophthora revealed by genome mining. PLoS Pathog. 12 (7), e1005685. Waqas, M., Khan, A.L., Kamran, M., Hamayun, M., Kang, S.M., Kim, Y.H., Lee, I.J., 2012. Endophytic fungi produce gibberellins and indoleacetic acid and promotes host-plant growth during stress. Molecules 17 (9), 10754–10773. Waqas, M., Khan, A.L., Kang, S.M., Kim, Y.H., Lee, I.J., 2014. Phytohormone-producing fungal endophytes and hardwood-derived biochar interact to ameliorate heavy metal stress in soybeans. Biol. Fertil. Soils 50 (7), 1155–1167. Zhang, H.W., Song, Y.C., Tan, R.X., 2006. Biology and chemistry of endophytes. Natural. Prod. Rep. 23 (5), 753–771.

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Microbial endophytes and their intellectual property rights

14

Ashutosh Raia, Avinash Chandra Raib a Division of Biotechnology, Indian Institute of Vegetable research, Varanasi, Uttar Pradesh, India; bInstitute of Plant Sciences, The Volcani Center, Agriculture Research Organisation, Bet-Dagan, Israel Chapter outline head 14.1 Introduction  349 14.2 Endophytes and their patents  352 14.3 Microbe’s intellectual properties-related conflicts  374 14.4 Role of WIPO governing intellectual properties of microbial organisms  376 14.5 Implementation of IP protection of microorganisms  378 14.5.1 Microorganisms submission for purposes of the patent process  378

14.6 The Budapest Treaty  379 14.6.1 Fundamental structure of the Budapest Treaty  380

14.7 Conclusion and prospects  384 References  385

14.1 Introduction Endophytes are microorganisms either bacteria, fungi, or actinomycetes that are commonly living in the plant tissue without causing any infection (Gouda et al., 2016; Kumar et al., 2016). In general, they are universally present and isolated from most of the plant species. Since the beginning of civilization, humans is exploiting various natural resources and probably uncovered almost whole plant kingdom but could explore less than 1% of total microbial populations associated with the plants (Jackson et al., 2015). Mycologists, environmentalists, ecologists, and plant biologists are now a days focusing on endophytic microbes, which are a fascinating assemblage of organisms coupled with a variety of tissues and organs of plants. The endophytic microbial associations are unobtrusive and at the starting phase of infestation plant parts are symptomless, but in later stages, these internal microbial associations can be visualized with histological means (Tadych and White, 2019). However, the modern science techniques have provided ease in characterization and identification of these microorganisms on the basis of conserved DNA sequences for each specific group (Emerson et al., 2008). In recent past years the trend of microbial deposition has been increased remarkably (Fig. 14.1). Microbial endophytes comprise of a vast untapped reservoir of genetic and metabolic diversity with the opportunity to explore them for human welfare. Microbial endophytes possess genes and derived compound, which is agriculturally important, Microbial Endophytes. http://dx.doi.org/10.1016/B978-0-12-819654-0.00014-4 Copyright © 2020 Elsevier Inc. All rights reserved.

350

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Figure 14.1  Top depositors in microbial depositories before and after year 2001.

particularly helping pest and disease management (Bacon and Hinton, 2007). The infections in plants due to these endophytes are not easily understood due to a huge number of fungal as well as bacterial infestations nevertheless the true symbiotic associations between these endophytes are host genus, species, and cultivar specific (Zhang et al., 2006). In general, the majority of the microbes that colonize plants and that can be readily isolated from any microbial or plant growth medium are the reservoirs of novel bioactive secondary metabolites (Shukla et al., 2014). These secondary metabolites, namely, alkaloids, phenolic acids, quinones, steroids, saponins, tannins, and terpenoids (Tan and Zou, 2001; Kaul et al., 2012) serve as a potential candidate for antimicrobial, anti-insect, anticancer and many more properties (Schulz et al., 2002). A high demand for secondary metabolites in pharma, medicine as well as agriculture sector makes them a precious and industrial demanding product. Inventors of these microorganisms and their byproducts have a right to protect their products in the form of patent under intellectual property rights (IPR) (Nair and Ramachandranna, 2010). Concept of IPR is not new, from the ancient time, concept and basis of IPR have been used for the trade secrets and/or for the privilege of monopoly, through which world has been promoted for various forms of civilization, science, and arts. A patent is the best proprietorship of an inventor, in fact the modern concept of patent has kept the balance between the invention and investment. If investor has right to get patent of their R&D products than it further provokes them to explore much more their research in an innovative way. Notwithstanding, if their credit will take by others without any intellectual as well as economic investment, then this would be an inappropriate result from the inventor/investor’s point of view (Williams, 2017). Recently, industries are investing their majority of money in the innovative R&D and IPR protects, their inventions in terms of legal issues. These innovative industrial inventions are now the major revenue generating resource of those investing industries. Owners of these IPR are free to practice or assign their patent rights to any interested parties through different instruments such as agreement, etc. These exclusive territorial rights usually have a life time of 20 years from the date of the grant/publication of inventions in public domains.(Greaves, 1994; Sangal et al., 2017). Patent is a trusted resource where vast

Microbial endophytes and their intellectual property rights

351

knowledge and talent can be assembled to convey as a reliable resource for levitating the economy of any nation. World Intellectual Property Organization (WIPO) is a United Nation agency, which controlled the world intellectual property rights in a common platform (Vagadia, 2007; Janke, 2003). It is estimated that more than 1,000,000 species of bacteria and approximately 600,000 species of Protoctists exist in this earth (Alam, 1991; Jiménez-García, 1989). However, it is assumed that “microorganisms” epitomize a major proportion of the world’s biodiversity and many countries greatly claims over these biodiversities. Every ascendant nation has the right to protect their genetic resources. Even if detentions of biological heterogeneity usage cannot be stopped by one country to others because of the human profit. So different countries of the world needs to get their IPR in a common platform and those countries also have a responsibility to respect international laws and rules. To cope with this problem many different international levels of meetings like General Agreement on Tariff and Trade (GATT), Trade-Related Aspects of Intellectual Property Rights (TRIPS), and Budapest and PCT have been organized in order to tackle International level of clashes (Sangal et al., 2017). Out of the above treaties, TRIPS agreement was the suitable and important form of agreement, which is especially important in biotechnological point of view, that provides patent of microbes and their processed byproducts as per microbiological operations in fellow countries (De Wet, 1995). TRIPS agreement of world trade organization (WTO) assigned several guidelines and their implementation time to time for the fellow integral nations in order to take homogeneity in the International law associated with IPR (Leal et al., 2014). In this way TRIPS agreements have provided a common platform for all the fellow countries to follow common law of IPR and most of the fellow countries have implemented their indigenous law according to said agreements. Most of the developed and developing countries now follow TRIPS agreement for their IP rights. According to TRIPS agreement, it is clearly specified that member countries have no rights to refuse an invention from patentability on giving an excuse that, their national native laws are not permitted/sanctioned that invention for the patent purposes. No country has the freedom to select the segment of law according to his personal profit/will or to escape from the other segment. At the beginning of the agreement various countries especially developing country have not a proper provision or law according to TRIPS agreement. For these countries, WTO provided an extended time period for implementing the uniform law (Correa, 2007). Microorganism concomitant patents and their shielding is a critical subject in the monarchy of biotechnology (Sangal et al., 2017). The wide credit and value given to it, because of the anticipated fact that in spite of research concern, microorganism has been used as a most important resource in bioprocess industries, however, their byproducts and secondary metabolites are the choice of concern in most of the food, beverages, and pharmacy industries (Gavrilescu and Chisti, 2005). The escalation in patenting related to microorganisms shows the importance of microorganisms in the form of rich and largely unexploited sources such as DNA, amino acids, and proteins for the production of enzymes that are used as a medicine, food, agriculture, and chemical for the industries (Singh et al., 2016). IPR literacy, especially in developing

352

Microbial Endophytes

countries, is in such a pathetic condition, where highly qualified microbiologists are also not cognizant of the basic criteria of microorganism patenting, which is really a matter of concern (Chaudhary, 2003). Apart from this, the nature of the patent area of microorganisms is filled with ambiguity.

14.2  Endophytes and their patents The use of combinatorial biochemical and biosynthetic techniques, novel natural product identification has been optimized on the basis of their involvement in various biological activities to yield effective chemotherapeutic and other bioactive agents. Plant endophytic fungi/bacteria are an important and novel resource of natural bioactive compounds with their potential applications in agriculture, medicine, and food industry (Zhao et al., 2010). From the last two decades, many precious bioactive compounds with insecticidal, cytotoxic, anticancer, and antimicrobial activities have been effectively revealed from endophytic fungi (Zhao et al., 2010). To till dates numbers of bioactive compounds and/or secondary metabolites in the form of different products are patented from the fungal and bacterial endophytes are listed in Tables 14.1 and 14.2. During the time of evolution, there is a closed relationship formed in between endophytes and their host plants (Zhao et al., 2011). Some endophytes can produce the same or similar bioactive compounds as those originated from their host plants. The Cinnamomum zeylanicum hosted the fungus Muscodor albus has been generated from a large number of volatile antimicrobial compounds, which are actively controlling soil-borne pathogens (Ezra et al., 2004). A plethora of such mycofumigants have now been available and patented, are now commercially used as soil fumigation. Ascocoryne sarcoides, an endophytic fungus that harvests a variety of hydrocarbons commonly found in diesel, petrol, and biodiesel, deals mankind a potential alternative to fossil fuels (Chowdhary et al., 2019). These purified products are patented in a different form are summarized in Table 14.1 and 14.2. Taxol is one of the commercially successful anticancer drugs and its structural analogs are well-known examples of medicinally important microbial endophytes (Fridlender et al., 2015). Apart from this preparation of natural irones, benzofuran, pestacin, and isopestacin are produced by fungal endophyte having a moderate antifungal activity (Strobel, 2018). Diosgenin, a plant steroid, isolated from rhizome of Chinese yam (Rao and Kale, 1992), Toosendanin, a well-known compound produced by endophytic actinomycetes recommended for its antibotulismic (Shi and Wang, 2004), antifeedant, and growth inhibitory effects of toosendanin (Chen et al., 1995). Recent developments in metabolic engineering and synthetic biology offer new possibilities for the overproduction of complex natural products through more technically amenable microbial hosts (Tyo et al., 2007). Some of the popularized drugs are now commercially available which are naturally derived from the endophytes and effectively used in the medical sciences for the cure of mankind as summarized in Table 14.3. After the discovery of penicillin almost all the bacterial infections which were fatal have become treatable with a different generation of antibiotics. Although first antibiotic penicillin was never patented but almost every newly discovered antibiotic after that has been patented (Katz et al., 2006). These antibiotics

Endophyte

Patent number

Title of published patent/application

Publication date

Acremonium sp.

AU-2017210482-B2

Seed-origin endophyte populations, compositions, and methods of use Microbial consortia

10/04/2018

Endophytic fungi from Pteromischum sp. plant, compounds and methods of use Co-incubating confined microbial communities

05/14/2009

Association of microorganisms for production of plant growth control agent Plant growth biostimulating agent symbiont and microorganisms consortium Bioactive fungi Biofumigant compositions and methods thereof Chromobacterium subtsugae genes Muscodor mengyangensis having biological prevention and control effects Muscodor malipoensis with biocontrol effect Antimicrobial compositions and related methods of use Compounds derived from muscodor fungi New polypeptides from endophytes of Empetrum nigrum and their fragments Muscodor sp. endophytic fungi ZJLQ024 and application thereof and fungicide Gliocladium isolate c-13 and methods of its use for producing volatile compounds Methods and compositions insect repellents from a novel endophytic fungus

01/20/2001

CN-107532139-A WO-2009061950-A2 WO-2009015390-A2 RU-2161884-C1 RU-2100932-C1 Muscodor sp.

US-2018305657-A1 WO-2018141020-A1 AU-2015312182-A1 CN-104419649-A CN-104419647-A CA-2904383-A1 US-2012058058-A1 WO-2011113999-A1 CN-101691541-A US-2009142816-A1 US-7267975-B2

Microbial endophytes and their intellectual property rights

Table 14.1  List of fungal endophytes and their patent application/publication.

10/02/2018

01/29/2009

01/10/1998 10/25/2018 08/09/2018 02/09/2017 03/18/2015 03/18/2015 09/18/2014 03/08/2012 09/22/2011 04/07/2010 06/04/2009 09/11/2007

353

(Continued)

Endophyte Aspergillus sp.

Patent number WO-2018218138-A1 US-10117841-B2 WO-2016044655-A2 WO-2016044548-A1 AU-2014302296-B2 WO-2015138361-A1 WO-2015034629-A1 US-2015020236-A1 WO-2014145828-A1 CA-2902946-A1 US-2014220150-A1 WO-2013138398-A1 US-8415294-B2

US-7022524-B1 WO-0029607-A1 CA-2336122-A1 US-5935570-A

Publication date 11/29/2018 11/06/2018 03/24/2016 03/24/2016 02/25/2016 09/17/2015 03/12/2015 01/15/2015 09/18/2014 09/12/2014 08/07/2014 09/19/2013 04/09/2013 08/18/2011 01/08/2009 10/25/2007 04/04/2006 05/25/2000 01/13/2000 08/10/1999

Microbial Endophytes

US-2011201642-A1 US-2009011476-A1 WO-2007120253-A2

Title of published patent/application Elicitor-derived peptides and use thereof Volatile organic compound formulations having antimicrobial activity Fusion proteins, recombinant bacteria, and methods for using recombinant bacteria Compositions comprising recombinant bacillus and another biological control agent Seed-origin endophyte populations, compositions, and methods of use Integrative fungal solutions for protecting bees Methods and compositions for control of mite infestations Engineered pesticidal proteins Antimicrobial compositions and related methods of use Epichloae fungal endophytes and combinations with Secale spp. Integrative fungal solutions for protecting bees and overcoming colony collapse Pesticidal flavobacterium strain and bioactive compositions, metabolites and uses Cyclodepsipeptides with antineoplastic activity and methods of using to inhibit cancer Bioactive compounds Gene cluster and method for the biosynthesis of terrequinone a Endophytic streptomycetes, compounds derived there from, and methods of their use Increasing plant growth with luminol Novel enhancers of plant growth Taxol production via generation of extrachromosomal DNAs in fungus pestalotiopsis Synthesis of immunologic, therapeutic, and prophylactic compounds

354

Table 14.1  List of fungal endophytes and their patent application/publication. (Cont.)

