Bacterial Endophytes for Sustainable Agriculture and Environmental Management [1st ed. 2022] 9811644969, 9789811644962

Table of contents :
Contents
About the Editors
1: Endophytic Bacteria: Application Against Biotic and Abiotic Stresses and Plant Health Improvements for Sustainable Agricult...
1.1 Introduction
1.2 Importance of Endophyte
1.3 Isolation of Endophytes from Plants
1.4 Application of Endophytes Microorganism in Biotic and Abiotic Stresses
1.4.1 Anti-Fungal Activity of Endophytes
1.4.2 Antibacterial Activity of Endophytes
1.4.3 Plant Growth Promoters (PGPR)
1.4.4 Applications of Endophytes in Soil pH Stress
1.4.5 Applications of Endophytes in Drought Tolerance
1.4.6 Applications of Endophytes in Soil Salinity Tolerance
References
2: Endophytic Bacteria: Mitigating Abiotic Stress from Inside
2.1 Introduction
2.2 Diversity of Endophytic Bacteria and Associated Abiotic Stresses
2.3 Endophytes and the Molecular Defence Response
2.4 The Role of Multi-Omics: A Better Understanding of Molecular Interactions
2.5 Genomics and Metagenomics
2.6 Transcriptomics and Meta-transcriptomics
2.7 Proteomics and Meta-proteomics
2.8 Interactomics and Metabolomics
2.9 Integration of Multi-omics and Discovery of New Data
2.10 Conclusion
References
3: Diversity and Bioactive Potential of Endophytic Bacteria from High-Value Medicinal Plants
3.1 Introduction
3.2 Diversity of Endophytic bacteria Associated to Medicinal Plants
3.2.1 Culture-Dependent Diversity
3.2.2 Diversity Based on Culture-Independent Methods
3.3 Applications of Endophytic bacteria
3.3.1 Pharmaceutical and Medical Applications
3.3.1.1 Alkaloids
3.3.1.2 Terpenoids
3.3.1.3 Flavonoids
3.3.1.4 Peptides and Their Derivatives
3.3.1.5 Quinones
3.3.1.6 Phenols
3.3.1.7 Antibiotics
3.3.2 Plant Growth Promotion Abilities
3.4 -Omics in Plant-Microbe Interactions Study
3.5 Conclusion and Prospects
References
4: Plant Growth Promoting Rhizobacteria (PGPR)-Assisted Phytoremediation of Contaminated Soils
4.1 Introduction
4.2 Rhizomicrobiome
4.2.1 Plant Growth Promoting Rhizobacteria
4.3 Role of PGPRs in Phytoremediation
4.3.1 Role of Rhizobacteria in Phytoremediation of Contaminated Soil
4.3.1.1 Metal-Contaminated Soil
4.3.1.2 Petroleum-Contaminated Soil
4.4 From the Lab to the Field and Commercialization
4.5 Genetics and Genomics of Heavy-Metal Resistance in PGPRs
4.5.1 Genetically Engineered PGPRs
4.6 Conclusion
References
5: Endophytic Bacteria: Role in Phosphorous Solubilization
5.1 Introduction
5.2 Colonization of Bacterial Endophytes
5.3 Role of Bacterial Endophytes in Phosphorous Solubilization
5.4 Mechanism of Soil P-Solubilization
5.4.1 Mechanism of Organic P-Solubilization
5.4.1.1 Phosphatases
5.4.1.2 Phytases
5.4.1.3 C-P Lyases and Phosphonatases
5.4.2 Inorganic P Solubilization
5.4.2.1 Role of Proton Liberation P- Solubilization
5.4.2.2 Role of Siderophores in Mineral P- Solubilization
5.4.2.3 Role of Exopolysaccharides in Phosphate Solubilization
5.4.3 Plant Growth Promoting Attributes of P-Solubilizing Endophytic bacteria
5.4.4 Genetics of Phosphate Solubilization
5.4.4.1 Genetics of Inorganic Phosphate Solubilization
5.4.4.2 Genetics of Organic P Mineralization
5.4.5 Bacterial Endophytes as Crop Bio-Inoculant
5.4.6 Conclusions
References
6: Endophytes of Medicinal Plants: Diversity and Bioactivity
6.1 Introduction
6.2 Entry and Colonization of Endophytes
6.3 Movement and Localization of Endophytes
6.4 Medicinal Plants
6.5 Endophytic Diversity in Medicinal Plants
6.6 Endophytic Microbes in Metabolites Production
6.7 Conclusion and Future Prospects
References
7: Biotechnological Applications of Bacterial Endophytes
7.1 Introduction
7.2 History of Endophytes
7.3 Endophytes and Their Mode of Association with Host
7.3.1 Symbiotic Association of Fungi with Host
7.3.2 Symbiotic Association of Endophytic Bacteria with Host
7.3.3 Cyanobacterial Association with Host
7.3.4 Interaction of Endophytes to Increase Resistance to Biotic and Abiotic Stress
7.4 Host Associated Biotic Factors and Endophyte Ecology
7.4.1 Endophytes Attracted by the Host Plants
7.4.2 Entry of Endophytic Microbes Through Root Into the Plants
7.4.3 Colonization of Endophytes by Roots of the Plant
7.4.4 Entry and Colonization of Endophytes by Aerial Parts of the Plants
7.5 Physiology and System Diversity in Association
7.5.1 Crucial Changes in Plant Physiology Due to Bacterial Endophytes
7.5.2 Diversity of Endophytic Bacteria and Their Association with Host
7.5.3 Factors Affecting the Diversity in Host Plants
7.5.3.1 Endophytic Species and Strains
7.5.3.2 Host Plant
7.5.3.3 Environmental Conditions
7.6 Molecular Events During the Endophytes Association
7.6.1 Molecular CounterAction Upon Endophytic Association in Plants
7.6.1.1 Defensive Response of Host Plant
7.6.1.2 Activation of Plants Immune System
7.6.1.3 Biotic and Abiotic Stress Remediator
7.6.1.4 Protection from Reactive Oxygen Species
7.6.1.5 Modulation of Protein Secretion Systems
7.6.1.6 Phytoremediation Mediated by Endophytic Microbes in Association with Host Plant
7.7 Application of Endophytes
7.7.1 Endophytes in Agriculture
7.7.2 Endophytes in Industry
7.7.3 Endophytes in Nanobiotechnology
7.7.4 Endophytes in Pharmaceutical
7.7.4.1 Antimicrobial Compounds
7.7.4.2 Anti-Cancerous Compounds
7.7.4.3 Antibiotics
7.8 Conclusion
References
8: Genetic Basis of Fungal Endophytic Bioactive Compounds Synthesis, Modulation, and Their Biotechnological Application
8.1 Introduction
8.2 The Genetic Basis of Secondary Metabolites Production
8.2.1 Important Biosynthetic Gene Clusters in Endophytes
8.2.1.1 NRPS Gene Clusters
8.2.1.2 Polyketide Synthases Gene Clusters for Maklamicin Biosynthesis
8.2.1.3 Indole-Diterpenes Gene Cluster
8.2.1.4 Loline Biosynthetic Gene Cluster
8.3 Potential of Endophytic Bioactive Compounds
8.3.1 Antioxidant Activity
8.3.2 Antimicrobial Activity
8.3.3 Antiviral Activity
8.3.4 Anticancer Activity
8.3.5 Antidiabetic Activity
8.3.6 Insecticidal Activity
8.4 Strategy for Production of Endophytic Secondary Metabolites
8.5 Major Obstacles to the Production of Bioactive Compounds from Endophytes
8.6 Conclusion and Future Prospects
References
9: Endophytic Bacteria for Plant Growth Promotion
9.1 Introduction
9.2 Ecology and Diversity
9.3 Mechanism of Plant Growth Promotion
9.3.1 Direct Mechanism
9.3.1.1 Nutrient Acquisition
9.3.1.2 Phytohormone Production
9.3.1.3 Ethylene Level Maintenance and Role of 1-Aminocyclopropane-1-Carboxylase (ACC) Deaminase
9.3.2 Indirect Mechanism
9.4 Gene Responsible for Plant Growth
9.5 Agricultural Application of Bacterial Endophytes
9.5.1 Growth Promotion
9.5.2 Phytoremediation
9.6 Conclusion
References
10: Bacterial Endophytes and Bio-nanotechnology
10.1 Introduction
10.2 Applications of Bacterial Endophytes
10.3 Biosynthesis of Nanoparticles Using Endophytic Bacteria
10.3.1 Isolation of Endophytic Bacteria from Plant Leaves
10.3.2 Biosynthesis of Nanoparticles Using Endophytic Bacterial Culture
10.4 Advantages Over Conventional Nanoparticles
10.5 Discussion
10.6 Future Prospects
References
11: Role of Endophyte Metabolites in Plant Protection and Other Metabolic Activities
11.1 Introduction
11.2 Endophytic Bacteria
11.3 Plant Colonization by Endophytic Bacteria
11.4 Endophytic Bacteria Diversity
11.5 Bioactive Compounds synthesized by Bacterial Endophytes
11.6 Modulation of Plant´s Defence by Endophytes
11.7 Endophytic Metabolites in Plant Protection
11.7.1 Direct Mechanism
11.7.1.1 Antibiosis
11.7.1.2 Synthesis of Lytic Enzymes
11.7.1.3 Essential Nutrients: Availability and Competition
11.7.2 Indirect Mechanism
11.7.2.1 Phytohormone Modulation
11.7.2.2 Stress Tolerance
11.7.2.3 Bioremediation
11.8 Conclusion
References
12: Role of Bacterial Endophytes in the Promotion of Plant Growth
12.1 Introduction
12.2 Endophytes Biodiversity
12.3 Isolation, Identification and Colonization of Endophytes
12.4 Strategies Employed by Endophytes for Plant Growth Promotion
12.5 Applications of Bacterial Endophytes
12.5.1 Bacterial Endophytes in Plant Growth Promotion
12.5.2 Endophytes-Mediated Biodegradation of Soil Contaminants
12.5.3 The Bacterial Endophytes Towards Inhibition of Plant Pathogens
12.5.4 Endophytes Maintain the Free Radicals in the Plant Tissue
12.6 Concluding Remarks and Future Perspectives
References
13: Bacterial Endophytes and Abiotic Stress Mitigation
13.1 Introduction
13.2 International and Indian Scenario of Various Abiotic Stress
13.3 Plant Responses to Abiotic Stress
13.3.1 Drought and Heat Stress
13.3.2 Salt Stress
13.3.3 Cold Stress
13.3.4 Heavy Metal Stress
13.3.5 Other Abiotic Stresses
13.4 Diversity and Colonization of Bacterial Endophytes
13.5 Endophytic Bacteria as Mitigants for Various Abiotic Stress
13.5.1 Production of Phytohormones
13.5.2 Production of Exopolysaccharides (EPS)
13.5.3 1-aminocyclopropane-1-carboxylate (ACC) Deaminase Production
13.5.4 ROS Production
13.6 Role of Endophytic Bacteria in Mitigation of Heavy Metal Stress
13.7 Gene Expression Under Abiotic Stress in Plants Inhabited with Endophytes
13.8 Conclusion
References