US-2016316760-A1 US-8728442-B2 AU-2015279557-B2 CN-1322861-C CN-108368125-A CN-106163281-A AU-2018200620-A1 CN-1926238-A US-2018153174-A1 AU-2016258268-A1 US-2018213800-A1 US-2017292108-A1 US-2018251776-A1 AU-2016274683-A1

08/20/2015 12/07/1993 09/12/2014 12/11/2001 04/24/2014 10/28/2010 01/19/1999 08/09/2016 12/22/2016 11/03/2016 05/20/2014 03/16/2017 06/27/2007 08/03/2018 11/23/2016 02/22/2018 03/07/2007 06/07/2018 12/14/2017 08/02/2018 10/12/2017 09/06/2018 02/01/2018

355

CN-108290902-A

Endophytic microbial symbionts in plant prenatal care Plant promoter glucuronidase gene construct Epichloae fungal endophytes and combinations with Secale spp. Taxol production by a microbe Screening methods Antimicrobial compositions and related methods of use Taxol production by a microbe Endophytes, associated compositions, and methods of use thereof Designed complex endophyte compositions and methods for improved plant traits Isolated complex endophyte compositions and methods for improved plant traits Method of controlling the growth of microorganisms by a composition Endophytes, associated compositions, and methods of use thereof Application of Cytospora bacterin B in preparation of antifungal medicine Acc inhibitors and uses thereof Fungicidal compositions of pyrazolecarboxylic acid alkoxyamides Antimicrobial compositions and related methods of use High efficiency peptide production in plant cells Modulated nutritional quality traits in seeds Microbial compositions and methods for bioprotection Penicillium endophyte compositions and methods for improved agronomic traits Beneficial microbes for delivery of effector peptides or proteins and use thereof Endophyte compositions and methods for improvement of plant traits Streptomyces endophyte compositions and methods for improved agronomic traits Esters acc inhibitors and uses thereof

Microbial endophytes and their intellectual property rights

Cladosporium sp. US-2015230478-A1 US-5268463-A CA-2902946-A1 US-6329193-B1 US-2014115731-A1 US-2010272690-A1 US-5861302-A US-9408394-B2 US-2016366892-A1

07/17/2018 (Continued)

Endophyte Patent number Colletotrichum sp. US-8080256-B2 CN-104004664-A US-2011182862-A1 CN-101974438-A US-7232565-B2 WO-2015100432-A2 EP-2676536-A1 US-2010272690-A1 WO-2015100431-A2 WO-2016044529-A1 US-9408394-B2 US-2014086879-A1 CN-106916853-A CN-103173359-B

CN-103173362-B CN-102002463-B

Publication date 12/20/2011 08/27/2014 07/28/2011 02/16/2011 06/19/2007 07/02/2015 12/25/2013 10/28/2010 07/02/2015 03/24/2016 08/09/2016 03/27/2014 07/04/2017 05/28/2014 07/30/2014 05/28/2014 05/30/2012

Microbial Endophytes

CN-103173360-B

Title of published patent/application Endophytic fungi from Pteromischum sp. plant, compounds and methods of use Endophytic fungus capable of improving photosynthesis of cedar Endophytic fungus and uses thereof Eucalyptus endophyte and application thereof Use of endophytic fungi to treat plants Method for propagating microorganisms within plant bioreactors and stably storing microorganisms within agricultural seeds Method for producing plant seed containing endophytic microorganisms Antimicrobial compositions and related methods of use Plants containing beneficial endophytes Compositions comprising recombinant bacillus cells and another biological control agent Endophytes, associated compositions, and methods of use thereof Muscodor albus strain producing volatile organic compounds and methods of use Method of preparing bioactive substance with co-cultivation of plantbased endophytes Endophytic fungus promoting Casuarina equisetifolia root system growth effect Endophytic fungus increasing Casuarina equisetifolia chlorophyll content Endophytic fungus promoting Casuarina equisetifolia photosynthesis Endophytic fungus for improving anti-freezing capability of eucalypt and application

356

Table 14.1  List of fungal endophytes and their patent application/publication. (Cont.)

CN-103173364-B CN-101993827-B CN-104004665-B US-2013252289-A1 US-2011262416-A1 US-2015157027-A1 US-2011287471-A1 US-2016330976-A1 US-2016262335-A1 US-8728442-B2 US-2011165649-A1

CN-103173363-B CN-103194396-B US-2013252313-A1 US-2014086878-A1 US-2013137131-A1

06/25/2014 07/09/2014 04/11/2012 04/06/2016 09/26/2013 10/27/2011 06/11/2015 11/24/2011 11/17/2016 09/15/2016 05/20/2014 07/07/2011

07/30/2014 07/09/2014 09/26/2013 03/27/2014 05/30/2013 11/26/2013 (Continued)

357

US-8592344-B2

Endophytic fungus promoting Casuarina equisetifolia nutrient element absorption Endophytic fungus promoting Casuarina equisetifolia biomass growth Functional endophytic fungus for prompting photosynthesis of eucalypt and application An endophytic fungi can relieve stress phosphorus fir Method of producing volatile organic compounds from microorganisms Bacillus subtilis strain having antagonistic activity for controlling plant diseases Nanosystems for formulation of effective minimum risk biocides System and method of producing volatile organic compounds from fungi Method for propagating microorganisms within plant bioreactors and stably storing microorganisms within agricultural seeds Improved fungal endophytes Method of controlling the growth of microorganisms by a composition containing isolated or in vitro synthesized nitrosoamides Methods and compositions to improve the health of plants, animals and microbes by manipulating protein entry into symbionts and their hosts Phyllosticta strain promoting Casuarina equisetifolia photosynthesis Aspergillus strain capable of promoting root growth of Casuarina Microorganisms for producing volatile organic compounds Muscodor albus strain producing volatile organic compounds and methods of use System and method of producing volatile organic compounds from fungi Pesticidal compositions comprising 4,5-dihydroxyindan-1-one

Microbial endophytes and their intellectual property rights

CN-103173361-B

Endophyte

Patent number CN-106317144-A

WO-2018027275-A1 US-2015040629-A1 WO-2015183003-A1

WO-2014175496-A1

AU-2016259449-B2 EP-2539432-B1 KR-101764184-B1 AU-2016259448-B2 KR-20050034000-A

AU-2011323740-B2 RU-2618873-C2 CN-102875375-A

Publication date 01/11/2017

02/15/2018 02/12/2015 12/03/2015

10/30/2014

07/26/2018 04/12/2017 08/02/2017 11/08/2018 04/14/2005 03/16/2017 11/07/2018 12/24/2015 05/11/2017 01/16/2013

Microbial Endophytes

AU-2015279557-B2 JP-6421115-B2

Title of published patent/application Compound or its salt, Colletotrichum aotearoa fermentation product, pharmaceutical composition or health food, and use of Colletotrichum aotearoa fermentation product Metabolite production in endophytes Novel mycorrhizae-based biofertilizer compositions & method for mass production & formulations of same Novel plant endophytic bacteria bacillus oryzicola isolated from rice rhizosphere, and development of agent for natural plant protection and plant enhancement using same Plant endophytic bacteria Bacillus methylotrophicus yc7077 strain, multi-functional biopesticide using same, and development of microbial fertilizer Designer endophytes (2) Isolated bacterial strain of the genus Burkholderia and pesticidal metabolites therefrom Streptomyces costaricanus HR391 having anti-fungal activity against pathogenic fungi Novel endophytes (2) Biocontrol of plant diseases using novel endophytic isolate of Burkholderia vietnamensis Endophytes, associated compositions, and methods of use thereof A plurality of host-selection of the symbiont by screening the symbionts association Method for high-throughput identification of microbial antagonists against pathogens Method of highly efficient selection of microbial antagonizers against pathogens Octadecen BHBB compound, its preparation method and application thereof

358

Table 14.1  List of fungal endophytes and their patent application/publication. (Cont.)

JP-2015528017-A WO-2014165174-A2 CN-104152385-A

AU-2016258268-A1 CN-102634462-A WO-2017156457-A1 EP-3193617-A1 CN-107338202-A US-2017283472-A1 AU-2015317715-A1 US-2018014547-A1

Curvularia sp.

CN-108368125-A CN-102311925-A US-6329193-B1 US-7192939-B2

09/24/2015 11/23/2016 07/17/2018 05/04/2017 07/21/2017 09/24/2015 10/09/2014 11/19/2014

12/14/2017 08/15/2012 09/14/2017 07/26/2017 11/10/2017 10/05/2017 04/06/2017 01/18/2018 08/03/2018 01/11/2012 12/11/2001 03/20/2007 03/15/2016

359

US-9282747-B2

Antimicrobial compositions and related methods of use Fungicidal compositions of pyrazolecarboxylic acid alkoxyamides Esters acc inhibitors and uses thereof Endophytes and related methods Compound separated from Colletotrichum gloesporioides and preparation method and application thereof Composition comprising a pest terpene mixture and insecticides kill Microorganisms for producing volatile organic compounds and methods using same Bacillus mojavensis QLY002 strain, microbial inoculum, preparation method of microbial inoculum and use of Bacillus mojavensis QLY002 strain and microbial inoculum Microbial compositions and methods for bioprotection Separation method for plant endogenetic fungi Multimeric defensin proteins and related methods Compositions comprising recombinant bacillus cells and a fungicide Bacillus amyloliquefaciens with broad-spectrum pathogen inhibition function Compositions comprising recombinant Bacillus cells and another biological control Compositions comprising recombinant Bacillus cells and a fungicide Uses of Daldinia sp. or volatile organic compounds derived there from Acc inhibitors and uses thereof Endophytic fungi Chaetomium globosum strain, microbial agent and application Taxol production by a microbe Pestalotiopsis microsporia isolates and compounds derived there from Antimicrobial and anti-inflammatory activity of switchgrass-derived extractives

Microbial endophytes and their intellectual property rights

AU-2014232433-A1 CN-106163281-A CN-108290902-A US-2017118943-A1 CN-106966887-A

(Continued)

Endophyte

Patent number EP-2633064-B1 US-2018251776-A1 US-2018153174-A1 AU-2016213956-A1

Penicillium sp.

EP-3288387-A1 AU-2018200620-A1 US-10144719-B1 AU-2018222918-B2 US-2018265402-A1 US-2018235236-A1 US-2018213800-A1 CN-105219651-B

CN-107475244-A

Publication date 08/01/2018 09/06/2018 06/07/2018 08/24/2017 03/07/2018 02/22/2018 12/04/2018 10/11/2018 09/20/2018 08/23/2018 08/02/2018 07/24/2018 07/12/2018 07/05/2018 07/05/2018 07/03/2018 05/10/2018 12/15/2017

Microbial Endophytes

US-2018193411-A1 US-2018186843-A1 US-2018186844-A1 CN-107232638-B AU-2014284267-B2

Title of published patent/application Method for high-throughput identification of microbial antagonists against pathogens Endophyte compositions and methods for improvement of plant traits Modulated nutritional quality traits in seeds Uses of Daldinia sp. or volatile organic compounds derived there from Microbial compositions and methods for bioprotection Antimicrobial compositions and related methods of use Antimicrobials from an epigenetics based fungal metabolite screening program Seed-origin endophyte populations, compositions, and methods of use Polyphosphate glass microspheres, methods of making and uses thereof Microbial consortia Penicillium endophyte compositions and methods for improved agronomic traits Improve drought-resistant species of rice, salt damage endogenous Aspergillus fumigatus stress capability and its application New antimicrobial peptides, their variants and uses New antimicrobial peptides, their variants and uses New antimicrobial peptides, their variants and uses Raisin seed extract purification method and its application upgrading Volatile organic compound formulations having antimicrobial activity Method for constructing metagenomic Fosmid library of soil microorganisms in tropical rainforest

360

Table 14.1  List of fungal endophytes and their patent application/publication. (Cont.)

US-9814242-B2 US-9790528-B2 WO-2017131821-A1 CN-105017203-B CN-106591156-A US-2016366892-A1 CN-106135143-A US-2016316760-A1 WO-2016135700-A1 WO-2016125153-A1 EP-2817323-B1 WO-2016044563-A1 EP-2967053-A1 CN-105176845-A CN-105177050-A WO-2015142186-A1 US-2015257394-A1 US-2015230478-A1 AU-2014200349-B2

11/16/2017 11/14/2017 10/17/2017 08/03/2017 06/09/2017 04/26/2017 12/22/2016 11/23/2016 11/03/2016 09/01/2016 08/11/2016 04/06/2016 03/24/2016 01/20/2016 12/23/2015 12/23/2015 09/24/2015 09/17/2015 08/20/2015 07/23/2015 (Continued)

361

Methods and compositions for the inhibition of quorum sensing in bacterial infections Bacillus subtilis isolate from corn and extracts that inhibit the growth of undesirable microorganisms in food products Production of lysergic acid by genetic modification of a fungus Microbial consortia Is a derivative of marine-derived fungus Azaphilones based compounds and their preparation method and application Epicoccum nigrum FXZ2 and application thereof Designed complex endophyte compositions and methods for improved plant traits Method of using carrot pure calluses to cultivate and breed aseptic Radopholus similis Isolated Complex Endophyte Compositions and Methods for Improved Plant Traits Microbial consortia Uses of Daldinia sp. or volatile organic compounds derived therefrom Antimicrobial cyclic peptide compositions for plants Compositions comprising recombinant bacillus cells and a fungicide Antimicrobial compositions and related methods of use Method for preparing Paecilomyces hepiali fermentation full-liquid instant particles Method for preparing Paecilomyces hepiali purpurin Screening methods for the selection of microorganisms capable of imparting a beneficial property to a plant Tea extracts and uses in promoting plant growth Endophytic microbial symbionts in plant prenatal care Synthetic mycotoxin adsorbents and methods of making and utilizing the same

Microbial endophytes and their intellectual property rights

WO-2017197303-A1

Endophyte

Patent number CN-104762341-A WO-2015091967-A1 US-2014242036-A1 US-2014115731-A1 WO-2014062929-A1 CA-2830541-A1 US-2014082771-A1 US-2013196013-A1 CN-103180453-A US-8455395-B2 US-2013071425-A1 KR-20120088495-A

CN-102595886-A CN-102168021-B CN-102168022-A

RU-2373276-C1

Publication date 07/08/2015 06/25/2015 08/28/2014 04/24/2014 04/24/2014 04/18/2014 03/20/2014 08/01/2013 06/26/2013 06/04/2013 03/21/2013 08/08/2012

07/18/2012 05/23/2012 08/31/2011 10/21/2010 09/22/2010 05/05/2010 11/20/2009

Microbial Endophytes

US-2010266717-A1 EP-2230235-A1 CN-101701193-A

Title of published patent/application Preparation method of low-molecular-weight Paecilomyces hepiali exopolysaccharides Mixtures comprising a super absorbent polymer (sap) and a biopesticide Process for potentiating the production of lingzhi mushroom (Ganoderma lucidum) substances and antifungal activity thereof Screening methods Tea extracts and uses in promoting plant growth Tea extracts and uses in promoting plant growth Fungi and products thereof Chemical and biological agents for the control of molluscs Method for high-throughput identification of microbial antagonists against pathogens Endophyte enhanced seedlings with increased pest tolerance Biopesticide and method for pest control Martelella endophytica YC6887 having biocontrol activity of plant diseases and nitrogen fixation effect and multifunctional bacterial inoculant containing the same Pesticidal compositions comprising 4,5-dihydroxyindan-1-one Diphyllria sinensis L.-derived endophytic fungus Geotrichum candidum and application thereof Endophytic fungus Penicillium ateckii from plant Chinese Umbrellaleaf rhizome and application thereof Chemical and biological agents for the control of molluscs Botryosphaerones, novel depsidones and their use as medicaments Method for preparing podophyllotoxin and/or astragaloside and special bacterial strain Penicillium verrucosum fungal strain, used for making agent with immunomodulating properties, and immunomodulator agent based on said strain

362

Table 14.1  List of fungal endophytes and their patent application/publication. (Cont.)

US-7070985-B2 WO-2006066184-A2 US-2005260182-A1 KR-100507041-B1 US-6911338-B2 US-2004141955-A1 US-6638742-B1 US-6372211-B1 CN-1046943-C

Phyllosticta sp.

Phomopsis sp.

US-5861302-A AU-2017201009-B2 ES-2565516-B1 WO-2016044533-A1 EP-3253864-A1 US-2017295798-A1 KR-101158503-B1

Stemphylium sp.