Citation preview

Amit Kishore Singh Vijay Tripathi Awadhesh Kumar Shukla Pradeep Kumar   Editors

Bacterial Endophytes for Sustainable Agriculture and Environmental Management

Bacterial Endophytes for Sustainable Agriculture and Environmental Management

Amit Kishore Singh • Vijay Tripathi • Awadhesh Kumar Shukla • Pradeep Kumar Editors

Bacterial Endophytes for Sustainable Agriculture and Environmental Management

Editors Amit Kishore Singh Botany Department Bhagalpur National College Bhagalpur, Bihar, India

Awadhesh Kumar Shukla Botany Department K.S. Saket P.G. College Ayodhya, Uttar Pradesh, India

Vijay Tripathi Department of Molecular and Cellular Engineering Sam Higginbottom University of Agriculture, Technology and Sciences Prayagraaj, Uttar Pradesh, India Pradeep Kumar Applied Microbiology Laboratory, Department of Forestry North Eastern Regional Institute of Science and Technology Nirjuli, Arunachal Pradesh, India

ISBN 978-981-16-4496-2 ISBN 978-981-16-4497-9 https://doi.org/10.1007/978-981-16-4497-9

(eBook)

# The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Contents

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Endophytic Bacteria: Application Against Biotic and Abiotic Stresses and Plant Health Improvements for Sustainable Agriculture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kute Lachu, Madhu Kamle, Rituraj Borah, Beauty Tiwari, and Pradeep Kumar

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Endophytic Bacteria: Mitigating Abiotic Stress from Inside . . . . . . Garima Malik and Rahul Arora

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Diversity and Bioactive Potential of Endophytic Bacteria from High-Value Medicinal Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Namita Ashish Singh and Rahul Jain

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Plant Growth Promoting Rhizobacteria (PGPR)-Assisted Phytoremediation of Contaminated Soils . . . . . . . . . . . . . . . . . . . . . Garima Malik, Samira Chugh, Sunila Hooda, and Ritu Chaturvedi

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Endophytic Bacteria: Role in Phosphorous Solubilization . . . . . . . . Neha

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Endophytes of Medicinal Plants: Diversity and Bioactivity . . . . . . . 117 Sandeep Kumar Singh, Vipin Kumar Singh, Dharmendra Kumar, Dinesh Prasad Gond, and Ajay Kumar

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Biotechnological Applications of Bacterial Endophytes . . . . . . . . . . 129 Mohit Mishra, Sudheer Pamidimarri, V. Balasubramanian, Sneha Kumari, Shalini Pandey, Bhairav Vaibhav, and Sushma Chauhan

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Genetic Basis of Fungal Endophytic Bioactive Compounds Synthesis, Modulation, and Their Biotechnological Application . . . 157 Anuj Ranjan, Abhishek Chauhan, Vishnu D. Rajput, Rupesh Kumar Basniwal, Tatiana Minkina, Svetlana Sushkova, and Tanu Jindal

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Contents

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Endophytic Bacteria for Plant Growth Promotion . . . . . . . . . . . . . 187 Nitin Bohra, Rupesh Kumar Singh, Raksha Jain, Lav Sharma, Eetela Sathyanarayana, Francisco Roberto Quiroz-Figueroa, and Vishnu D. Rajput

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Bacterial Endophytes and Bio-nanotechnology . . . . . . . . . . . . . . . . 201 Shruti Rathore, Mansi Ujjainwal, Ajeet Kaushik, and Jyoti Bala

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Role of Endophyte Metabolites in Plant Protection and Other Metabolic Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Neha Singh, Santosh Kumar Mishra, Priya Ranjan Kumar, Narendra Kumar, and Dhirendra Kumar

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Role of Bacterial Endophytes in the Promotion of Plant Growth . . . 235 Isha Kohli, Swati Mohapatra, Prashant Kumar, Arti Goel, Ajit Varma, and Naveen Chandra Joshi

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Bacterial Endophytes and Abiotic Stress Mitigation . . . . . . . . . . . . 255 Sonali Jaiswal, Anupama Ojha, and Sarad Kumar Mishra

About the Editors

Amit Kishore Singh is currently working as Assistant Professor (Botany) in Bhagalpur National College, Bhagalpur. He obtained his doctoral thesis award while working on Bt Brinjal-associated microflora from Banaras Hindu University, Varanasi, India. Then after, he gained his postdoctoral research from the Department of Postharvest Science at Agricultural Research Organization, Israel. In the span of more than 8 years, he has been actively engaged in various aspects of plant–microbe interactions and published research and review articles in journals of international repute. Currently, he has edited five books based on PGPR applications in sustainable research. His basic research interest includes rhizosphere-associated microbial dynamics, host–pathogen interactions and microbial applications for sustainable environment. During his research career, he has published more than 12 research articles, 10 book chapters and recognized his research work at international level. He has been felicitated by several fellowships and awards like CSIR-NET JRF, CSIR SRF, Best poster presentation and ARO postdoctoral fellowship. Dr. Singh is a lifetime member of the Association of Microbiologists of India (AMI) and Biotech Research Society, India (BRSI). Vijay Tripathi is currently working as Assistant Professor at the Department of Molecular and Cellular Engineering, Sam Higginbottom University of Agriculture, Technology and Sciences, Prayagraj, India. He was previously awarded an ARO Postdoctoral Fellowship at the Department of Soil, Water, and Environmental Science, Agricultural Research Organization, Bet Dagan, Israel. He has also received two prestigious postdoctoral fellowships (Indo-Israel Government Fellowship and PBC Outstanding Postdoctoral Fellowship) to work as a postdoctoral fellow at the Institute of Evolution, University of Haifa, Israel. Dr Tripathi began his research career as a doctoral student at the Centre of Bioinformatics, University of Allahabad, India. During his doctoral thesis work, he was also awarded a MUIR fellowship and visited the University of Cagliari, Italy. He has published more than 40 research and review papers in reputed national and international journals and nine book chapters and edited two books. He has received the Early Career Research grant from SERB, Government of India, and Co-PI of GlobalStar-DBT, India sponsored project.

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About the Editors

Awadhesh Kumar Shukla is currently working as Assistant Professor (Botany) at K.S. Saket P.G. College, Ayodhya, Uttar Pradesh, India. He obtained his doctoral degree in Botany while working on biodegradation of trichloroethylene using bacterial community from Banaras Hindu University, Varanasi, India. Then after, he has been awarded CSIR-Research Associate, New Delhi, and worked as postdoctoral research from Department of Botany, Banaras Hindu University, Varanasi, India. After completion further, he has also been awarded Dr. D.S. Kothari Postdoctoral fellowship (higher fellowship category) by UGC. In the span of more than 10 years, he has been actively engaged in various aspects of plant–microbe interactions and exploitation of microbial community for the removal of xenobiotics and published more than dozens of research and review articles in journals of international repute to his credit. His basic research interest includes rhizosphereassociated microbial community and their potential utilization in environmental clean-up and sustainable agriculture. During his research career, he has published more than 12 research articles and more than six book chapters, and his research work gained recognition at international level. He has been felicitated with several academic fellowships and awards like Young Scientist Award, CSIR-RA, UGC-Dr. D.S. Kothari postdoctoral fellowship, UGC-NET-JRF, CSIR-NET JRF and CSIR SRF. Dr. Shukla is a lifetime member of the Association of Microbiologists of India (AMI) and Biotech Research Society, India (BRSI). Pradeep Kumar is currently working as Assistant Professor in the Department of Forestry, NERIST, Nirjuli, India. He served as Assistant Professor at the Department of Biotechnology, Yeungnam University, South Korea. He was awarded PBC Outstanding Post-Doctoral Fellowship and worked for more than 3 years at the Ben Gurion University of the Negev, Israel. Dr. Kumar research interests are biocontrol and PGPR, nanotechnology, phytochemistry, bioremediation, food microbiology, phytopathology and gene expression studies. He has published more than 70 research and review articles in peer-reviewed journals, 16 book chapters and 6 edited books (Springer-Nature; Taylor & Francis Group) with a total of 2485 citations and h-index 24 and i10-index 35. He has been awarded Early Career Research Award from the SERB, Government of India, and is currently handling four projects as PI and Co-PI funded by DBT, DST-SERB and GBPNIHESD. He was awarded with Narasimhan Award from the Indian Phyto pathological Society, Emerging Scientist Award, Research Excellence award, Young Scientist Award by various societies. Dr. Kumar served as guest editor, associate and academic editor, and editorial board member of several peer-reviewed journals.

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Endophytic Bacteria: Application Against Biotic and Abiotic Stresses and Plant Health Improvements for Sustainable Agriculture Kute Lachu, Madhu Kamle, Rituraj Borah, Beauty Tiwari, and Pradeep Kumar

Abstract

Endophytes are an endosymbiotic group of microorganisms that colonize in plants and microbes that can be readily isolated from any microbial or plant growth medium. They act as reservoirs of novel bioactive secondary metabolites, such as alkaloids, phenolic acids, quinones, steroids, saponins, tannins, and terpenoids, that serve as a potential candidate for antimicrobial, anti-insect, anticancer, and many more properties. Endophytes are known to produce metabolites of utility value for various applications. Endophytes can also be beneficial to their host by producing a range of natural products that could be harnessed for potential use in medicine, agriculture, or industry. In addition, it has been shown that they have the potential to remove soil contaminants by enhancing phytoremediation and may play a role in soil fertility through phosphate solubilization and nitrogen fixation. In the present chapter we briefly summarize the importance of endophytic bacteria and their role in abiotic and biotic stresses. Keywords

Phytopathogenic activity · Growth promotion · Antioxidant · Plant health

Author Contribution: P.K. conceived and designed the manuscript; P.K., K.L. and M.K. wrote the manuscript; RB and B. T. helped in editing; P.K. critically reviewed the manuscript and did the required editing. K. Lachu · M. Kamle · R. Borah · B. Tiwari · P. Kumar (*) Applied Microbiology Laboratory, Department of Forestry, North Eastern Regional Institute of Science and Technology, Nirjuli, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. K. Singh et al. (eds.), Bacterial Endophytes for Sustainable Agriculture and Environmental Management, https://doi.org/10.1007/978-981-16-4497-9_1