US-7259004-B1 ES-2610186-B1

11/11/2009 03/11/2008 07/04/2006 06/22/2006 11/24/2005 08/08/2005 06/28/2005 07/22/2004 10/28/2003 04/16/2002 12/01/1999 01/19/1999 09/27/2018 01/17/2017 03/24/2016 12/13/2017 10/19/2017 06/20/2012 08/21/2007 09/06/2018 12/02/2010 12/02/2010

363

WO-2010135758-A1 WO-2010135759-A1

Aspergillus fumigatus and application thereof Application of Muscodor albus to control harmful microbes in human and animal wastes Application of volatile antibiotics and non-volatile inhibitors from Muscodor spp. to control harmful microbes in human and animal wastes Methods and compositions for ultra-high throughput screening of natural products Coronamycins Antimicrobial compounds, periconicin A, B and the composition comprising the same Endophytic fungi and methods of use Compositions related to a novel endophytic fungi and methods of use Methods for obtaining taxanes Methods and compositions for controlling insects Potycyclic antiparasitic agent, its process and composition containing same Taxol production by a microbe Endophytes, associated compositions, and methods of use thereof Biocidal products and their use for controlling phytopathogenic yorganismos plague Compositions comprising recombinant bacillus cells and a fungicide Uses of Daldinia sp. or volatile organic compounds derived therefrom Compositions comprising recombinant bacillus cells and a fungicide Compound isolated from Phomopsis longicolla and method for preparing thereof Endophytic streptomycetes from higher plants with biological activity Natural broad spectrum biocides endophyte fungus from Solani stemphylium Use of anthracene derivatives as anti-infectives Novel anthraquinone derivatives

Microbial endophytes and their intellectual property rights

CN-101575576-A US-7341862-B2

364

Table 14.2  List of bacterial endophytes and their patent application/publication. Endophyte

Patent number

Title of published patent/application

Publication date

Bacillus sps.

US-2018318207-A1 CN-107058182-B

11/8/2018 10/23/2018

AU-2018214127-A1

Compositions and methods comprising yeast organisms and extracts thereof One kind of Bacillus amyloliquefaciens and Bacillus Death Valley complex microbial agent Volatile organic compound formulations having antimicrobial activity

WO-2018152411-A1

Compositions comprising recombinant bacillus cells and an insecticide

8/23/2018

RU-2664252-C1

8/15/2018

RU-2662992-C1

Strain of microfungus Fusarium equiseti, containing biologically active substances Method of pre-plant treatment of seeds of agricultural plants

CN-108143764-A

Preparation method for sijunzisan fermented by composite strains

6/12/2018

WO-2018081194-A1

Insecticidal proteins

5/3/2018

US-2018099999-A1

Fusion proteins, recombinant bacteria, and methods for using recombinant bacteria Novel plant endophytic bacteria bacillus oryzicola isolated from rice rhizosphere A special preparation of the medium and the bacteria isolated insect

4/12/2018

CN-107849518-A CN-104531556-B CN-107400675-A

8/30/2018

7/31/2018

3/27/2018 2/23/2018

US-2017311594-A1

Antimicrobial compositions and related methods of use

11/2/2017

US-2017291924-A1

Hypersensitive response elicitor-derived peptides and use thereof

10/12/2017

US-2017290339-A1

Compositions comprising recombinant bacillus cells and an insecticide

10/12/2017

CN-104419649-B

Having the biocontrol mengyang gas infestans

9/15/2017

CN-107090414-A

Pesticidal Flavobacterium strain

8/25/2017

Microbial Endophytes

WO-2017196850-A1

Cloning and sequence analyzing method for active product gene for fusarium 11/28/2017 wilt Gnotobiotic rhizobacterial isolation plant systems and methods of use thereof 11/16/2017

Antifungal Paenibacillus strains, fusaricidin-type compounds, and their use

8/18/2017

KR-101757350-B1

7/12/2017

CN-104419647-B

Endophytic Bacillus thuringiensis KB1 strain having controlling activity against pathogen Having biocontrol Malipo gas infestans

CN-106754478-A

Paenibacillus mealiensis and applications thereof

5/31/2017

US-2016326174-A1

11/10/2016

WO-2016178086-A1

Modulators of Clavibacter michiganensis and methods of making and using thereof Microbial compositions and methods for bioprotection

US-2016316759-A1

Mixtures comprising a superabsorbent polymer (sap) and a biopesticide

11/3/2016

CA-2976866-A1

Microbial consortia

9/1/2016

AU-2011302135-B2

Bacillus subtilis isolate from corn

8/25/2016

ES-2579683-T3

Antimicrobial compositions for plants cyclic peptides

8/16/2016

US-2016174570-A1

Endophytic microbial symbionts in plant prenatal care

6/23/2016

US-2016145558-A1

Co-incubating confined microbial communities

5/26/2016

WO-2016044542-A1

Compositions comprising recombinant bacillus cells and an insecticide

3/24/2016

WO-2016039961-A1

Chromobacterium subtsugae genome

3/17/2016

WO-2016036635-A1

Chromobacterium subtsugae genes

3/10/2016

US-2016007614-A1

1/14/2016

US-2015353852-A1

Bacillus subtilis isolate from corn and extracts that inhibit the growth microorganisms Microorganisms for producing volatile organic compounds

CN-104962490-A

Enteromorpha microbial agent and preparation method thereof

10/7/2015

US-2015237807-A1

Continuous bioprocess for organic greenhouse agriculture

8/27/2015

WO-2015118516-A1

Soil bacteria for inoculating stress soils

8/13/2015

CN-104419648-A

Musodormenghaiensis having biological prevention and control effect

3/18/2015

6/9/2017

11/10/2016

Microbial endophytes and their intellectual property rights

CN-107075458-A

12/10/2015

365

(Continued)

Endophyte

366

Table 14.2  List of bacterial endophytes and their patent application/publication. (Cont.) Patent number WO-2014161902-A1

Title of published patent/application Antimicrobial peptide produced by marine sponge-derived bacillus subtilis

Publication date 10/9/2014

CN-103232940-B

8/6/2014

CA-2884135-A1

Bionectria ochroleuca Bo-1 strain, cultures thereof, and applications of the strain Compositions and methods for controlling plant-parasite nematode

CN-102703328-B

Hypocrellin high-yielding Shiraia bambusicola strain

8/21/2013

US-2013212748-A1

Secreted insecticidal protein and gene compositions from Bacillus thuringiensis Ultra-high throughput screening of natural products

8/15/2013

Wastewater treatment process and plant comprising controlling the dissolved oxygen Insecticidal proteins secreted from Bacillus thuringiensis and uses therefor

10/13/2010

US-2010311107-A1 CN-101861286-A AU-2004267355-B2 WO-2009056901-A1

3/20/2014

12/9/2010

4/8/2010

EP-1948799-A1

Use of bacteriocins for promoting plant growth and disease resistance

7/30/2008

US-2007269542-A1

Endophytic streptomycetes from higher plants with biological activity

11/22/2007

CN-101072864-A

11/14/2007

EP-1711519-A1

Method of growing bacteria to deliver bioactive compounds to the intestine of ruminants Proteins inducing multiple resistance of plants to phytopathogens and pests

CN-1704120-A

Compound biological product conposition and preparation method thereof

12/7/2005

WO-2005040406-A1

High throughput screening of antibody libraries

5/6/2005

WO-2005010169-A2

High throughput or capillary-based screening for a bioactivity or biomolecule 2/3/2005

WO-2008138129-A1

10/18/2006

Microbial Endophytes

US-2008261815-A1

Cyclodipeptide synthases (cdss) and their use in the synthesis of linear 5/7/2009 dipeptides Thuricin 17 for promoting plant growth and disease resistance and transgenic 11/20/2008 plants 10/23/2008 Bioherbicide from Festuca spp.

US-2018291397-A1 US-2017283814-A1 AU-2016389840-A1 AU-2016204568-A1 KR-20170009704-A AU-2016224902-A1 AU-2016224901-A1 AU-2016224903-A1 US-9826743-B2 AU-2015317724-A1 CA-2957803-A1 JP-6377262-B2 US-9732335-B2 RU-2641916-C2 WO-2013026105-A1 DK-2539432-T3 CN-101861286-B

10/11/2018 10/5/2017 7/19/2018 7/21/2016 1/25/2017 8/31/2017 8/31/2017 8/31/2017 11/28/2017 4/6/2017 3/17/2016 8/22/2018 8/15/2017 1/23/2018 2/28/2013 7/3/2017 9/25/2013 5/24/2007 2/22/2007 7/7/2005 10/18/2006 2/6/2003 3/4/2004 5/7/2003 6/9/1998 5/29/1996 3/20/1996 (Continued)

367

WO-2007056848-A1 WO-2007022447-A1 AU-2004303536-A1 CN-1849397-A US-2003026795-A1 JP-2004506432-A JP-2003515608-A US-5763245-A CN-1123560-A CN-1119027-A

Engineered pesticidal proteins Biological controls of coleopteran pests Microbial consortia Fungi and products thereof (2) Endophytic Bacillus thuringiensis KB1 strain having controlling activity against pathogen Microbial consortia Microbial consortia Microbial consortia Compositions comprising recombinant bacillus cells and another biological control agent Compositions comprising recombinant bacillus cells and an insecticide Chromobacterium subtsugae genome The development of rice rhizosphere bacterium bacillus origination Kola Methods of screening for microorganisms that impart beneficial properties to plants Heterocyclic compounds as pesticides Pest-controlling agents isolated from spider venom and uses thereof An isolated bacterial strain of the genus Burkholderia and pesticide metabolites Wastewater treatment process and plant comprising controlling the dissolved oxygen Use of bacteriocins for promoting plant growth and disease resistance Insecticidal compositions and methods of using the same Proteins inducing multiple resistance of plants to phytopathogens and pests Insecticidal proteins secreted from Bacillus thuringiensis and uses therefor Methods and compositions for controlling insects New insecticidal toxin from Bacillus thuringiensis insecticidal crystal protein Amino heterocyclyl amides as insecticides and anthelmintics Method of controlling insects Biological control agents containing mollusc toxins Method of controlling insects in plants

Microbial endophytes and their intellectual property rights

B. thuringiensis

Endophyte Burkholderia cepacia

Patent number CN-104508116-B

Publication date 5/24/2017

US-5518908-A

Title of published patent/application Insecticidal bioactive Flavobacterium strains and compositions, metabolites and uses Beneficial microbes for delivery of effector peptides or proteins and use thereof Methods and compositions for ultra-high throughput screening of natural products Hypersensitive response elicitor-derived peptides and use thereof Î2-glucuronidase and glucuronide permease gene system Use of bacteriocins for promoting plant growth and disease resistance Insecticidal proteins secreted from bacillus thuringiensis and uses therefor Secreted insecticidal protein and gene compositions from bacillus thuringiensis and uses therefor Biological control of coleopteran pests Streptomyces endophyte compositions and methods for improved agronomic traits in plants Proteins inducing multiple resistance of plants to phytopathogens and pests Toona sinensis endophytic fungus TS8, secondary metabolite thereof as well as preparation method and application of secondary metabolite Chromobacterium subtsugae genes Method for high-throughput identification of microbial antagonists against pathogens Method of controlling insects

US-2012184030-A1 JP-2004529934-A US-2010092493-A1 US-9359275-B2 CN-1082543-C

Methods for establishing symbioses The use of anti-microtubule agents Complement system activation for treatment of bleeding-related inflammation Natural product antibiotics and analogs thereof Polycyclic antiparasitic agents, process and strain for their prepn. and use

7/19/2012 9/30/2004 4/15/2010 6/7/2016 4/10/2002

WO-2017176588-A1 CN-101120084-A

Erwinia sp.

WO-2017176587-A1 US-5599670-A US-2008248953-A1 US-2006191034-A1 WO-2005107383-A2 WO-2013192256-A1 WO-2016200987-A1 EP-1711519-B1 CN-106754396-A EP-3189145-A1 US-2012107915-A1

10/12/2017 2/6/2008 10/12/2017 2/4/1997 10/9/2008 8/24/2006 11/17/2005 12/27/2013 12/15/2016 2/25/2009 5/31/2017 7/12/2017 5/3/2012 5/21/1996

Microbial Endophytes

Microbacterium sps.

368

Table 14.2  List of bacterial endophytes and their patent application/publication. (Cont.)

7/30/2014

US-2017290334-A1 CA-2999782-A1

Method for high-efficient extraction of 3 beta-acetyl-15 alpha, 22-dihydroxyhopane Novel plant-growth promoting bacteria and the use thereof Dressing for caring skin ulcer Total nutrient formulation food for patients with rheumatoid arthritis Application of butyrolactone compounds to preparation of antituberculosis drugs Cyclodipeptide synthases (cdss) and their use in the synthesis of linear dipeptides Novel anthraquinone derivatives Compositions comprising recombinant bacillus cells and another biological control agent Plant-endophyte combinations and uses therefor Lipopolysaccharide isolated from pyrularia tissue and/or pyrularia-associated bacteria Seed-origin endophyte populations, compositions, and methods of use A bacillus methylotrophicus strain and method of using the strain to increase resistance Lipopolysaccharide isolated from Pyrularia tissue and/or Pyrularia-associated bacteria Chromobacterium subtsugae genes Bioactive fungi

US-2003049841-A1 US-2004241759-A1 WO-0231203-A2 US-2005070005-A1 WO-9421805-A2 US-2009061006-A1

High throughput or capillary-based screening for a bioactivity or biomolecule High throughput screening of libraries High throughput or capillary-based screening for a bioactivity or biomolecule High throughput or capillary-based screening for a bioactivity or biomolecule Method of controlling insects in plants Layered nanoparticles for sustained release of small molecules

3/13/2003 12/2/2004 4/18/2002 3/31/2005 9/29/1994 3/5/2009

WO-2018073454-A1 CN-106620831-A CN-104146201-A CN-107286120-A US-2010279334-A1 WO-2015172171-A2 CN-107205404-A Pantoea sps.

WO-2015200852-A2 US-2011070269-A1 AU-2016202480-B2 AU-2015292194-A1 AU-2010298191-A1

Pseudomonas sps.

4/26/2018 5/10/2017 11/19/2014 10/24/2017 11/4/2010 11/19/2015 9/26/2017 12/30/2015 3/24/2011 6/16/2016 3/16/2017

Microbial endophytes and their intellectual property rights

CN-103290089-B

4/19/2012 10/12/2017 3/30/2017

369

(Continued)

Endophyte

Patent number US-6613738-B1 WO-2013078365-A1 WO-0109175-A2 CN-102002469-A WO-2006062946-A2 KR-100574346-B1 KR-100566264-B1 RU-2553547-C2 KR-101711091-B1 WO-2014043058-A1 US-2016185717-A1 US-9896450-B2 JP-6316810-B2 US-8633154-B2

CN-104603260-B CN-108503616-A CN-104904750-A AU-2016306469-A1

Publication date 9/2/2003 5/30/2013 2/8/2001 4/6/2011 6/15/2006 4/27/2006 3/29/2006 6/20/2015 2/28/2017 3/20/2014 6/30/2016 2/20/2018 4/25/2018 1/21/2014 4/18/2017 9/22/2009 9/7/2018 9/7/2018 9/16/2015 2/22/2018

Microbial Endophytes

US-9622487-B2 US-7592509-B2

Title of published patent/application Cyclic lipopeptide from Cryptosporiopsis quercina possessing antifungal activity Highly efficient organic fertilizer and components thereof Plant defensin variants with modified cystein residues Bacterial strain for producing prodigiosin and method thereof Incorporation of bone marrow derived stem cells in tumors Bacillus subtilis eb072 strain, microorganism formulation for controlling plant diseases Biocontrol of plant diseases using novel endophytic isolate of Pseudomonas fluorescence Bacillus subtilis strain, producing peptide with antimicrobial activity Endophytic V. paradoxus KB5 strain having controlling activity against plant pathogen Compositions and methods for controlling plant-parasite nematode Compounds with antibacterial activity Modulators of Clavibacter michiganensis and methods of making and using thereof Method of increasing the production of phenolic compounds from cocoa Cyclodepsipeptides with antineoplastic activity and methods of using to inhibit cancer Annual brome control using a native fungal seed pathogen Isolated DNA sequences and polypeptides inducing multiple resistance of plants For improving crop productivity and reducing nitrous oxide emissions of nitrogen-fixing Dicumarol derivative as well as extraction method and application thereof Biological antiseptic, and preparation method and application thereof Compositions and their use for pest control and to induce plant hormone

370

Table 14.2  List of bacterial endophytes and their patent application/publication. (Cont.)