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1.1

K. Lachu et al.

Introduction

Endophytes are found in plants of most ecosystems and are of agricultural importance since they help to improve crops yields, by stimulating plant growth and immune response, excluding plant pathogens by niche competition, and actively participating in phenylpropanoid metabolism and antioxidant activities (Pandey et al. 2018). Endophytes are bacteria or fungi that live within a plant within intercellular spaces, tissue cavities, or vascular bundles without harming the host and often benefit the host. The endophytic bacteria can be found in most plant species and can be recovered from roots, leaves, stems, and a few from flowers, fruits, and seeds (Lodewyckx et al. 2002); they have the potential to produce a variety of secondary metabolites with application in agriculture and pharmaceutical and industrial biotechnology. It is known that endophytic bacteria are located in the apoplast, and plant roots are proposed to be the entry point (Paungfoo-Lonhienne et al. 2013). It is also suggested that they are transmitted using an alternative vertical strategy due to their presence in flowers and seeds (Tamosiune et al. 2017). Endophytes are facultative or obligate symbiotic microorganisms, mainly bacterial and fungal species that live in apparently healthy internal plant tissues, without causing disease (Schulz and Boyle 2006). The use of chemical fungicides against fungal pathogens adversely affects soil and plant health thereby resulting in overall environmental hazards. Therefore, biological source for obtaining antifungal agents is considered as an environmentfriendly alternative for controlling fungal pathogens. Fungal phytopathogens are challenging to control because of their diverse host spectra and their soil-borne nature. Chemical fungicides are commonly used in higher doses to manage the phytopathogens. However, the increasing use of chemical fungicides results in several undesirable effects, such as development of resistance in pathogens and non-targeted environmental impacts. Therefore, alternative measures are essential for long-term and environment-friendly control of the fungal phytopathogens. The use of antagonistic microbes in biological control not only will provide an efficient control of the plant pathogens but is also harmless to the environment. Endophytes are an endosymbiotic group of microorganisms that colonize in plants and microbes that can be readily isolated from any microbial or plant growth medium. They act as reservoirs of novel bioactive secondary metabolites such as alkaloids, phenolic acids, quinones, steroids, saponins, tannins, and terpenoids that serve as a potential candidate for antimicrobial, anti-insect, anticancer, and many more properties. While plant sources are being extensively explored for new chemical entities for therapeutic purposes, endophytic microbes also constitute an important source for drug discovery. The endophytic bacteria play important roles in plant growth, such as a plant growth promoter with the production of plant growth regulators such as IAA and GA3, and they can supply the nutrients that are essential for the growth and development of plants. Endophytic bacteria have been found in virtually every plant studied, where they colonize the internal tissues of their host plant and can form a range of different relationships including symbiotic, mutualistic, commensalistic, and trophobiotic. Most endophytes appear to originate from the

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rhizosphere or phyllosphere; however, some may be transmitted through seeds. Endophytic bacteria can promote plant growth and yield and can also act as a biocontrol agent. Endophytic actinomycetes act as a promising resource of biotechnologically valuable bioactive compounds and secondary metabolites. Endophytic Streptomyces sp. produce some novel antibiotics which are effective against multi-drug-resistant bacteria. Antimicrobial agents produced by endophytes are eco-friendly, toxic to pathogens, and do not harm humans. Endophytic inoculation of the plants modulates the synthesis of bioactive compounds with high pharmaceutical properties besides promoting growth of the plants. Hydrolases, the extracellular enzymes produced by endophytic bacteria, help the plants to establish systemic resistance against pathogen invasion. Phytohormones produced by endophytes play an essential role in plant development and drought resistance management. The high diversity of endophytes and their adaptation to various environmental stresses seem to be an untapped source of new secondary metabolites. Endophytes can also be beneficial to their host by producing a range of natural products that could be harnessed for potential use in medicine, agriculture, or industry. In addition, it has been shown that they have the potential to remove soil contaminants by enhancing phytoremediation and may play a role in soil fertility through phosphate solubilization and nitrogen fixation. There is increasing interest in developing the potential biotechnological applications of endophytes for improving phytoremediation and the sustainable production of non-food crops for biomass and biofuel production. Plants have served as a source of medicinal bioactive compounds against numerous forms of ailments for centuries. Ironically, in recent years, microorganisms associated with plants rather than plants themselves have proved to offer materials and products with high therapeutic potential (Subbulakshmi et al. 2012). Endophytes are an endosymbiotic group of microorganisms—often bacteria or fungi—that colonize the intercellular and/or intracellular locations of plants (Pimentel et al. 2011; Singh and Dubey 2015). For these organisms, whole or a part of their life cycle occurs within their hosts, without causing any apparent symptoms of disease. They are ubiquitous in nature and exhibit complex interactions with their hosts, which involve mutualism, antagonism, and rarely parasitism (Nair and Padmavathy 2014). Endophytic bacteria form a large proportion of the indigenous microbial communities in plants. Their internal colonization is often assisted by a wide array of enzymes, which are also present in plant pathogenic bacteria. Endophytic bacteria have become adapted to the plant’s selective environment and can be beneficial, neutral, or deleterious for the plant by affecting plant growth and or the defense of the plant against pathogens. In contrast to pathogenic bacteria they do not produce visual symptoms. Pleban et al. (1995), reported the inhibition of plant pathogens Rhizoctonia solani (46–56%, in bean), Pythium ultimum, and Sclerotium rolfsii (26–79%) by using Pseudomonas and Bacillus sp. as introduced endophytes, comes from studies conducted on crops such as cotton, oilseed rape, tomato, cucumber, and peas

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(Alstrom 2001). In some of these studies, the introduced endophytic bacterium caused both growth promotion and biological control.

1.2

Importance of Endophyte

Endophytes are known to enhance host growth and nutrient gain. They may enhance the plant’s resistance to insects and pests along with tolerance to various abiotic and biotic stresses. They produce phytohormones and other bioactive compounds of biotechnological interest (enzymes and pharmaceutical drugs) (Joseph and Priya 2011). In recent past, researchers defined endophytes as ‘endo-symbionts’ which inhabit the inner parts of plant tissues and do not damage or inflict diseases which could be isolated through adherence of aseptic methods (Arnold and Lutzoni 2007; Khan et al. 2015). Plant interiors are occupied by a large variety of microorganisms, which interact towards the development of a relatively stable microbial community. The number of reports on bacteria being isolated from healthy plant tissues is increasing fast, and several reviews have been published recently (Kobayashi and Palumbo 2000). Fisher et al. (1992) studied the distribution of bacterial endophytes in fieldgrown Zea mays plants and found that the plant parts closer to the soil were more heavily colonized by bacteria than those near the top of the plants. In general the basal part of the stem and the root contain the largest numbers and biodiversity of endophytic bacterial species. Several groups known as plant growth promoting rhizobacteria have been isolated also as endophytes, including the nodulating and nonnodulating diazotrophs. Endophyte populations usually range from 103 to 106 and rarely exceed 107 cfu/g plant matter, as have been reported for tissues of various plant species (Chanway 1998). The high biodiversity among these bacterial endophytes is evident from the studies made by Mundt and Hinkle (1976) and McInroy and Kloepper (1995). The endophytic bacteria most commonly isolated from various plant parts of different crops are summarized by Hallmann et al. (1997) and Kobayashi and Palumbo (2000). The study of Tjamos et al. (1999) with two effective root-tip Bacillus isolates provided evidence that their ability to colonize both epiphytically and endophytically can be an important factor determining their effectiveness in controlling Verticillium wilt in planta. Redman et al. (1999) studied the biochemical analysis of plant protection afforded by a nonpathogenic endophytic mutant of Colletotrichum magna. Studies had shown previously to protect watermelon (Citrullus lanatus) and cucumber (Cucumis sativus) seedlings from anthracnose disease elicited by wild-type C. magna. Plant biochemical indicators of a localized and systemic (peroxidase, phenylalanine ammonia-lyase, lignin and salicylic acid) “plant-defense” response were investigated in anthracnose-resistant and susceptible cultivars of cucurbit seedlings. Results indicated disease protection in path-1- colonized plants were correlated with the ability of inducing plant defense mechanism in the host. Strobel et al. (2007) discovered a novel endophytic fungus Muscodor vitigenus from the liana Paullinia paullinioides that produced naphthalene, an insect repellant. The extracted naphthalene has chromatographic and mass spectral properties that are