Rhizobium sps.

EP-3311669-A1 CN-106801014-A CN-107949568-A CN-108640896-A CN-1393558-A CN-108383824-A WO-2016023106-A1 WO-2017001730-A1 CA-2977191-A1 CN-101805216-A AU-2011204749-B2 CN-1513972-A AU-2015261618-B2 CN-106171101-A WO-2013008974-A1 US-2019029263-A1 WO-2016011562-A1

Staphylococcus sps.

US-2017295797-A1 US-2017295785-A1 WO-2018002955-A1 US-2008201085-A1 US-9364005-B2

A non-nematicidal composition and use thereof A bacillus methylotrophicus strain and method of using the strain to increase drought Novel plant-growth promoting bacteria and the use thereof Endophytic fungi for improving salviae miltiorrhizae yield and content New antimicrobial peptides, their variants and uses Desertorin B, and extraction method and application thereof Process for producing vital catalyst compound by controlling reactive temp Benzopyrone dipolymer and extracting method and application thereof Bis-indole alkaloids for use in the treatment of infections New antimicrobial peptides, their variants and uses Microbial consortia Method for locally culturing and producing arbuscular mycorrhiza fungus biological agent Endophytes and related methods Natural inductive non bean family plant tumor forming nitrogen fixation Endophytes and related methods (2) Method of using joint action of medicago sativa and piriformospora indica Martelella endophytica yc6887 microbial strain having a plant-pathology biocontrol Microbial consortia A bacillus methylotrophicus strain and method of using the strain to increase resistance Compositions comprising recombinant bacillus cells and a fungicide Compositions comprising recombinant bacillus cells and an insecticide A formulation of bio-inoculants for agriculture with enhanced shelf life Reciprocal symmetry plots as a new representation of countercurrent chromatograms Plant-endophyte combinations and uses therefor

9/25/2014 5/22/2018 4/25/2018 6/6/2017 4/20/2018 10/12/2018 1/29/2003 8/10/2018 2/18/2016 1/5/2017 9/1/2016 8/18/2010 8/27/2015 7/21/2004 11/30/2017 12/7/2016 1/17/2013

Microbial endophytes and their intellectual property rights

WO-2014147199-A1 CN-108064271-A

1/31/2019 1/28/2016 10/19/2017 10/19/2017 1/4/2018 8/21/2008 6/14/2016 371

(Continued)

Endophyte

Patent number CN-1845676-A WO-2017001731-A1 WO-2017001732-A1 US-10010558-B2 KR-101788585-B1

Micrococcus sps. RU-2614126-C1 CN-104277099-B CN-104419648-B CN-103667093-A CN-106858588-A BR-102012005283-A2

Citrobacter sps.

EP-3262152-A1 US-9532572-B2 US-2019008158-A1

WO-2015200902-A2 US-2013150240-A1

Publication date 10/11/2006 1/5/2017 1/5/2017 7/3/2018 10/27/2017 3/22/2017 4/5/2017 6/9/2017 3/26/2014 6/20/2017 3/29/2016 1/3/2018 1/3/2017 1/10/2019 1/26/2017 3/12/2015 4/20/2018 2/2/2012 4/16/2009 12/30/2015 6/13/2013

Microbial Endophytes

US-2017020138-A1 WO-2015035099-A1 CN-107926430-A US-2012028342-A1 WO-2009048673-A2

Title of published patent/application Compositions related to a novel endophytic fungi and methods of use New antimicrobial peptides, their variants and uses New antimicrobial peptides, their variants and uses Frankiamicin A compositions and methods Novel Strain of Fusarium solani JS-169 having anti-bacterial and anti-fungal activity Fungi strain from sordariomycetes class - producer of eremoxylarin a antibiotic One kind of preparation and application of entomogenous fungi antimicrobial peptides Having biocontrol Menghai gas infestans Lactobacillus plantarum 929-2 strain having food preservative and freshkeeping effect Preparation method and application of flazine in fermented food obtaining bioactive compounds of endophyte side Phoma herbarium with broad spectrum Microbial consortia Methods of use of seed-origin endophyte populations Endophyte compositions and methods for improvement of plant traits in plants Agricultural endophyte-plant compositions, and methods of use Agricultural endophyte-plant compositions, and methods of use High-yield method of Shatian pomelo Slip chip device and methods Stochastic confinement to detect, manipulate, and utilize molecules and organisms Endophytes, associated compositions, and methods of use thereof Enterobacter sp- 638 and methods of use thereof

372

Table 14.2  List of bacterial endophytes and their patent application/publication. (Cont.)

CN-101287381-A US-2015247190-A1 US-2010226876-A1 US-2013109568-A1 CN-102994431-A CN-101695306-B JP-3726132-B2 EP-3215631-A2 WO-2018058078-A1 US-2018291432-A1 WO-2018132774-A1 US-2015203880-A1 CN-107427528-A CN-107667288-A EP-2654432-A2 CN-107988186-A CN-107708704-A JP-2015504312-A CN-106944473-A

Microbial engineering for the production of chemical and pharmaceutical products Stable, durable granules with active agents Methods and systems for microfluidics imaging and analysis Composition for controlling fish Compositions and methods for therapeutic delivery with microorganisms Microbial agent for repairing petroleum-polluted saline alkali soil and preparation method Compound bactericide of active crude extract of baumannii bacteria liquid Bacteria monitoring method that the chitin-degrading enzyme was used in gene marking Microfluidic measurements of the response of an organism to a drug Digital quantification of dna replication and/or chromosome segregation Rapid Identification of Microorganisms Methods and compositions for improving plant traits Co-culture based modular engineering for the biosynthesis of isoprenoids, aromatics Glycan therapeutics and related methods thereof Spectrometric analysis of microbes Volatile organic compounds from bacterial antagonists for controlling microbial growth Cold-adaptation beta-1,4-glucan endonuclease as well as expressed genes and application Microbiome regulators and related uses thereof Aldehydes, antimicrobial mixture of organic acids and organic acid esters Method for remediation of uranium contaminated soil through pasturemicroorganisms

8/20/2013 10/15/2008 9/3/2015 9/9/2010 5/2/2013 3/27/2013 10/19/2011 12/14/2005 9/13/2017 3/29/2018 10/11/2018 7/19/2018 7/23/2015 12/1/2017 2/6/2018 10/30/2013

Microbial endophytes and their intellectual property rights

US-8512988-B2

5/4/2018 2/16/2018 2/12/2015 7/14/2017

373

374

Microbial Endophytes

Table 14.3  Commercially important bioactive compounds produced by microbial endophytes. Compund Taxol

Podophyllotoxin

Camptothecine Vinblastine Hypericin Emodin Diosgenin Toosendanin Huperzine A α-Irone and β-Irone Pestacin and Isopestacin Calbistrin A Terpestacin Peramine

Applications Taxol and various taxane derivatives, including cephalomannine, are highly cytotoxic and possess strong in vivo activity in a number of leukemic and tumor systems. As raw material for semi-synthesis of potent anticancer compounds etoposide, teniposide, and etopophos High efficiency and low toxicity anticancer drugs Used for treatment of Hodgin's disease, leukemias and solid tumors. Used for its anti-viral and anti-retroviral activity As a broad spectrum pesticide Used as antioxidative and hypolipidemic Used as a pollution-free biopesticide For treatment of Alzheimer's disease As fragrances

Patent number US4814470A; US6329193B1

As antioxidant and antimycotic agents.

US7192939B2

As antimicrobial agent Inhibits tumor angiogenesis Improves resistance to invertebrate pests and drought

CN102368998A CN104195053B US6072107A

US20040248265A1

CN1500797A US3520778A US5120412A CN101362702A CN1394869A CN102342307A CN101602727A FR2620702A1

have either gram-positive or gram-negative like clarithromycin, ciprofloxacin, vancomycin, erythromycin, azithromycin, andrifampin. The pharmaceutical and biotech companies are the leading industries, which generate most of the antibiotic and the majority of the patent has been given to these industries. Some of the recent antibiotic and their patent have been concise in Table 14.4. However, the last decade has witnessed a remarkable drop in the development of new generation antibiotics. For example in the United States, very less patent applications have been filed in the Trademark Office (USPTO) for the development of new generation antibiotics, and among them only a few patents were granted from those applications (Katz et al., 2006).

14.3  Microbe’s intellectual properties-related conflicts The increasing information on biological activities of endophytes provides a better resolution on mutualism to the pathogenicity nature of plant microbiome (Strobel, 2018). Endophytic microorganisms inhabiting the intercellular parts of host plants, helping them to cope with different environmental stresses (Tadych and White, 2019). These

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Table 14.4  Important antibiotics derived from microbial endophytes. Antibiotics

Endophyte

Patent number

Ecomycins Pseudomonad Pseudomycins Methylalbonoursin Munumbicins Echinomycins Cryptocandin A Cryptocin Jesterone Pestaloside Ambuic acid

Pseudomonas viridiflava Pseudomonas fluorescens Pseudomonas syringae Streptomyces spp. Streptomyces spp. Streptomyces spp. Cryptosporiopsis cf quercina Muscodor sp. Pestalotiopsis microspora Pestalotiopsis microspora Pestalotiopsis microspora

US6103875A CN102002468A US6630147B1 US7259004B1 US7259004B1 US7259004B1 US6613738B1 CN101691540A US7192939B2 US7192939B2 CN107129936A

qualities to produce a large number of bioactive substances attract researchers to characterize and utilize important biomolecules. The commercial exploitation of these biologically produced substances can be better protected by patent enforcements. In some countries, plant pathogens are described and indexed in the plant quarantine databases in the common names of their native language. When any modification in taxonomy is proposed for plant pathogens, the national officials enforce listed quarantine practices followed by interpretation of new system, sometimes conflict arise in recognizing the obnoxious pathogens (Sikes et al., 2018). In 1980, the US Supreme Court made more authentic decisions regarding the patentability of microorganisms. Chakrabarty, a microbiologist in 1972, filed a patent application, assigned to the General Electric Co., the patent application enclosed three primaries of claims; First—process claims for the method of producing the bacteria; Second—an inoculum composed of carrier molecule and the bacterium; Third—genetically engineered bacterium themselves. The United States patent examiner allowed the claims on the ground of the first two categories but rejected the claims for the bacteria. The examiner gave their decision on two ground; (1) Microorganisms are the products of nature, (2) Living things that are not patentable subject matter under 35 U.S.C. 101. Chakrabarty again appealed against this rejection and the court gave their decision in favor of him and stated that “microorganisms are alive without legal significance for the purposes of patent law.” So, court decided that genetically altered microorganisms were indeed patentable as they are man-made and have industrial application. Legal position is unconditionally clear that microorganism is patentable if it has industrial applications; the patent office was granted a patent of Chakrabarty genetically modified bacterium, but at that time it was the big question that, is microorganism a patentable subject matter? So, interoperating many conflicts of different patent laws like EPC Art.52 (fifty two) (PILA 2005), U.S.C. Art.-101 (one hundred one), and Diamond v. Chakrabarty (Mazzola, 2014) case, we can say that the basic principle is that in-

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vention should not be frivolous or against morality, on the ground of this court also reaffirmed their previous decision like the Chakrabarty case. A good deal of misunderstanding with regard to ambit and scope of microbe patent per se have been avoided in TRIPS agreement which allows microorganism patent (Gruner, 2001). The more authentic decisions regarding the patentability of microorganisms were made by the US Supreme Court in 1980 (Diamond v. Chakrabarty case), when the genetically modified bacterium was granted a patent, on the basis of that, genetically modified microorganisms were undeniably patentable because they were man-made and have industrial use. The legal situation is unquestionably clear that microorganism is patentable if it has industrial use, but there is still silence on the question, what is microorganism? There is a great deal of ambiguity in defining the subject matter.

14.4  Role of WIPO governing intellectual properties of microbial organisms As discussed earlier, the utilization history of microbial organisms starts in ancient civilization. In our day to day life microorganisms play roles in various aspects, in therapeutics–microbial control agents or antibiotics, vaccines; metabolism-related agents, insulin, and an array of diagnostic tools; in farming system microbial strains are used in improving crop yield, conferring resistance to various stresses; food industry for different kinds of enzymes and fermenting agents (Vitorino and Bessa, 2017; Kumar et al., 2018). Microbial endophytes are also being used in waste management of industrial and residential waste. In fuel industry microbial endophytes are used in green fuel production. These tiny potent organisms are improving life of almost all human beings. In the 20th century, industrial development and 21st century biotechnological interventions made necessary rules for the protection of microbial organisms. Budapest Treaty deals a precise topic in the International patent system: inventions concerning microorganisms (Nair and Ramachandranna, 2010). All signatories party to the treaty are bound to be acquainted with microorganisms deposited as a part of the patent disclosure system with an international depositary authority (IDA), irrespective of the location of depositary authority (Table 14.5). In fact, this means that the prerequisite to submit microbes to each and every national authority in which patent protection is required no longer exists. The TRIPS agreement under Article 27 Section 5 patents, deals on the patentable subject matter on sub sec 3 (b), defines that, “Plants and animals other than microorganisms, and essentially biological processes for the production of plants or animals other than nonbiological and microbiological processes. However, members shall provide the protection of plant varieties either by patents or by an effective Sui generis system or by any combination thereof” (Rao, 2002). The provision of this paragraph shall be reviewed after every four year by WTO agreement.

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Table 14.5  Name and locations of international depositary authorities functioning under WIPO. S.No. Name of the International Depositary Authority

Location

 1.  2.  3.

Australia Australia Belgium

 4.  5.  6.  7.  8.  9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.

Lady Mary Fairfax Cell Bank Australia (CBA) National Measurement Institute (NMI) Belgian Coordinated Collections of Microorganisms (BCCMTM) National Bank for Industrial Microorganisms and Cell Cultures (NBIMCC) International Depositary Authority of Canada (IDAC) Colección Chilena de Recursos Genéticos Microbianos (CChRGM) China Center for Type Culture Collection (CCTCC) China General Microbiological Culture Collection Center (CGMCC) Guangdong Microbial Culture Collection Center (GDMCC) Czech Collection of Microorganisms (CCM) VTT Culture Collection (VTTCC) Collection nationale de cultures de micro-organismes (CNCM) Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) National Collection of Agricultural and Industrial Microorganisms (NCAIM) Microbial Culture Collection (MCC) Microbial Type Culture Collection and Gene Bank (MTCC) Advanced Biotechnology Center (ABC) Industrial Yeasts Collection (DBVPG) Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna “Bruno Ubertini” (IZSLER) National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) International Patent Organism Depositary (IPOD), National Institute of Technology and Evaluation (NITE) Microbial Strain Collection of Latvia (MSCL) Colección de Microorganismos del CNRG (CM-CNRG) Moroccan Coordinated Collections of Microorganisms (CCMM) Westerdijk Fungal Biodiversity Institute (CBS) IAFB Collection of Industrial Microorganisms Polish Collection of Microorganisms (PCM) Korean Agricultural Culture Collection (KACC) Korean Cell Line Research Foundation (KCLRF) Korean Collection for Type Cultures (KCTC) Korean Culture Center of Microorganisms (KCCM) Russian Collection of Microorganisms (VKM) Russian National Collection of Industrial Microorganisms (VKPM) Culture Collection of Yeasts (CCY)

Bulgaria Canada Chile China China China Czech Republic Finland France Germany Hungary India India Italy Italy Italy Japan Japan Latvia Mexico Morocco Netherland Poland Poland Republic of Korea Republic of Korea Republic of Korea Republic of Korea Russian Federation Russian Federation Slovakia (Continued)

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Table 14.5  Name and locations of international depositary authorities functioning under WIPO. (Cont.) S.No. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47.