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identical to authentic naphthalene. In a preferred embodiment the naphthalene in the gas phase of M. vitigenus is useful in the repellency of unwanted insect pests. This unique biological activity of this novel endophyte suggests a wide range of potential practical applications particularly in the area of insect repellents, insecticides, antimicrobials, anti helminthics, and vermicides. Pimentel et al. (2011) reviewed on the use of endophytes for the production of bioactive compounds and their use in biotransformation process. The role of endophytes on the production of anticancer, antimicrobial, and antioxidant compounds illustrating their potential for human use was inferred. It also describes biotransformation as an auspicious method to obtain novel bioactive compounds from microbes. Biotransformation allows the production of regio and stereo selective compounds under mild conditions and that using endophytic fungi have been reviewed for e.g. biotransformation of grandisin by the endophytic fungus Phomopsis sp. to a tetrahydrofuran which showed trypanocidal activity.

1.3

Isolation of Endophytes from Plants

Lodewyckx et al. (2002) elaborated the methods used for the isolation and characterization of bacteria and reported at least 81 bacterial species which were found to be associated with crop plants. Moore et al. (2006) isolated endophytic bacteria from the popular tree grown on contaminated site and studied the effectiveness of these bacteria in phytoremediation. Ryan et al. (2008) indicated that endophytic bacteria can be isolated from all kinds of plants in the plant kingdom irrespective of the nature of plants like trees, herbs, and shrubs. Taghavi et al. (2010) analyzed the bacterial species in different parts of plants and observed that Azoarcus, Acetobacter (renamed as Gluconobacter), Bacillus, Enterobacter, Burkholderia, Herbaspirillum, Pseudomonas, Serratia, Stenotrophomonas, and Streptomyces were the predominant bacterial endophytes colonized in plant tissues. Malfanova (2013) reviewed in depth the diversity of endophytic bacteria and reported that three major phyla were studied predominantly by the researchers, namely, Actinobacteria, Proteobacteria, and Firmicutes. Hallmann and Berg (2006) reported that the species of the above genera were found to form colonies in most of the soil and rhizosphere of the plants, whereas Compant et al. (2010) confirmed the presence of endophytes above the root zone, flowers, and also seeds. Jesus and Lugtenberg (2014) reported the presence of bacterial endophytes and their identification from various parts of the plant sap. Endophytes populations are always greater in the roots than any other organ of plants. In the root the average density is 105 cfu per g fresh weight, whereas average values of 104 and 103 are reported for stem and for leaf, respectively. Endophytes can be easily isolated on any microbial or plant growth, such as agar, potato dextrose agar, and any nitrogen- or carbon-containing media. The most frequent method used to detect and enumerate endophytes involves isolation from surface-sterilized host plant tissue. The main factors that may regulate entophyte colonization within a plant or microbial species, include the genotype of the plant, the growth stage of the plant, the physiological status of the plant, the type of plant

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tissues, the environmental condition of the soil in which it is grown, the sampling season, the surface sterility, selective media, and culture conditions as well as different agricultural practices (Gaiero et al. 2013; Golinska et al. 2015) Ecological awareness on the role of endophytes in nature can also provide the best clues for targeting a particular type of endophytic bioactivity with the greatest potential for bioprospecting (Strobel and Daisy 2003). Das et al. (2017) reported the antimicrobial potentials of endophytic bacteria isolated from leaf tissues of Hyptis suaveolens against some clinically significant pathogens. The results indicated that 60% of the isolates showed antimicrobial activity inhibiting at least one of the test pathogens in preliminary screening. Among them two isolates showed considerable antimicrobial activity against the test pathogens. The isolates were identified as Bacillus and Pseudomonas species by morphological and biochemical characterization. Sharma and Roy (2015) reported a total number of 536 bacterial and fungal endophytes isolated from root, stem, and leaves of the plant Amaranthus spinosus. Roots supported more number of bacterial endophytes than stem or leaves, whereas stem supported more number of fungal endophytes than either roots or leaves. The plant harboured more of gram negative compared to gram positive bacterial endophytes. The fungal endophytes isolated from root, stem, and leaves of the plant A. spinosus revealed the presence of Penincillum, Aspergillus Cladosporium, Phoma, Bipolaris, and Fusarium spps. Etminani and Harighi (2018) demonstrated the production of plant growth hormone auxin and gibberellins by the strains isolated from leaves and stems healthy wild Pistachio trees (Pistacia atlantica L.). The isolated strains belong to the genus Pantoea, Bacillus, Pseudomonas, Serratia, and Stenotrophomonas and also showed biocontrol activity. Endophytes can either be fungal or bacterial in nature and are capable of producing biologically active compounds, some of which are used by the plant as part of its arsenal in its defense against pathogens, while some promote plant growth (González-Teuber et al. 2014). Most of the bioactive compounds extracted from endophytes have shown a plethora of bioactivities including but not limited to antimicrobial, immunosuppressant, and anticancer (Nair and Padmavathy 2014).

1.4

Application of Endophytes Microorganism in Biotic and Abiotic Stresses

1.4.1

Anti-Fungal Activity of Endophytes

Hazarika et al. (2019) reported that seven endophytic bacteria were isolated from sugarcane leaves and screened for its antifungal activity against 10 fungal isolates belonging to the genera Alternaria, Cochliobolus, Curvularia, Fusarium, Neodeightonia, Phomopsis, and Saccharicola isolated from diseased leaves of sugarcane. They concluded that the antifungal potential of isolate Bacillus subtilis SCB-1 was established against taxonomically diverse fungal pathogens including the genera Saccharicola, Cochliobolus, Alternaria, and Fusarium, and the potent

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antifungal compound surfactin as well as volatiles produced by the bacterial isolate which could be responsible for its bio-control activity against fungal infections. Abdallah et al. (2018) studied that Fusarium graminearum can cause Giberella Ear Rot (GER) and seedling blight in maize, resulting in major yield losses. Besides GER, the infected grains are consequently contaminated with multiple mycotoxins of F. graminearum. They explored the effect of the endophytic fungal genera of Epicoccum and Sordaria to control F. graminearum infection in comparative trials with Piriformospora spp., an elusive endophytic genus. The results showed a considerable variability in the antifungal activity, both among species and among isolates within one species. Endophytic fungi (EF) are an important source for novel, potential, and active metabolites. Plant–endophyte interaction and endophyte–endophyte interactions study provide insights into mutualism and metabolite production by fungi. Bioactive compounds produced by endophytes main function are helping the host plants to resist external biotic and abiotic stress, which benefit the host survival in return. Plants lack immune response to certain pathogens, but the endophytes that reside inside the plant tissue enhance the immune response of the plants to fight against invading pathogens (Melotto et al. 2008). Endophytes commonly increase plant biomass under stressful conditions but the cellular mechanisms involved in stress tolerance and growth enhancement are poorly characterized. The “balanced antagonism” hypothesis was initially proposed to address how an endophyte controls host defense mechanisms to be activated against it, ensures self-resistance before being incapacitated by the toxic metabolites of the host, and manages to grow within its host without causing visible manifestations of infection or disease (Arnold and Lutzoni 2007). Liang et al. (2014) reported a total of 83 endophytic fungi strains isolated from the root, stem, leaf, and fruit of Brucea javanica. About 34 strains were obtained from the stem, 32 strains were obtained from the leaf, 15 strains were isolated from the root, and 2 strains came from the fruit, and it was concluded that 14 strains had antifungal activities against at least one pathogenic fungi and 9 strains showed antibacterial activities against one or more bacteria. Tashi-Oshnoei et al. (2017) demonstrated the samples of roots, leaves, and stems of healthy oak trees collected from various locations in the Baneh and Marivan regions, Iran. In total, 63 endophytic bacteria were isolated and grouped according to phenotypic properties. The isolates have the ability to produce plant hormone such as auxin and gibberellin along with siderophore production, phosphate solubilization, atmospheric nitrogen fixation, and protease and hydrogen cyanide production. Premjanu et al. (2017) explained the isolation of endophytic fungi from Lannea coromandelica having antifungal activity potential and the isolation of the secondary metabolite from the dominant fungi and predict the probable mechanism behind its activity. They have isolated Aspergillus flavus, Aspergillus niger, Alternaria alternata, and Colletotrichum gloeosporioides identified based on their morphological features as endophytic fungi and concluded that among the four dominant fungi, the antifungal activity of Aspergillus flavus showed the maximum activity with an

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inhibitory zone of 26.22 mm against Candida albicans and 16.72 mm against Malassezia pachydermis. The endophytic bacteria were isolated from the root tissues of Talinum triangulare by surface sterilization method. The isolates were cultured in Trypticase Soybean agar (TSA) and antagonist activities were evaluated by dual culture assay against Fusarium oxysporum, Trichoderma reesei, and Candida albicans. The result reveals that 4 of 23 endophytic bacterial isolates demonstrated great antifungal potentiality against many tested fungi. Ali and Rante (2018) isolated and identified bacteria with antifungal properties on the basis of morphological, physiological, biochemical and 16S rRNA analysis as member of genus Bacillus and Brevibacillus. Osama et al. (2018) isolated endophytic bacteria from Chinese traditional medicinal plant Glycyrrhiza uralensis (licorice) and evaluated their in vitro antimicrobial activities against common fungal pathogens of tomato (Fusarium oxysporum f. sp., Fulvia fulva, Alternaria solani), cotton (Fusarium oxysporum f. sp. vasinfectum, Verticillium dahliae), pomegranate (Ceratocystis fimbriata), Cymbidium (Colletotrichum gloeosporioides), and Tsao-ko (Pestalotiopsis microspora and Fusarium graminearum). And, they reported that the antimicrobial activities of natural endophytes, particularly Bacillus atrophaeus, suggest this species may be a promising candidate as a biocontrol agent to confer resistance to Verticillium wilt disease and other phytopathogens in cotton and other crops. Wild-type Arabidopsis used to test the effect of the endophytic bacterium on inhibition of seed surface mycoflora. The seeds were treated with a bacterial suspension and allowed to germinate. The bacterium-treated seedlings were healthy without disease symptoms while a majority of the untreated A. thaliana seedlings showed evidence of overgrowth by fungi and necrosis. The bacteria either antagonized fungal spores and mycelia on the seed surface directly or they released antifungal lipopeptides that suppressed fungal growth. An endophytic Bacillus species isolated from a paddy field showed similar activity in protecting maize and horsebean from infection of Bipolaris maydis and R. solani, respectively (Wang et al. 2009). Bacon and Hinton (2002) characterized two strains of B. subtilis (ATCC55422 and ATCC55614) as endophytic to maize plants having in vitro inhibition activity of F. moniliforme. Endophytic fungi of maize have also been shown to have antagonistic activity against fungal pathogens including Aspergillus flavus and Fusarium verticillioides (Wicklow et al. 2005).

1.4.2

Antibacterial Activity of Endophytes

Manganyi et al. (2019) determined the antimicrobial properties and identified the chemical compounds of secondary metabolites produced by endophytic fungi isolated from Sceletium tortuosum. A total of 60 endophytic fungi produced secondary metabolites that were detected after fermentation and extraction. Antibacterial properties of the secondary metabolites were determined using the disc diffusion

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assay against pathogenic environmental Gram-positive and Gram-negative bacteria as well as control stain. They found that 15% of fungal extracts displayed a narrow spectrum of activity against the bacteria strains. Despite this, none of the fungal extracts inhibited growth of Enterococcus faecalis (ATCC S1299) and Enterococcus gallinarum (ATCC 700425) while Bacillus cereus (ATCC 10876) was the most susceptible against the fungal extracts. Fusarium oxysporum (GG 008) with accession no. KJ774041.1 displayed significant antibacterial activity that was linked to high levels of 5-hydroxymethylfurfural (HMF) and octadecanoic acid as revealed by GC-MS and concluded that endophytic fungi from S. tortuosum L. produced secondary metabolites that exhibited highly effective antibacterial activity against multi-drug-resistant bacterial strains, and these isolates could serve as potential sources for the isolation of novel antimicrobial agents that may contribute in the fight against antibiotic strains. Beiranvand et al. (2017) demonstrated the molecular identification and measuring the antimicrobial activity of endophytic actinomycetes isolated from medicinal plants of Iran. About 16 out of 23 bacterial isolates (69%) exhibited antimicrobial activity against the selected pathogenic bacteria Bacillus cereus, Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae, Citrobacter freundii, Proteus mirabilis, Shigella flexneri, and Escherichia coli. About 2 out of 23 endophytic bacterial isolates, EB4 and EB7, showed inhibitory activity against Bacillus cereus. In addition, EB9 showed inhibitory activity against Staphylococcus aureus, Citrobacter freundii, and Shigella flexneri. About 16 isolates (69%) obtained by hot method showed strong activity against selected pathogenic organisms and two of them (EB7 and EB69) had broad spectrum antibacterial activity. Ultrasonic method showed that 13 out of 23 isolates (46%) inhibited microbial growth. Among the 5 endophytic bacteria only 4, except Staphylococcus spp. LCP, showed antimicrobial activity. Pseudomonas spp. SSRN1 and Enterobacter spp. SSRP1 were considered as the most active strains as they both had a moderate activity against S. aureus. High zone of inhibition was by Pseudomonas spp. SSRN1 and Enterobacter spp. SSRP1, followed by Lysinibacillus spp. HSRN, then lastly Bacillus spp. Endophytic bacteria have the potential to produce novel natural compounds with antibacterial and antifungal activity (Christina et al. 2013). Bacterial endophytes (Pseudomonas spp. and Bacillus spp.) isolated from Plectranthus tenuiflorus have shown great antimicrobial activity against some human pathogenic strains such as Salmonella typhi, S. aureus, E. coli, Klebsiella pneumoniae, Streptococcus agalactiae, Proteus mirabilis, and Candida albicans (EI-Deeb et al. 2013). Enterobacter spp. isolated from Raphanus sativus L. also showed antibacterial activity against a few human pathogenic bacteria including E. coli, Salmonella enteritidis, Shigella sonnei, Salmonella typhimurium, P. aeruginosa, Shigella flexneri, and B. cereus (Seo et al. 2010). Pseudomonas spp. has been proven to possess antimicrobial compounds called ecomycins and pseudomycins (Christina et al. 2013). Secondary metabolites from C. molle were also reported to possess antimicrobial activity (Kaleab et al. 2006; Fankam et al. 2015). Marcellano et al. (2017) reported the antibacterial activity of endophytic fungi associated with the bark of Cinnamomum mercadoi. The pure isolates were