Name of the International Depositary Authority Banco Español de Algas (BEA) Colección Española de Cultivos Tipo (CECT) Culture Collection of Switzerland (CCOS) CABI Bioscience, UK Centre (IMI) Culture Collection of Algae and Protozoa (CCAP) European Collection of Cell Cultures (ECACC) National Collection of Type Cultures (NCTC) National Collection of Yeast Cultures (NCYC) National Collections of Industrial, Food and Marine Bacteria (NCIMB) National Institute for Biological Standards and Control (NIBSC) Agricultural Research Service Culture Collection (NRRL) American Type Culture Collection (ATCC) Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA)

Location Spain Spain Switzerland United Kingdom United Kingdom United Kingdom United Kingdom United Kingdom United Kingdom United Kingdom USA USA USA

14.5  Implementation of IP protection of microorganisms Budapest treaty proposed a competent and productive means of compulsory revelation related to patenting microbes. On 28th April 1977, Budapest Treaty was signed for the protection of microorganism for the international recognition of microorganisms for the patent purposes procedure, and further, it was amended on 26th September (Rai and Chatterjee, 2013). The main feature of the treaty “contracted state which allows or requires for the submission of microorganisms must be recognized for the purposes of the patent process, to fulfilling such purposes, the deposit of a microorganism with any IDA,” nevertheless of whether such an authority is on or outside the zone of the said State. The Budapest Treaty provides a detailed description of microbial intellectual properties. The following main points to ponder in regard to microbial organisms.

14.5.1  Microorganisms submission for purposes of the patent process 14.5.1.1  Revelation and requisite for submission The primary necessity of patent law requires complete disclosure of the invention sought for patent protection. Each and every detail related to a patent should be clearly mentioned in the patent application. This essential requirement has been enabled the skilled person to do the task in a similar manner as mentioned in the patent application (Giugni and Giugni, 2010). So providing every tiny detail is very essential for the further working with the same patent efficiently. The description should be supported by drawings, if needed. However, inventions related to novel microbes, that are not

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available to the public even though disclosed in patent applications cannot ensure the same invention to happen. When a microbe is isolated, characterized, and improved, it is impossible to repeat the same process without having direct access to that microbe. In that case, microbe disclosure itself becomes the essential part of the document (Parashar, 2017). These conditions led the intellectual property organizations to consider about microbial depositories or culture collection units (Parashar, 2017). The description of culture submission has been enabling a prescript to obtain that improved microbial culture to the public domain.

14.5.1.2  The requirement for an identical international deposit system In 1970s, it was common to submit the microbial culture for patent procedures, but there was no such identical procedure of submission, and/or, perhaps more precisely, the recognitions of deposits are also not established. The contribution made by such countries in “recognized collection” for patent proceedings were going on, but the minimum requirement for these depositories was not up to the mark according to the international framework (Parashar, 2017). The collections were not made in such a legal framework that can be shared culture requesting parties. The lack of definite guidelines has given depositors almost full control over their microorganisms, which has ultimately created various anomalies to the local culture deposition system. For instance, many application submissions have been done for the same culture in more than one culture collection centers located in different countries. This way of same culture deposition in different collection center increases the risk in a greater extent to the users as well as the owner. This time exhausting, wasteful and sometimes costly, practices lead to the requirement of submission of cultures in all countries where patent protection must be soughed. To sojourn the submission of such multiple depositions of microorganism, in 1973 two governments of the United Kingdom proposed an option to the WIPO, to deposit and allocation of single submission for a single microorganism worldwide. This proposal was taken in consideration and agreed by the WIPO Governing Bodies.

14.6  The Budapest Treaty The director general (DG) of WIPO organized a team of specialists in 1974 for the discussion with international communities on the opportunities submission of microorganism for patent purposes (Introduction to the Budapest Treaty; https://www.wipo. int/export/sites/www/treaties/en/registration/budapest/guide/pdf/introduction.pdf). The squeezing of the discussion made by this committee was that; the culture collection should be identified by the depository authorities and submission made to any of the depository authorities should be considered for all patents by the region. In this way, they can get the necessary protection for related invitations. The expert team also found that the decision of the treaty would be compulsory to implement this recommended resolution in to effect. Further bureau of WIPO had prepared a draft for the treaty and regulations for the international recognition of microorganism’s submission for patent purposes. Treaty was thoroughly studied by the expert team in 1975–76.

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On April 14–28, 1977, a third and actually the streamline draft of the treaty has been approved. This was based on consideration of diplomatic conference, agreed, and organized by the DG of WIPO, in collaboration with Government of Hungary held in Budapest (WIPO, 1977). A total of 29 representatives of 29 state, one member of Paris Union (for the protection of industrial property), observer from the two member states, two of the Paris Union, the interim committee of the European patent organization and eleven nongovernmental international organizations had been attended this diplomatic conference. Out of them three conceived the treaty with title “Budapest Treaty on the international recognition of submission of microorganism for the purpose of patent procedure” (WIPO, 1977). Regulations passed after this meeting has been shielded under the treaty. Finally in 1980, it was approved or appropriated by the required minimum number of states (five) (WIPO, 2017). Further again in 1981 and 2002, the regulations under the Budapest Treaty were revised. Presently, more than eighty countries are part of this treaty and a total of 39 IDAs in the 22 countries have been functioning (Parashar, 2017). To avail the membership of Budapest Treaty, every country must have endorsed the Paris convention, which is related to the protection of industrial application. This treaty has vast adequacy and it is not only accepted the microorganisms but also animal cells, plants, cell lines, hybridoma cells, RNA, and plasmids (Parashar, 2017).

14.6.1  Fundamental structure of the Budapest Treaty 14.6.1.1  IDA and recognition of one submission Certain culture collections are identified as “IDAs” in the treaty (WIPO, 2017). Now a days various culture collection centers are working worldwide under IDA that collects the microorganism for the purpose of patent (Table 14.5). These IDA units are the key place for the culture supply, sample deposition, as well as characterization and preservation of biological samples (Parashar, 2017). The key feature of IDA is that all the member of the countries will recognized any IDA as a submission center and that submission will be recognized internationally. The IDA accepts the submission of microorganism from all the member countries and the IDA member is not bounded with the territorial boundaries. In modern patent system, IDA is the single recognized platform for uniformly accepted patent application from any of the member countries. In the same way, any intergovernmental organization, such as the European Patent Office, can submit a proper affirmation with the DG of the WIPO for their own patent purposes. Such organizations has also bounded with the provisions of the treaty and rules of WIPO for their international recognition. Patent application is only accepted after all the rules have been followed by the applicant. In such a case that patent office has also been recognized for the submissions in any IDA (https://www.wipo.int/treaties/en/registration/budapest/guide/pdf/introduction.pdf). One of the fiery questions that resolved in this treaty was to sojourn multiple submissions of microorganism in more than one IDA for patent purposes. In the Budapest Treaty also it has been clearly mentioned that single submission of sample for patent purpose in IDA will be enough to give recognition by other member countries (Parashar, 2017) and it has been clearly

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mentioned that any contracted country that fulfills the terms and conditions of WIPO agreements becomes a member of IDA (WIPO, 2017). The contracting country legally deposits/nominates a culture collection within the territory of their own IDA depository. The depository location lies within IDA territory of the contracting state, has to provide assurance that their collection meets all the terms and conditions. Along with that they have to be fulfilling the necessities as well as conventions defined in the treaty. (https://www.wipo.int/treaties/en/registration/budapest/guide/pdf/introduction. pdf). Another important feature of IDA is to their availability to any depositors in similar terms as mentioned in the treaty. IDA is accepted and store deposit microorganisms with the full-term specified in the treaty. It is also provided the sample of submitted microorganism to the people who entitled to obtain that microorganism. In a similar fashion any intergovernmental industrial property organization which has filed the affirmation with the fulfillment of the term and condition mentioned in paragraph 6 of Budapest Treaty, that organization will get a secure guarantee in respect of a culture collection situated in the region of any contracting States. They are also having the right to obtain the culture collection from any of the contracting states.

14.6.1.2  Deposit and furnishing of samples Depositors should have to follow the detail procedures of IDAs, which are mentioned in the regulations under the treaty (WIPO, 2015). The period of storage of submitted microorganisms (minimum 30 years; maybe extended for five years after the most current demand for a sample) and the depositor has to provide methods for securing the samples. Although under the treaty regulation do not submit the time of deposition, however, the national law should have the responsibility to follow and keep the complete information related to the particular submission. So, to a large extent, there are many more conditions to equip the samples. The depositor can provide the sample at any time, to someone, who has written authorization by any “interested” IP offices and/or the IDA authority, (i.e., a deposit is working with a patent application related to microorganisms and which make available IDA with the affirmation of that effect), but apart from this for any other cases the national law has the rights to whom, when, and in which circumstances they will provide the samples to the requesting third party (WIPO, 1977). However, different countries have their own laws to provide the samples on the request of a third party and IDA may not aware of the laws of those countries. In such cases, the regulation has provided the platform that the requesting third party makes a request from the IDA on a form on which the respective IP office certifies that the third party has entitled to avail the sample of that particular microorganism. Apart from this the IP offices are also provided information time to time to the IDAs, of the accession numbers of the microorganism, which are actually outlines and published in their own patent office. They have provided the patent of those accessions, in such case those microorganism become available to anyone without the need for authentication (WIPO, 2015).

14.6.1.3  Safeguard of deposits The treaty has various rules and regulations to protect the loss of the deposited microorganism, due to the nonavailability of deposited microorganism in the IDAs. Treaty

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has strictly stated that IDA must have the skill and abilities required to retain the microorganism viable and uncontaminated throughout the storage period (WIPO, 2015). If for some reason an IDA is not capable of presenting the samples of microorganisms, then in such cases the depositor can submit the new fresh of the same sample and he/ she will be benefited from the date of initial submission. In the treaty, there is also a provision that if by some reason an IDA is no longer functioning, in that case treaty provides a facility to transfer the deposited microorganism from ceased IDA to another functional IDA. (WIPO, 2015).

14.6.1.4  Implications of the term microorganism Detail description of “microorganisms” has not been given in the treaty to broaden the scope and applicability of microorganisms (https://www.wipo.int/export/sites/www/ treaties/en/registration/budapest/guide/pdf/part_i.pdf). It has been not clearly mentioned the group, sub-group, or the category of the microorganism in the treaty. For the patent purposes, part or unit of the microorganism is necessary for the disclosure of submission. Consideration of biological material under the microorganism category such as plasmids and tissue culture products provide a broader definition of the microorganism (WIPO, 2015). These submitted biological products under the term of the treaty have been eventually accepted by IDA. None of the internationally recognized gatherings like Budapest Treaty, Paris Convention, TRIPS, PCT explains or defines the term of microorganism. In all these agreements and treaties, the microbes have been defined broadly and the plasmid, tissue culture, as well as transgenic products, are also considered under the category of microorganism. Globally, none of the patent law has provided a proper and uniform definition of microorganism. There are widespread confusion and speculation in defining the leis of microorganism. Consequently, many countries taking advantage of the unclear or unspecific definition of microorganism in international laws. However, according to the convention of law, if the relevant treaty is not able to provide a proper definition than the general meaning of that term should be used by any country in their native law. The concise definition of microorganism by the oxford dictionary is “any of the microscopic organisms, including algae, bacteria, fungi, protozoa, and viruses” (Adcock and Llewelyn, 2000). This definition has not described microorganism accurately and scientifically for the purpose of providing a clear definition according to the requisite of Article 27.3 (b) of TRIPS agreement. Broadly “microorganism” comprises of bacteria and cyanobacteria, slime moulds, bacteriophages, plasmids, viruses, archaebacteria, algae, protozoa, actinomycetes, virus strains, yeasts, filamentous fungi and mushrooms, fused cells, vectors, cell lines, tissue cultures, and plant cells (Unson et al., 1994). A microorganism or a microbe is generally a small living thing that is visible under microscope. The European patent convention (EPC) wide Article 53-b, has mentioned that European patents shall not be granted in respect of plant or animal varieties or essentially biological processes for the production of plants or animals; this provision

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shall be not applied for microbiological processes or the products (https://www.epo. org/law-practice/legal-texts/html/epc/2016/e/ar53.html). The European Union (EU) has introduced the new term “biological material” for the microorganism. This term acts as a substitute for microorganism for the purpose of patent and denotes that any things which is holding the genetic information and have the ability to replicate and multiplied itself in the biological system have been considered as biological material (Ryan et al., 2006). Thus, EU also included the genetic information which has passed generation to generation in the replicating microorganism are considered under the category of biological material (Ryan et al., 2006). Brazilian laws describes the term “transgenic microorganism” for the patent purposes, which explain transgenic microorganisms are those organisms, which express in certain part of the plant or animals and that manipulation of the genetic material are carried out by the human being using the genetic engineering. (Giust, 1997). In another term microorganism can also define as “The biological material having genetic constitutes and replication capability without human interference” (Sangal et al., 2017). In the current scenario, the role of IPR professionals should not be undervalued in the explanation of the microbiological definition. In the absence of a clear definition, the ability to define microorganism, thus becoming a key factor to achieve the desired purpose (Sangal et al., 2017). Microorganism identification, characterization, and submission for patenting as well as granting for patent is one of the major hurdles of the evolving field of microbial patent. Before PCR technology, the DNA (deoxyribonucleic acid) replication and amplification were performed manually and this way it is a time-consuming process that makes microbe’s identification and their characterization much more complicated and tedious (Chowdhury et al., 2009). Now it is much easier to characterize the microbes, firstly isolating the microbes from their natural environment using suitable technique and further identified through the DNA fingerprinting techniques. This technique facilitates the clear identification and characterization of microbes at the level of genetic level. Now specific markers are available which map the most variable region present in the genetic materials of the microbes. Scientifically, these regions are known as mini and microsatellite regions. These region of the microbe’s genetic material has most diverse and variable information. Narrative of the above para has been discussed that microbial diversity and their conservation are a valuable sources for culture collection. Correct characterization and identification of culture is the key factor for the differentiation of the microbes (Tripathi et al., 2007). The current advancement of molecular biology has to open of the different strata of the microbes rapidly. Discovery of new strains and microbial taxa increases the necessity to preserve those newly discovered microbes for further use of other researchers (Sharma and Shouche, 2014). Culture collection is a manpower consuming and expensive procedure, so individual laboratories have not been capable to maintain all the culture collections (Sharma and Shouche, 2014). Developed infrastructure and skilled to the long term preservation of culture collection are necessary for the good culture collection (Daniel and Prasad, 2010). IDA has been recognized in the different contracting states based on the skilled

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and advancement of the culture collection center. For the sustainable conservation of microbial assets, culture collection plays a key role (Daniel and Prasad, 2010). It is also a very suitable and authentic material for high-quality microbial research (Smith, 2003). Before the Budapest Treaty, insufficient constant instructions were messed up with different patent indigenous laws of different countries, which ultimately managed the culture collection in big confusion (Sangal et al., 2017). The breakthrough comes when Budapest comes into existence after that problem related to the submission of microbial culture and their identification had been reduced. Thus, the Budapest Treaty has recognized the various IDAs worldwide as a depository’s center for the microbial culture collections. Therefore, depository centers are also known as a microbial bank. If an applicant remarks biological material in innovation and is not capable to produce the specimen for the description, then the disclosure can be made by submitting such material in the IDA under the Budapest Treaty. These biological materials shall not be submitted after the date of filing; however, for the deposition within 3 months from the date of application submission, references number will be given for the specification. Details of deposition, source, and geographical origin of the biological material shall be mention in the complete specification (Schlosser,  1980).