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identified through their morphological characteristics. Agar plug diffusion assay was employed in the primary screening of their antibacterial activity against Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa. Twelve (12) endophytes were isolated from the bark of C. mercadoi. All endophytes exhibited antibacterial activity on at least one of the test pathogens. However, only 2 of the 4 endophytes subjected to the secondary screening showed wide-spectrum activity and inhibited the growth of all test bacteria. Fusarium sp. 2 was identified to have the most promising activity with MIC values ranging from 2.1 to 4.2 mg/mL. They concluded that C. mercadoi harbours endophytes, particularly Fusarium sp. 2, which possess antibacterial activity and thus a potential source of antibacterial compounds. Indrawati et al. (2018) reported that the bacterial endophytes from the tropical plant Syzygium polycephalum (Kupa) can be used as an alternative solution to reduce the utilization of synthetic antibiotics. A total of 9 isolates of bacterial endophytes have been successfully obtained. From these isolates, a total of 4 species of endophytic bacteria were identified: Bacillus sp. (1), Bacillus sp. (2), Bacillus pumilus, and Bacillus amyloliquefaciens. Antibacterial tests revealed that Bacillus sp. (2) derived from the leaves appeared to be the most potent antibacterial isolates against pathogenic bacteria with 22 and 9 mm of inhibitory zone to methicillinresistant Staphylococcus aureus (MRSA) and to Bacillus cereus, respectively. On the other hand, endophytic isolate Bacillus sp. (1) derived from stem was able to inhibit Klebsiella pneumoniae and B. cereus with inhibitory zones as much as 10 and 7 mm. They concluded that the results strongly indicated that the antibacterial effect of bacterial endophytes from the study was species-specific and indeed the bacterial endophytes in this study could serve as a potential source of novel natural antibiotics. Francielly et al. (2017) analyzed the antimicrobial potential of 10 actinomycetes isolated from the medicinal plant Vochysia divergens located in the Pantanal sul-mato-grossense, an unexplored wetland in Brazil. Strains were classified as belonging to the Aeromicrobium, Actinomadura, Microbacterium, Microbispora, Micrococcus, Sphaeris Sporangium, Streptomyces, and Williamsia genera, through morphological and 16S rRNA phylogenetic analysis. All conditions were analyzed for active metabolites, and the best antibacterial activity was observed from metabolites produced with SG medium at 36
C. They concluded that LGMB491 (closely related to Aeromicrobium ponti) extract showed the highest activity against methicillin-resistant Staphylococcus aureus (MRSA), with an MIC of 0.04 mg/mL, and it was selected for SM identification. Strain LGMB491 produced 1-acetyl-β-carboline (1), indole-3-carbaldehyde (2), 3-(hydroxyacetyl)-indole (4), brevianamide F (5), and cyclo-(L-Pro-L-Phe) (6) as major compounds with antibacterial activity.

1.4.3

Plant Growth Promoters (PGPR)

A growing body of literature indicated an array of advantages of endophytes. Kang et al. (2007) detailed the growth-promoting characteristics of endophytes, while

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Kloepper et al. (2004) demonstrated the disease- inhibiting traits of endophytes. The nature of endophytes in strengthens the defense mechanism of crops to various plant diseases. Anti-herbivory products were found to be instigated by endophytes (Sullivan et al. 2007). Backman et al. (1997) discussed various factors influencing endophytes as biocontrol agents against various plant diseases like specific bacterial species colonizing in a particular crop species, the changing population in different seasons, the pattern with which they have been colonizing and their capacity to mobilize inside the tissues and to stimulate systemic resistance. Endophytic bacteria seem to be distributed in most plant species and have been isolated from roots, leaves, and stems, and a few from flowers, fruits, and seeds (Lodewyckx et al. 2002). Endophytic bacteria may accompany certain metabolic properties, such as promoting plant growth, controlling soil-borne pathogens, or helping host plants to defeat stress responses to environmental abuse (Mastretta et al. 2006; Taghavi et al. 2007; Ryan et al. 2008). Furthermore, the interactions between plants and bacteria help plants to settle in ecosystem restoration processes (Glick et al. 1995). These interactions may increase the ability of plants to utilize nutrients from the soil by increasing root development, nitrate uptake or solubilizing phosphorus, and to control soil-borne pathogens (Whipps 2001). The improvement of the growth and health of the plants cannot be separated from the role of endophytic bacteria. They supply nutrient elements through the process of fixing the nutrient elements from the air (Hirano and Upper 2000), improve the mobilization of P, trapped Fe (Ryan et al. 2008), fight against plant pathogens through the induction of systemic resistance and produce secondary metabolic compounds that are antagonists (Kloepper and Ryu 2006; Sturz and Nowak 2000), as well as reduce plants’ biotic or abiotic stress without pathogenicity (Lugtenberg and Kamilova 2009). Shen et al. (2019) demonstrated the seedlings of rice (Oryza sativa) planted in soil with 1, 5, or 10 times as the recommended rates of the fungicides etridiazole, metalaxyl, and tricyclazole. Endophytic Bacteria were isolated from roots of rice seedlings. The bacterial 16S rDNA sequences and related PGP characteristics including potential nitrogen fixation, phosphorus-solubilizing and indole acetic acid (IAA) production ability were examined. They found that 17 different strains were obtained from rice seedling roots; five strains with both nitrogen fixation potential and IAA production ability included Rhizobium larrymoorei E2, Bacillus aryabhattai E7, Bacillus aryabhattai MN1, Pseudomonas granadensis T6, and Bacillus fortis T9. With further test, they concluded that Bacillus aryabhattai MN1 showed high tryptophan dose-dependent IAA production ability, tolerance towards etridiazole and metalaxyl application and should be considered a potential bacterial biofertilizer. Microorganisms play a key role in the health and development of crops (Tikhonovich and Provorov 2011; Cory and Franklin 2012) and the relationship between rhizobacteria and endophytes with their plant hosts has been reviewed extensively (Ryan et al. 2008; Hayat et al. 2010; Blanco and Lugtenberg 2014). Such plant growth promoting rhizobacteria (PGPR) have considerable potential as biological inoculants in sustainable agriculture (Saharan and Nehra 2011; Glick