14.7  Conclusion and prospects Microorganisms especially endophytes and their different metabolites or byproducts in the purified form and how to maintain their protection have been discussed at international level, moreover clarifications of different global assemblies, generally TRIPS, Paris Convention, Budapest has conversed. Along with developed countries, after TRIPS agreement, various developing countries also seriously implemented their law and provided patenting of microorganisms. This chapter highlights the need for describing microorganisms at global level with reference to intellectual rights as well as the patent provided to the different endophytes to date. Moreover, the chapter skins the submission of culture in the IDAs of several countries with universal standards of patentability. We are also doing efforts to discuss the balanced level of laws and science in terms of microorganism. However, more emphasis is given over the laws related to possible objections with infringement issues related to the microbial patent, which will later assist in building cognizance to scientific and legal community. Improvement in technology enhances unexpected possibilities and increases the new encounters to legal system, which forced the legal authorities for frequent laws updates according to the advancement in technology. Although there are lots of improvements and transparency has been imposed by different world patent regulatory authorities. However, there is a requirement of big decorative microorganism associated patent law at worldwide level with the establishment of identical patent code which will uniformly enforceable at the universal level.

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Index

A Abiotic stress, 37, 58, 59, 112, 118, 125, 127, 138, 262, 327, 328 alleviation by endophytes, 164 drought stress, 164 alleviation by endophytes, 166 endophyte mediated tolerance in plants, 166 tolerance by plants, mechanism of, 166 heavy metal stress, 172 endophyte mediated metal stress tolerance in plants, 175 metal stress alleviation by endophytes, 174 nutrient starvation, role in endophyte mediated nutrient uptake in plants, 177 salinity stress endophyte mediated salt stress tolerance in plants, 170 plant mechanisms to tide over soil salinity, 169 salinity stress alleviation by endophytes, 169 temperature stress cold stress alleviation by endophytes, 171 endophyte mediated temperature stress tolerance in plants, 172 heat stress alleviation by endophytes, 171 mitigation of genomics aspects of, 237 legume crops, 199 plant tolerance to, 196 ABI/SOLiD sequencing, 257 Abscisic acid (ABA), 88, 135, 145, 163, 238, 263 Acetoin, 46 N-Acetyl glucosamine, 4 Achnatherum inebrians, 114

Achnatherum sibiricum-Neotyphodium Sp., 235 “N” and “P” limiting and nonlimiting conditions, 235 Achromobacter xylosoxidans AUM54, 87 Acid phosphatase, 235 Aciphyllene, 261 Acremonium coenophialum, 138 Actinobacteria, 11, 58, 229 S-Adenosylmethionine decarboxylase, 262 Aedes aegyptii, 314 Aegle marmelos, 274, 292 Aerugine, 66 Affymatrix wheat genome array chip, 262 Agriculture, 76, 77, 94 bioproducts, 35 food production, 195 productivity, 116, 161 Agrochemicals, 35, 57, 195 Agroecosystems, 127, 195 Alcaligenes denitrificans, 79 Alcaligin, 79 Alcohol dehydrogenases, 7 Alfalfa, 10 Alginate, 68 Alkaloids, 137, 142 Alkylating agents, 291 Alternaria alternata, 88, 137, 274, 282 1-Aminocyclopropane-1-carboxylate (ACC), 236 1-Aminocyclopropane-1-carboxylate (ACC) deaminase, 44, 59, 63, 175 activity, 235, 328 production, 63 1-Aminocyclopropane-1-carboxylate oxidase, 88 Ammodendron bifolium, 84 Ammonia, 42 α-Amylase activity, 108 Anamorphic fungi, 251 Anhydrofusarubin, 178 Antagonism, 329

390 Index

Anthocyanin, 263 Antibiotics, 66 derived from microbial endophytes, 375 production, 88 Anticancerous compounds, 286 aldehyde, 286 alkaloids, 286 chromones, 287 cyclohexanones, 287 depsidones, 287 depsipeptides, 288 ergochromes, 288 esters, 288 lactones, 289 lignans, 289 peptides, 289 polyketides, 290 quinone, 290 spirobisnaphthalenes, 290 Antifungal products, 66 Antifungal volatiles P. chlororaphis (PA23), 66 Antimicrobial compounds, 41 Antimicrobial metabolites, 240 Antioxidant defense system, 114 Antitumor antibiotics FR901463, 66 Apiospora montagnei, 287 Aquaporins, 116, 263 Arabinogalactan proteins (AGPs), 5, 263 l-Arabinose, 7 Arable lands, 195 Arachnid oil, 196 Arbuscular mycorrhizal fungi (AMF), 133 associated salinity tolerance in host plant, 237 Arbuscule forming mycorrhiza (AMF), 212 Arogenate dehydratase (ADT), 263 Arthrinium phaeospermum (AP) toxin, 89 Artificial inoculation methods, for colonization, 333 Ascocoryne sarcoides, 352 Ascomycetes, 251 asexual filamentous, 229 Asexual spores, 127 Aspergillus flavus, 206, 274, 276 Aspergillus fumigates, 236 Aspergillus japonicus EuR-26, 171 Aspergillus niger, 274, 276 Aspergillus tamarii, 138

Aspergillus terreus, 311 strain 2aWF (2aWF), 117 Aspergillus versicolor, 138 Aspergillus wentii EN-48, 91 Atmospheric nitrogen fixation, 64 Atractiella rhizophila (SML-TX-18), 117 Autoinducer N-acylated homoserinelactone, 240 Autophagy, 116 Auxin, 145, 236, 261, 324 Auxin-like compounds (ACCS), 164 Azadirachta indica, 81 Azithromycin, 374 Azoarcus sp. BH72, 6, 7, 39 Azomycin, 66 Azorhizobium caulinodans ORS571, 2, 39 Azospirillum brasilense, 3, 39, 209 Azotobactin, 79 B Baccharoides anthelmintica, 82 Bacillus amyloliquefaciens, 88 strain RWL-1, 82 Bacillus anthrasis, 79 Bacillus brevis, 206 Bacillus cereus strain BS 03, 90 strain NRL2, 81 strain S42, 90 Bacillus halotolerans Y6, 86 Bacillus megaterium (BmBP17), 178 Bacillus mycoides EC18, 39 Bacillus oryzicola YC7007, 88 Bacillus pumilus AM11, 170 Bacillus pumilus INR7, 88 Bacillus spp. (RCh6), 88 Bacillus subtilis, 45 strain 11BM, 114 strain CBR05, 90 strain EPC016, 81 strain LK14, 236 strain NIIST B580, 92 strain NIIST B595, 92 strain NUU4 with Mesorhizobium cicero IC53 enhanced root and shoot biomass in, 238 strain SV41, 88 strain V26, 91 strain YC7010T, 86

Index

Bacillus thuringiensis, 206 Bacillus thuringiensis (Bt)-based biopesticides, 215 Bacillus velezensis YC7010, 88 Bacterial-based plant bio-stimulants, 209 Bacterial bioformulation, for agricultural applications, 46 Bacterial endophytes, 58 and biodiversity, 58 entry and colonization of plants by, 5 mutant strains, 227 and patent application/publication, 374 plant growth promotion mechanisms, 59 properties of, 60 Bacterial genera, as phosphate solubilizers, 64 Bacterial surface components, 39 Bacteroidetes, 11, 58 Bakanae disease, 88 Barley, 5 Barley1 genome array chip, 262 Basidiospores, 84 Beauveria bassiana, 181, 206, 288 Benzene acetic acid, 235 Benzene, ethylbenzene, toluene, and xylene (BTEX) compounds, 181 Benzoates, 2 Benzothiadiazole to induce systemic resistance, 88 2-Benzoxazolinone (BOA), 145 Beta-proteobacteria, 11 Bgy6 overexpression, 86 Bikaverin, 178 BIK1(botrytis-induced kinase1), 88 Bioactive compounds commercially important, 377 for human health, 291 Bioactive metabolite production by endophytes, through statistical approach, 91 Biochar, 115 Biocompatible nanoparticles, 308 Biocontrol, 8, 65 ability of endophytic microbes, 94 agents, 206 plant diseases, 45 efficiency of endophytic bacteria, 45 products, 336 Met52, 336

391

Biodiversity, 275, 351 Biofertilizers, 209, 211, 215, 335 Biofilm, 39 formation, 199, 230 Bioformulations, 92 as biocontrol agents, 206 defined, 198 formulation and application methods, 207 liquid formulations, 208 solid bioformulation, 207 Bioinoculants, 215 Biological control, 137, 138, 143 Biologically active compounds, 164 Biological methods, 308 Biological nitrogen fixation (BNF), 235 Biomarket, 334 Biopesticide, 215 Bioprimed plants, 68 Bio-priming, 115 Biostimulant, 209 Biosurfactant, 199 Biotechnological applications, 283 Biotic stress, 37, 41, 58, 117, 177 in host plants by endophytes, genomics of alleviating, 239 role of endophytes in biotic tolerance, 177 tolerance, 138 Bio vaccine, 335 Blast, 76, 81 Blight, 86, 88 Botrytis cinerea, 88 Box-Behnken Design (BBD), 91 Bradyrhizobium japonicum, 38 USDA110, 8 Brassica napus, 12, 64 Bruguiera gymnorhiza, 282 Budapest treaty, 376, 379 fundamental structure, 380 deposit and furnishing of samples, 381 IDA and recognition of one submission, 380 implications of term microorganism, 382 safeguard of deposits, 381 Burkholderia cenocepacia 869T2, 89 Burkholderia cepacia strain G4, 181 strain L.S.2.4, 174 strain VM1468, 174

392 Index

Burkholderia gladioli HDXY-02, 82 Burkholderia glumae, 2 Burkholderia kururiensis, 163 Burkholderia phymatum strain STM815A, 8 Burkholderia phytofirmans PsJN, 2, 39, 68, 115, 171 Burkholderia plantarii, 2 2-Butanone, 261 Butylated hydroxytoluene, 286 Butyrolactones, 66 C Cadophora sp., 234 Caffeic acid O-methyl transferase (COMT), 263 Callose, 133 Callus, 40 Calvin cycle, 263 Camellia sinensis, 287 Camptosar (anticancer agents), 286 Camptotheca acuminata, 286, 287 Camptothecin, 291 Cankers, 76 Cannabis sativa L., 68 Carbohydrate active enzymes (CAZymes), 234 Carbon, 2 utilization, 199 Carboxylate ions, 235 Carboxylates, 65, 79 Carboxymethylcellulose (CMC), 198 Catalases (CAT), 38, 86, 88, 143, 265 Catecholates, 65, 79, 80 Catharanthus roseus, 274 cDNA/EST library, 257 Cell to cell communication, 68 Cellular homeostasis, 237 Cellulases, 6, 84, 129, 261 Cellulose, 39 Cell wall-degrading enzymes, 47 Centella asiatica, 314 Central composite design (CCD), 91 Cepaciamide A, 66 Cepafungins, 66 Ceratocystis paradoxa, 180 Chaetomium globosum, 276, 286, 288 Chaetopyranin, 286 Chartreusin, 82 Chelation of metal ions, 59

Chemical communication, 35 Chemical fertilizers, 69, 198 Chemical pesticides, 198 Chemoattractants, 2 Chemo-priming, 108 Chemotaxis, 38, 39 agents, 2 Chitinase, 47, 67, 84, 129 Chlorinated solvents, 181 Chlorophyll biosynthesis, 263 Chondrostereum purpureum, 206 Chromium toxicity, 172 Chromobacterium violaceum, 68, 240 Cinnamic acid 4-hydroxylase (C4H), 260, 263 Cinnamomum zeylanicum, 352 Cinnamoyl-CoA reductase (CCR), 263 Cinnmoyl alcohol dehydrogenase (CAD), 263 Ciprofloxacin, 374 Citrate, 235 Citric acid, 235 Cladosporium resinae, 290 Clarithromycin, 374 Claviceps purpurea, 288 Clerodendrum colebrookianum, 84 Clethra barbinervis, 239 Climate change, 35 Climate resilient agricultural technologies, 227 Climatic variation, 161 Cobalt (Co)toxicity, 172 Coffee, 10 Colletotrichum falcatum, 180 Colletotrichum gloeosporioides, 274 Colletotrichum magna, 166 Colletotrichum orbiculare, 47 Colletotrichum protuberate 4666D, 166 Colletotrichum tropicale, 261 Colonization, 2, 5 aerial surfaces (phyllosphere), 35 fungal. See Fungal colonization internal tissues of plants (endosphere), 35 of nitrogen-fixing microorganism, 21 in phyllosphere, 7 Rhizobium strain only in symbiotic zone of root nodule of legume, 7 root-associated soils (rhizosphere), 35 route of strain Paraburkholderia phytofirmans PsJN, 6

Index

site of xylem, 6 stomata as entry site during, 7 Streptomyces galbus on Rhododendron wax-degrading enzyme helping in, 6 Commensal microorganisms, 227 Commercialization biocontrol formulations, 95 endophyte products for sustainable agriculture, 334 Communication, 2 Competition, 65 colonization completely depends on, 66 effectiveness of PGPB mediated processes, 65 Complementary DNA (cDNA) libraries, 254 Conidia, 196 Coniothyrium minitans, 206 p-Coumaroyl-CoA synthase (4CL), 263 C-P lyases, 235 CRISPR/Cas9 technology, 95 Crop diseases, management of, 76 growth, 108 losses, 76 production, 57, 107 Cross talk processess, 35 Cucumis sativus, 47 Culex quinquefasciatus, 314 Curvularia, 137, 146 Cutinase, 261 Cyanea acuminate, 286 Cyclin dependent protein kinases, 263 Cyclosis, 7 Cytokinin, 44, 127, 145, 236, 324 gene, 45 Cytotoxic secondary metabolites, 291 D Damage associated molecular patterns (DAMPs), 261 Danshen rot disease, 81 Deciphering disease suppressive mechanisms, 329 antagonism, 329 competition for niche and nutrition, 329 induced systemic resistance (ISR), 330 plant growth promotion, indirect mechanisms of, 329

393

Defensins, 261 Defensive mutualism, 229 Dendryphion nanum, 274 Denitrification genes, 235 Desferrioxamine-like siderophore, 79 2, 4-Diacetylphloroglucinol (DAPG), 66, 177 Diacetylphloroglucinols, 199 Dianthus caryophillus, 47 1, 4-Diaza-2, 5-dioxo-3isobutylbicyclo[4.3.0]nonane (DDIBN), 81 Diazotrophs, 108, 164, 235 Dicerandra frutescens, 288 Dichanthelium lanuginosum, 146 Diepoxin, 290 Dietzia natronolimnaea, 238 Differential display reverse transcription PCR (DDRT), 254 Dihydro cinnamic acid, 82 Dihydrozeatin (DZ), 135 Dikaryomycota, 229 Diketopiperazines, 137 N-N, Dimethylformamide, 309 Diversity, 325 microbial endophytes, 325–327, 331 DNA repair, 263 DNA synthesis, 263 Drechslera tritici-repentis (Dtr), 143 Drought, 108, 143, 195 Drought stress tolerance, 144 2, 4-D Toxicity, 180 DXR (deoxyxylulose-4-phosphate reductase), 260 E Ecological niche, 127 Ecomycins, 66 Ecosystem, 75 (2E,5E)- phenyltetradeca-2,5-dienoate, 82 Effector-triggered immunity (ETI), 40 Efflux pumps, 38 eglA gene, 6 eglS gene, 84 Electron transport pathway, 116 Electrostatic charges, 3 Endo-β-1, 4-glucanase, 84 Endoglucanase, 6, 84

394 Index

Endophytes. See also Endophytic bacteria; Endophytic fungi association, 116 based bioproducts market, regulation of, 337 as biocontrol agents against pests, 331 bio-formulation of challenges related to development, 93 colonization. See Colonization commercialization, challenges in, 337 defined, 1 diazotroph, 64, 235 endophytic-plant associations, 127 help in stress tolerance, 162 and host plant surfaces, 3 inoculants, development of, 332 microbiomes, 274 microorganisms, 36, 43, 116, 237, 239, 240 N2 fixing bacteria, 235 and patents, 352 plant growth promoting activities, 165 proteobacteria, 227 providing disease resistance, and mode of action, 77 providing ISR against wilt diseases, 89 and rhizospheric microbes, 227 transmission of, 10 Endophytic bacteria, 2 bacterial strains Bacillus and Variovorax species disrupting QS and associated virulence factors, 240 Cannabis sativa L. intercepting in QS signals of biosensor strain Chromobacterium violaceum, 240 benefits on plant growth promotion, 41 colonization in plants, 37 effect of endophytic bacterial priming on plants, 69 mode of entry of, 58 molecular mechanisms interactions with plants, 40 adhering to plant root surface, 38 chemotaxis, 38 penetration and colonisation in internal parts of plants, 38 plant hormone-signaling pathways, 41 plant receptors, 40 plant disease protective property

plant-endophytic interactions, benefits of, 41 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, 44 in biocontrol of plant diseases, 45 cell wall degrading enzymes, 47 induced systemic resistance, 47 production of antimicrobial compounds, 46 mobilization of nutrients, 42 production of phytohormones, 44 plant growth-promoting mechanisms, 63 properties, 60 transition from rhizospheric bacteria to, 38 Endophytic entomopathogenic fungi (EEPF), 130 Endophytic fungi, 229, 263 abiotic stress, 262–265 tolerance, 143 drought stress tolerance, 144 heavy metal stress tolerance, 146 salinity stress tolerance, 143 temperatures stress tolerance, 144 biodiversity and distribution of, 275 biotechnological applications, 283 biotic stress, 261 tolerance, 138 defense against herbivores, 142 defense against insects, 138 defense against pathogens, 143 classification of, 253 colonization. See Fungal colonization entry into host plant, 253 gene expression analysis, 254 in plant-microbe interactions, 259 groups, 229 historical evolution, 252 importance in agriculture, 252 medicinal plants from, 274, 284 and patent application/publication, 353 plant growth through fungal secondary metabolite, 259 promote growth of plant, mechanisms used by, 133 direct growth promotion mechanism, 134 enhance the absorption of nutrient, 136 signaling compounds, 134 indirect growth promotion mechanisms, 137

Index

Endophytism, 126 Endosperm, 10 Endosymbiotic microorganisms, 41 Enterobacter cloacae, 60 Enterobacter sp. (FD17), 115 Enterobacter sp. SA187 alleviate salt stress tolerance in Arabidopsis, 238 yield and related parameter of alfalfa plant under salt stress, 238 Enterobactin, 79 Entomopathogenic fungi, 196 mode of action by, 331 Entrophosphora inferquens, 286 Environmental stresses, 108 Environmental toxicity, 35 Environment friendly fertilizers, 57 Environment friendly pesticides, 57 Ephedra fasciculata, 288 Epibrassinolides, 167 Epichloë festucae, 115, 263 Epichloë-induced stimulation, 115 Epicoccum nigrum, 313 Ergots alkaloids, 145 Erythromycin, 374 Ethylene, 41, 63, 67, 68, 85, 86, 88, 133 biosynthesis, 108 Eukaryotic organisms, 273 European biofertilizer market, 215 Exophiala pisciphila, 146 Exopolysaccharides (EPS), 3, 39, 199 Expressed sequence tag (EST) sequencing, 254 Extracellular enzymes, 59 F Facultative endophytes, 37 Fe deficiency, 236 Ferric iron (Fe3+), 79 Ferrous iron (Fe2+), 79 Fertilizers, 75 requirements, 234 Festuca arundinacea, 114, 138, 144 Festuca rubra, 115 Festuca sinensis, 115 Ficus religiosa, 274 Fimbriae, 3 Firmiculates, 58 Firmicutes, 11

395

Flagella, 3 deficient mutants, 3 Flagellin, 5, 41 Flagellin Sensing 2 (FLS2), 41 Flavonoids, 2, 81, 86, 87, 138, 199, 263 facilitates chemotactic response, 2 Fluorescence experiments, 95 GFP tagging gene mutants, 95 Foliar inoculation, 333 Food and Agriculture Organization, 75 Food crisis, 76 Food production, 57 Forsythia suspensa, 349 F. oxysporum f.sp. carthami, 90 F. oxysporum f.sp.ciceris, 90 F. oxysporum f. sp. lycopersici, 88 F. oxysporum f.sp zingiberi, 76 Frankia spp. strain CcI3, 8 F. udum, 90 Fungal based bio-stimulants, 211 Fungal biological control, 196 Fungal cell wall, lysis of, 59 Fungal colonization, 127 characterization confocal laser scanning microscopy (CLSM), 131 fourier transform infrared spectroscopy (FTIR), 131 wheat germ agglutinin alexa fluor (WGA-AF), 131 host recognition, 128 spore germination, 128 tissue colonization/multiplication, 130 tissue penetration, 129 Fungal endophytes. See Endophytic fungi Fungal formulations, 196, 213 Fungal-host interaction, 126 Fusarium equiseti, 126 Fusarium fujikuroi, 88 Fusarium graminearum, 88 Fusarium oxysporum, 134, 206, 276, 282, 286, 288, 308 Fusarium verticillioides, 180, 265 Fusarubin, 178 G Gaeumannomyces graminis var. tritici, 66 Gamma-proteobacteria Ganoderma boninense, 132

396 Index

GA3ox2 expression, 259 GA3-oxidase 2 (GA3 biosynthesis), 259 Gaseous phytohormones, 68 GDP dissociation inhibitor protein (GDI), 262 General Agreement on Tariff and Trade (GATT), 351 Genetic flexibility, 217 Genetic resources, 351 Genomic analysis, 81 Genomic constituent, 227 Genomic potential of microbial endophytes, 227 Genomics, 230 Gibberellic acid, 110, 259, 263 Gibberellin 3β-hydroxilase, 135 Gibberellins (GAs), 127, 163, 236, 324 Gibberellins-3-acid, 167 Glomus mosseae, 146 β-Glucanase Bgy6, 86 Glucanases, 84 β-1, 3-Glucanases, 47, 129 Gluconacetobacter diazotrophicus, 4, 7, 241 mutants of SOD and GR genes, 227 strain PAL5, 38, 43 Gluconate dehydrogenase (GAD), 42 Gluconic acid (GA), 42, 235 Glucose dehydrogenase (GCD), 42 Glutathione peroxidase, 38 Glutathione reductase, 227, 265 Glutathione S- transferase, 38, 265 Glycoprotein, 5 Green revolution, 57 Growth promotion, 59 aspects, due to symbiosis, 327 Guignardia mangiferae (BiosPTK4), 314 gumD gene, 39 Gymnema sylvestre, 314 H Halo-priming, 110 Harpophora oryzae, 256, 261 Heavy metal toxicity, 63 Hedypates betulinus, 206 Herbaspirillum seropedicae, 4 strain LMG2284, 174 strain SmR1 colonization, 39 Herbivore toxicity, 142 Hordeum vulgare, 112, 180 postanthesis drought stress

Host plant-endophyte relationship., 231 Host range and host-specificity entomopathogenic endophytes, 332 Human population, 195 Hydrogen cyanide (HCN), 65, 239 Hydrogen peroxide (H2O2), 260 Hydro-priming, 114 Hydroxamate, 65, 79 siderophores, 79 Hydroxycinnamoyl- CoA skimimate/ quinate, 263 Hydroxyl, 265 Hydroxyl cinnamoyltransferase (HCT), 263 N-(2-Hydroxy-4-methoxyphenyl)malonamic acid (HMPMA), 130 (S)-2-Hydroxy-N-((S)-1-((S)-8-hydroxy1-oxoisochroman-3-yl)-3methylbutyl)-2- ((S)-5-oxo-2, 5-dihydrofuran-2-yl)acetamide, 82 N-(2-Hydroxyphenyl) malonamic acid (HPMA), 130, 145 Hypersensitive response (HR), 40, 261 Hypoxylon anthochroum, 240 I IAA microbial production. See Indole-3acetamide (IAM) Illumina/Solexa sequencing, 257 Immunity, 8 Imperata cylindrica, 287 Indole-3-acetaldoxime pathway, 45 Indole-3-acetamide (IAM) microbial production, 59 pathway, 45 Indole acetic acid (IAA), 44, 145, 163, 166, 167, 169, 171, 174–176, 236 Indole-3-acetonitrile (IAN), 59 pathway, 45 Indole-3-pyruvate (IPyA), 59 pathway, 45 Induced systemic resistance (ISR), 37, 47, 65, 77, 239, 261, 330 mediated by endophytes, mechanism of, 86 Innate immune system, 40 Inoculation for delivery, types of, 333 Inorganic phosphate, 42 solubilisation, 42, 235 Intellectual property rights (IPR), 350

Index

implementation of IP protection of micro-organisms, 378 microbe’s intellectual properties related conflicts, 374 WIPO role in, 376 International Depositary Authority (IDA), 376 functioning under WIPO, 377 Inter transcribed spacer (ITS), 230 Intracellular microbes in root cells, 7 In vivo expression technology (IVET), 95 IPR. See Intellectual property rights (IPR) Irinotecan, 286 Iron (Fe), 7, 65, 79 chelators, 79 Fe deficiency, 236 Fe-siderophore receptors, 65 sequestration of, 59 Isopentenyladenine (iP), 135 ISR. See Induced systemic resistance (ISR) Iturin, 46 J Jasmonate, 68 regulated defenses, 88 Jasmonate/salicylic acid, 86 Jasmonic acid (JA), 41, 88, 136, 167 signalling pathways, 88 K Kanosamine, 66, 199 Karalicin, 66 2-Keto-4-methylthiobutyric acid (KMBA), 238 Kinetin, 110 Klebsiella oxytoca, 64 Klebsiella pneumoniae, 64 strain 342 (Kp342), 176 L Laccase, 132 Laser ablations, 308 Lasiodiplodia theobromae, 292 Lecanicillium lecanii, 130 Lectin receptor-like kinases (LecRLKs), 40 Legonoxamines A and B, 79 Legume-rhizobium endophytic association, 2 Lenticels, 37

397

Leptochloa fusca, 64 Leptosphaeria maculans, 88 Leucine-rich repeat receptor-like kinases (LRR-RLKs), 40 Leucine rich repeats (LRRs), 263 Lignin, 87, 132, 263 Lipase, 84, 261 Lipo-chitooligosachharides (LCOs), 199 Lipopolysaccharide, 3, 39, 66 role in colonization, 66 Lipoxygenase 2, 88 Lolium perenne (cv Atlas), 114, 174 Lupin, 2 Lyophilization, 94 Lysinibacillus xylanilyticus Chi-04, 84 Lys-motif receptors (LysM), 40 Lysozymes, 6 Lytic enzymes, 67, 84 M Macrophomina phaseolina, 87 Magnaporthe oryzae Guy11, 81, 261 Magnesium, 7 Maize, 10 Major intrinsic protein (MIP), 263 Malate, 2, 235 Malic acid, 235 MAMP-triggered immunity, 46 Mass spectrometry (MS), 231 Medicago truncatula, 8 Medicinal plants, endophytic fungi from, 274 Melatonin, 89 Meloidogyne incognita, 178 Mentha pulegium, 290 MEP (Methyl-Erythrose-4-Phosphate) pathway, 260 Meristem cells, 7 Metabolites, leakage of, 57 Metagenome analysis of rice roots, 235 Metagenomics, 95, 231 Metallothionein (MTs), 262 Metal nanoparticles effect of nanoparticles against plant pathogenic fungi, 313 extracellular synthesis of, 310 factors affecting mycosynthesis of, 315 intracellular synthesis of, 309 Metal toxicity, 143, 146 Metarhizium anisopliae, 181, 206

398 Index

Metatranscriptomics, 231 L-Methionine, 6 6-Methoxy-2-benzoxazolinone (MBOA), 130, 145 Methyl-accepting chemotaxis proteins (MCPs), 39 Methyl cellulose, 198 2-C-Methyl-D-erythritol-4-phosphate (MEP) pathway, 135 2-Methyl furan, 261 8-O-Methyl fusarubin, 178 Methylobacterium oryzae CBMB20, 88 MG RAST, 231 Micro and macro nutrient, 328 Microbes associated molecular pattern (MAMP)-triggered immunity, 240 Microbe’s intellectual properties related conflicts, 374 Microbial bioformulations, 199 based plant biostimulants, 209 current scenario/market trends, 215 microbial species employed in, 198 production and marketing constraints, 200 regulatory framework, 216 REACH regulation, 216 Microbial communities, 11 Microbial depositories, 378 Microbial diversity, 229 Microbial endophytes recent developments and applications, 338 auto fluorescent protein (AFP) technique, 338 genetic engineering, 338 genome studies, 338 Microbial interaction, 227 Microbial metabolites, 198 Microbial population, associated with soil ecosystem, 195 Microbiota, 2 Micrococcus yunnanensis SMJ12, 174 Micromonospora, 88 Microorganisms submission, for purposes of patent process, 378 requirement for identical international deposit system, 379 revelation and requisite for submission, 378 microRNAs (miRNAs), 231 Mimosops elengi, 288 Mineral nutrients, depletion of, 195

Mitogen-activated protein kinase (MAPK) cascad, 41 Moniliophthora perniciosa, 84 Monoamine oxidase, 260 Montmorrilonite, 68 Morinda citrifolia, 292 Moringa peregrine, 236 M. oryzae, 87 Mucor sp., 3 Musa acuminata, 64 Muscodor albus, 352 Mutants devoid of endoglucanase activity, 39 of RRLJ 04 and BS 03, 90 superoxide dismutase and glutathione reductase, 38 of T3SSs of Typhimurium, 8 Mutations genes for flagellin and LPS, 5 in Sphingomonas affecting Arabidopsis plant protection against, 239 MVA pathway, 135 Mycelium radices atrovirens, 229 Mycobacterium aurum, 292 Mycobacterium tuberculosis, 79, 275, 292 Mycobactin, 79 Mycorrhizal fungi, 209, 211, 212, 216 colonization, 229 N NADPH oxidases, 7 Nanoparticles, in plant disease management, 316 Nanotechnology, 316 Native vs. alien endophyte inoculants, 326 Neotyphodium coenophialum, 262 Next generation sequencing (NGS), 252 Nickel (Ni) contamination, 172 Nicotiana attenuata, 135, 240 Nitric oxide (NO), 260 Nitric oxide reductase, 38 Nitrogen (N), 7, 136 cycling, 235 deficiency, 42 fixation, 64, 199, 209 fixation by endophytes, 164 fixation (nifH) gene, 235 fixing bacteria, 42, 216, 231

Index

Nitrogenase, 136 Nod genes, 8 Nodulating bacteria, 5 Nothapodytes foetida, 276, 286 Nty gene, 135 Nutria-priming, 108 Nutrient acquisition, genes involved in, 234 extraction, 7, 108 limited conditions, 127 resources, 161 uptake, 227 use efficiency (NUE), 216 O Obligate endophytes, 37 Oligomers, 3 Omics techniques, 38, 227, 232 analysis to revealed plant-endophytes interaction, 232 Oncovin, 286 Oomycin A, 66, 199 Ophiosphaerella herpotricha, 137 Organic farming, 57 Organic phosphorus, 235 Ornithine decarboxylase, 263 Oryza sativa, 64, 168, 180 Osmo-priming, 108 Osmoprotectants, 108, 263 Osmotic shock, 199 Outer membrane porin F (OprF) proteins, 5 Oxalate, 2, 235 Oxalotrophy, 2 Oxford nanopore sequencing (ONT), 257 Oxidative stress, 116, 165, 170 P Paclitaxel, 291 PAD4 (phytoalexin deficient 4), 88 Paecilomyces farinosus, 130 Paecilomyces formosus, 135 Paecilomyces lilacinus, 143 Paenibacillin A, 82 Paenibacillus sp. Xy-2, 82 Palmarumycin CP17, 290 PAMP-triggered immunity (PTI), 40 Pantoea ananatis VERA8, 82 Papulaspora pallidula, 313 Parapediasia teterrella, 138

399

Passive endophytes, 37 Patent Budapest treaty and, 376 European patent convention (EPC) wide Article 53-b, 382 micro-organism identification, characterization and submission for, 383 Pathogen associated molecular patterns (PAMPs), 261 Pathogen-derived avirulence (Avr) genes, 40 Pattern recognition receptor (PRRs), 40, 261 P. brassicacearum subsp. neoaurantiaca, 81 P. chlororaphis (PA23), 66 Peanibacillus elgii NIIST B578, 92 Peat, 68, 198 Pectinase, 84, 132, 133 Pectins, 39 Pencillium thomi, 282 Penicillium bilaii, 199 Penicillium lilacinum, 289 Penicillium oxalicum, 288 strain 5aWF (5aWF), 117 Pennisetum glaucum, 64 Peptidase, 261 Periconia macrospinosa, 234 Peroxidase (POD), 47, 86, 114, 261 Pestalotiopsis clavispora, 290 Pestalotiopsis fici, 287 Pestalotiopsis microspora, 290 strain VJ1/VS1, 314 Pestalotiopsis pauciseta, 292 Pesticides, 57, 92 Petrobactin, 79 Phaseolus vulgaris L, 178 Phellodendron amurense, 313 Phenazine-1-carboxamide, 66 Phenazine-1-carboxyclic acid, 66 Phenazines, 66, 178, 199 Phenolates, 79 Phenolics, 86, 87 l-Phenylalanine ammonia lyase (PAL), 86, 260 Phialocephala fortinii, 239, 289 Phlogacanthus thyrsiflorus, 314 Phoma glomerata LWL2, 166 Phoma sorghina, 126 Phoma terrestris, 288 Phomopsis liquidambari, 136 strain SA1, 314

400 Index

Phomopsis longicola, 288 Phomopsis oblonga, 137, 138 Phosphatases, 235 Phosphate solubilisation, 64, 108, 235 by endophytes, 163 Phosphate solubilizing microorganism, 42 Phosphorous, 42 immobilized, release and cycling of, 64 Phosphorus (P), 136 Photosynthesis-related signal, 236 Phyllosphere, 239 Physocnemum brevilineum, 137, 138 Phytases, 235 Phytoalexins, 261 Phytohormone, 3, 35, 37, 59, 108, 127, 133, 134, 199, 235, 236, 238, 263 composition, 45 production, 44, 327 by endophytes, 163 Phytopathogens, 5, 37, 40, 41, 59, 65, 67, 68, 77, 89, 198, 211 Phytophthora capsici, 178 Phytoremediation, 329 Phytotoxicity, 181 Pilli, 7, 39 Piriformospora indica, 112, 135, 168, 180, 235, 265 genes responsible for nitrogen uptake and transfer, 235 Plant associated microorganisms, 35, 47 Plant cell wall degrading enzymes (PCWDE), 234 Plant derived resistance (R) genes, 40 Plant disease protective property, 46 Plant-endophyte interactions, 37, 40, 41, 231 methods to access, 230 Plant fitness, 234 Plant growth, 7, 41, 162 promoting activity (PGPR), 230 promoting bacteria (PGPB), 63, 66 promoting rhizobacteria (PGPR), 57, 75, 196, 210 promotion by endophytes, 163 promotion, direct mechanisms of, 327 promotion, endophytic microorganisms on, 43 through fungal secondary metabolite, 259 Plant host interaction, 252 Plant immune system, 239

Plant internalization, 7 Plant meristematic tissues, 45 Plant-microbe interaction, 8, 35, 57, 162 Plant-microbial association, 234 Plant microbiota, 35 Plant nutrient, 7 Plant-pathogen-endophyte interactions, 127 Plant pathogenic toxins, 89 Plant probiotics, 68 Plant productivity, 108 Plant tissue, 7 PLANT TONIC, 92, 93 Plasmodium falciparum, 290 Plasmopara viticola, 137 P. mallei (RBG4, ET17), 90 Podophyllum hexandrum, 289 Podophyllum peltatum, 289 Polyethylene glycol (PEG), 110 Polyhydroxybutyrate (PHB), 39 Polyketide, 82 Polymers, 198 Polyphenol oxidase (PPO), 86 Possible toxicity assessment, 338 Potassium (K), 137 pqq operon, 42 Productivity, 35, 44, 107, 108, 112, 116, 162, 196, 209, 211, 213, 339 Products, efficacy of, 337 Proline, 169 Proteases, 84 Proteinase inhibitor, 261 Protein PR-10, 262 Proteobacteria, 11, 58, 229 Proteomics, 230, 231 Protoplasts, 7 PR proteins, 47, 86, 261, 265 Pseudoalteromonas luxO and robp gene act as main regulatory genes, 240 QS regulation in, 240 Pseudomonads, 7 Pseudomonas aeruginosa, 41, 79, 206, 210 strain PAO1 pctA gene, 2 strain PNA1, 178 strain RRLJ 04, 90 strain UICC B-40, 82 Pseudomonas fluorescens, 87, 206 strain PCL1205, 66 strain Pf0-1, 2

Index

strain PICF7, 88 phenotypes, 95 strains Endo2 and Endo35, 87 strain WCS374, 66 strain WCS417r, 66 Pseudomonas frederiksbergensis OS261, 237 Pseudomonas indica, 166 Pseudomonas pseudoalcaligenes, 86, 169 Pseudomonas putdia, 5 strain VM1441 (pNAH7), 174 Pseudomonas strains RRLJ 134 and RRLJ 04, 87 Pseudomonas syringae DC3000, 239 Pseudomonas syringae pv. passiflorae, 240 Pseudomonas trivialis X33d, 207 Pseudomonas vancouverensis OB155, 237 Pseudomonas viridiflava, 88 Pseudomonic acid, 66 Pseudomonine, 79 Psychrotolerant endophytes, 237 pTOM toluene-degradation plasmid, 181 Pumpkin, 10 Pyochelin, 79 Pyoluteorin, 66, 199 Pyoverdine, 79 Pyrenochaeta terrestris, 288 Pyrophyllite, 68 Pyrrolnitrin, 66 Pyrroloquinoline quinone (PQQ), 42 Pythium myriotylum, 178 Pythium splendens, 178 Q Qiime, 231 Quality control of endophytes bioproduct, 337 Quantitative real time PCR (qRT-PCR), 258 Quantitative Trait Loci (QTL) mapping, 95 Quenching quorum signals, 45 Quercus variabilis, 289 Quercus virginiana, 117 Quinoa, 10 Quinolobactin, 79 Quorum quenching, 68 Quorum sensing (QS), 239 R Radopholus similis, 178 Ralstonia solanacearum, 6 Reactive nitrogen species (RNS), 38

401

Reactive oxygen species (ROS), 38, 110, 165, 261, 315 Receptor-like kinases (RLK), 40 Recombination in vivo expression technology, 95 Region compartmentalization, 35 Registration of product, 337 Representational difference analysis (RDA), 254, 256 Resistance gene (NPR1), 117 Response surface methodology (RSM), 77 Restriction fragment length polymorphism (RFLP), 11 Rhamnolipid-biosurfactants, 178 Rhamnolipids, 66 Rhizobia-legume association, 199 Rhizobium leguminosarum, 4 Rhizoctonia solani, 76, 86, 206 Rhizodermea veluwensis, 239 RhizoMyco, 199 RhizoMyx, 199 Rhizophagy cycle, 7, 108 Rhizoplane, 5, 39 Rhizoplex, 199 Rhizosphere, 37, 115, 227, 229 role in endophytic associations, 2 Rhodiola crenulata, 260 Rhopalosihum padi, 138 Rifamycin, 374 RLK receptor, 41 flagellin sensing 2 (FLS2), 41 RNA polymerase, 263 RNS. See Reactive nitrogen species (RNS) Roegneria kamoji, 116 Root dipping, 334 Root exudates, 2, 230 ROS. See Reactive oxygen species (ROS) Rust, 76 S SA. See Salicylic acid (SA) Saccharum officinarum, 60, 180 Salicylic acid (SA), 41, 79, 110, 133, 166, 235, 260 Salicylic and jasmonic acid signalling pathways, 88 Salidroside, 260 Salinicola peritrichatus SMJ30, 174 Salvia miltiorrhiza, 81

402 Index

SAR. See Systemic acquired resistance (SAR) Sarocladium kiliense strain 10aWF (10aWF), 117 Sawdust, 68 Sclerotinia sclerotiorum, 88, 180 Sclerotium rolfsii, 87 Sebacina vermifera, 135 Secondary metabolites, 66, 164, 170, 171, 180, 350, 352 Secretion systems (Type I-VIII), 40 Seed bio-priming, 108 Seed dipping, 334 Seed dormancy, 108 Seed germination, 108 Seedling development, 108 Seed priming bio-priming, 111 chemo-priming, 111 factors affecting processes, 112 hormonal priming, 110 hydro-priming, 109 osmo-priming, 110 role of endophytes in, 112 solid matrix priming (SMP), 110 thermo-priming, 111 Serendipita indica, 117 Serial analysis of gene expression (SAGE), 254 Serratia entomophila, 207 Serratia sp. EDA2, 2, 39 Setosphaeria rostrata, 137 Sexual spores, 127 Shelf life, 207 Shizaphis graminum, 138 Siderophores, 44, 79, 108, 164, 174, 176, 198, 235, 236 production, 65, 66 by endophytes, 164 Silver nanoparticles (AgNPs), 307, 315, 316 from endophytic fungi and efficacy in biocontrol, 311 from fungal endophytes, antimicrobial activity of, 314 mechanism of antimicrobial action by, 315 mycosynthesis, 309

Simple sequence repeat (SSR) markers, 90 Sinorhizobium meliloti CCNWSX0020 enhanced antioxidative defense response in Medicago lupulina, 239 Smut, 76 Sodium borohydride, 309 Soil ecosystem, 196, 209 environment, 206 fertility, 57, 161 health, 195 microbes, 7 microbiostasis, 206 salinity, 161, 195 spray, 334 Solanum lycopersicum, 170 Solanum melongena, 84 Solanum torvum, 240 Solanum trilobatum, 240 Solid matrix priming, 110 S. parvulus Av-R5, 91 Sphingomonas sp. LK11, 236 Spiropreussione, 290 Spiropreussomerin, 290 Spore germination, 127, 128 18S rRNA expression level, 260 16S rRNA gene, genome sequencing, 36 Stachybotrys chartarum, 274 Staphyloferrin, 79 Starch, 198 Stem and root rot, 76 Stem inoculation, 333 Stemphylium globuliferum, 290 Stenotrophomonas, 11 Steroids, 138 Streptomyces sp. GMKU 336 significantly promoted plant growth, 238 Streptomyces sp. GMKU 3100, 44 Streptomyces sp. NIISTA32, 90 Streptomyces sporocinereus OsiSh-2, 81 Streptomyces sp. SKH1-2, 82 Stress tolerant endophytes, commercial applications of, 180 Strigolactones, 3, 133 Suoeroxide, 265 Supercooling, 171 Superoxide, 7

Index

Superoxide dismutase (SOD), 114, 143, 227, 261 Suppression subtractive hybridization (SSH), 262 Surface adhesion, 230 Surface of soils, 108 Symbiosis, 10, 327 Symbiotic interaction, 108 Synthetic compounds, usage, 75, 94 Systemic acquired resistance (SAR), 41, 47, 65, 85, 239, 261 T Tabebuia pentaphylla, 292 Tabebula argentea, 274 Talaromyces flavus, 206 Talaromyces radicus, 286 Talc, 198 TATA-box-binding proteins, 263 Taxus baccata, 289 Terpenoids (3, 11, 12-trihydroxy cadalene), 138, 261 pathway, 143 Tetrapyrrole pathway, 263 Tetrohydofuran, 261 Theobroma cacao, 261 Thermo-priming, 111 Tilletia indica, 316 Tobacco, 10 Topoisomerases-I and II, 291 Toxin neutralization, 199 Toxoflavin, 82 Trade related aspects of intellectual property rights (TRIPS), 351 Trametes hirsuta, 289 Transcription factors, 263 Transcriptome sequencing (or RNA-Seq), 254 Transcriptomics ananlysis of B. phytofirmans PsJN colonizing potato in response to drought stress, 237 Trichoderma asperellum, 206 Trichoderma harzianum, 108, 206 application in NaCl affected Indian mustard, 237 colonization in rice alleviated drought tolerance by modulating, 237

403

Trichoderm viride, 117 Triticum aestivum, 176 Tryptamine pathway, 45 L-Tryptophan, 199 T3SSs and T6SSs genes, 8 Tubulin inhibitors, 291 TwinN, 334 Tyrosinase, 261 Tyrosine decarboxylase, 260 Tyrosol, 260 U UDP-glucosyltransferase, 260 V Vaccinium myrtillus, 292 Vancomycin, 374 Vermiculate, 68 Vermiculite, 198 Verrucosispora sp. FIM060022, 79 Verticillium dahliae, 206 Verticillium lecanii, 206 Verticillium wilt-resistant cotton, 86 Vibriobactin, 79 Vibrio cholerae, 79 Vibrio sagamiensis SMJ18, 174 Vinblastine, 276, 286 Vinca alkaloids, 286 Vincristine, 276, 286 Viscosinamide, 66 Volatile organic compounds (VOC), 46, 236, 240 W Wall-associated kinases (WAK), 40, 261 Wheat-endophyte interactions, 39 Wheat head blight, 88 Whole genome sequencing, 230 Wilt, 89 Withania somnifera (Indian Ginseng), 88, 117 World Intellectual Property Organization (WIPO), 351 role in governing intellectual properties of microbial organisms, 376 World trade organization (WTO), 351 Wounds, 57

404 Index

X

Y

Xantham gum, 198 Xanthomomas albilineans, 180 Xanthomonas axonopodis pv. vesicatoria, 88, 316 Xanthomonas campestris, 41, 88 X.campestris pv. vesicatoria, 90 Xylanase, 132 Xylulose, 39

Yersiniabactin, 79 Yield of crops, 107, 108 Z Zea mays, 64, 146 Zeatin (Z), 135, 145 Zeolite, 68 Zinc (Zn), 7, 137, 146, 169 Zwittermycin, 66, 199