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2012; Sivasakthi et al. 2014). Plants that are inoculated with PGPR benefit from the resulting plant–microbe interaction as the bacteria contribute to plant growth and health by multiple mechanisms including nitrogen fixation, synthesis of phytohormones, modulation of plant ethylene levels, solubilization of unavailable soil phosphate and suppression of pathogens through niche exclusion, and the production of anti-microbial metabolites (Fuentes-Ramirez and Caballero-Mellado 2005; Franche et al. 2009; Babalola 2010; Compant et al. 2010). A collection of plant-associated bacteria from various plant hosts (Miscanthus giganteus, B. napus, and Iris pseudacorus) have been isolated and partially characterized (Otieno et al. 2013). Strains were isolated following standard procedures from rhizosphere and internal plant tissues including root, leaves, and stem (Germaine et al. 2004; Otieno et al. 2013). Many of these strains have multiple plant growth promotion (PGP) characteristics including siderophore production, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity (for the regulation of plant stress hormone ethylene caused by abiotic stress conditions), phosphate solubilization, and in some cases biocontrol against fungal plant pathogens. Khan et al. (2020) explained that Paenibacillus polymyxa is a plant growthpromoting rhizobacterium that has immense potential to be used as an environmentally friendly replacement of chemical fertilizers and pesticides. Paenibacillus polymyxa SK1 was isolated from bulbs of Lilium lancifolium. The isolated endophytic strain showed antifungal activities against important plant pathogens like Botryosphaeria dothidea, Fusarium oxysporum, Botrytis cinerea, and Fusarium fujikuroi. The highest percentage of growth inhibition, i.e., 66.67  2.23%, was observed for SK1 against Botryosphaeria dothidea followed by 61.19  3.12%, 60.71  3.53%, and 55.54  2.89% against Botrytis cinerea, Fusarium fujikuroi, and Fusarium oxysporum respectively. They concluded that the isolated strain SK1 showed plant growth-promoting traits such as the production of organic acids, ACC deaminase, indole-3-acetic acid (IAA), siderophores, nitrogen fixation, and phosphate solubilization. IAA production was strongly correlated with the application of exogenous tryptophan concentrations in the medium and revealed that P. polymyxa SK1 may be utilized as a source of plant growth promotion and disease control in sustainable agriculture.

1.4.4

Applications of Endophytes in Soil pH Stress

Ngwene et al. (2016) studied endophytic Sebacinales member Piriformospora indica, which was isolated a decade ago from an Indian desert, and which is known for increasing plant resistance and tolerance to stress and for promoting plant growth. The authors hypothesized its ability to support plant nutrition and showed that P. indica growth was higher in the presence of inorganic phosphate than in organic phosphate sources. The related genes were all repressed by higher amounts of inorganic phosphate, but mostly expressed when the fungus received phytate. Interestingly, a pH decrease was observed in the presence of P. indica irrespective of the phosphate source. Ngwene et al. (2016) indicated that P. indica is

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able to solubilize phosphate from inorganic, but not from organic sources, and that phosphate solubilization could not be due to enzymatic activities, but rather to the lowering of the medium pH. Postma et al. (2007) demonstrated colonization by root endophytes that can be beneficial to plants growing on acid, nutrient-poor soils. Arbuscular mycorrhizal (AM) fungi can supply herbs with nutrients and may give protection against aluminium toxicity. AM fungi are the most prevalent symbionts in herbs at neutral to acidic soil pH. At extremely low pH, fungal growth can be limited and AM colonization is usually rare. They investigated root colonization by AM, FE and DSE in Maianthemum bifolium, Galium odoratum, Mercurialis perennis and Stellaria nemorum, from a range of acidic beech forests to relate endophyte colonization to a gradient in soil pH. With decreasing pH, colonization by AM decreased, whereas the other two endophytes increased. AM and FE colonization were inversely correlated in Maianthemum bifolium. They concluded with the compared changes in root colonization with those in chemical composition of soil and leaf samples and found a positive correlation between leaf magnesium concentrations and the presence of DSE in Galium odoratum and found aluminium concentration in Maianthemum bifolium tended to be lower when FE colonization was high, suggesting a possible role for the fungi in plant protection against Al. They suggested that FE and DSE may replace AM fungi in herbaceous vegetation at extremely low pH, counteracting some of the negative effects of high soil acidity on plants. Tall fescue is grown on soils where low pH and limited nutrient supply restrict plant productivity, caused by interactions among prior land use practices, relatively high rainfall amounts, and soil geochemistry. Plants that are adapted to acidic soils possess a variety of mechanisms that enable them to tolerate or overcome adverse soil chemical conditions. Soil acidity affects plant growth through a complex of chemical changes in the rhizosphere involving increased H0 , Al00 , and Mn 2. These include inhibition of metal cation (Ca20 , Mg2) uptake, a decrease in P and Mo solubility and increased efflux of nutrients and metabolites from roots. Root morphology and function also change when soil chemical conditions are less than ideal for plant growth, when soil water is scarce, some species can explore large areas of soil, and, in other instances, mineral challenges can elicit or suppress the production of fine root structure (Huang 2001).

1.4.5

Applications of Endophytes in Drought Tolerance

Ullah et al. (2019) demonstrated that endophytic bacteria which survive within plant tissues are among the most appropriate technologies improving plant growth and yield under drought conditions. These endophytic bacteria live within plant tissues and release various phytochemicals that assist plants to withstand harsh environmental conditions, i.e., drought stress. Their plant growth-promoting characteristics include nitrogen fixation, phosphate solubilization, mineral uptake, and the production of siderophore, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, and

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various phytohormones. These plant growth promoting characteristics of endophytic bacteria improve root length and density, which leads to enhanced drought tolerance. The plant-endophytic bacteria assist plants to withstand drought stress by producing drought-tolerant substances, for instance, abscisic acid, indole-3-acetic acid, ACC deaminase, and various volatile compounds. Indirectly, endophytic bacteria also improve osmotic adjustment, relative water content, and antioxidant activity of inoculated plants. Altogether, these bacterial-mediated drought tolerance and plant growth-promoting processes continue even under severe drought conditions which lead to enhanced plant growth promotion and yield. Vigani et al. (2018) determined the two endophytic bacteria endowed with an array of in vitro plant growth promoting traits. Their genome sequences confirmed the presence of traits previously shown to confer drought resistance to plants, such as the synthesis of nitric oxide and of organic volatile organic compounds. The two strains of pepper (Capsicuum annuum L.) were used because of their high sensitivity to drought. Under drought conditions, both strains stimulated a larger root system and enhanced the leaves’ photosynthetic activity. By testing the expression and activity of the vacuolar proton pumps, H+ -ATPase (V-ATPase) and H+ -PPase (V-PPase), they found that bacterial colonization enhanced V-PPase only and, therefore, concluded that the enhanced expression and activity of V-PPase can be favoured by the colonization of drought-tolerance-inducing bacterial endophytes. Dastogeer et al. (2018) reported that the fungal endophytes and a virus confer drought tolerance to Nicotiana benthamiana plants through modulating osmolytes, antioxidant enzymes, and expression of host drought responsive genes. They evaluated how the colonization of two fungal endophytes isolated from wild Nicotiana species from areas of drought-prone northern Australia, and a plant virus, yellowtail flower mild mottle virus (genus Tobamovirus), improved water stress tolerance in N. benthamiana plants. Inoculation with both of the two fungal strains used and the virus significantly increased plants tolerance to water stress as manifested by their significant delay in wilting of shoot tips. The water stress tolerance of fungus-inoculated plants was correlated with increases in plant biomass, relative water content, soluble sugar, soluble protein, proline content, increased activities of the antioxidant enzymes catalase, peroxidase and polyphenol oxidase, decreased production of reactive oxygen species, and decreased electrical conductivity. They concluded that the influence of the virus was similar to the fungi in terms of increasing the plant osmolytes, antioxidant enzyme activity, and gene expression. Although separate infection of fungi and virus increased plant water stress tolerance responses, their co-infection in plants did not have an additive effect on water stress responses. Hubbard et al. (2013) demonstrated the impact of fungal endophyte symbiosis on the growth, eco-physiological and reproductive success of wheat exposed to heat and drought. The resistance of pot-grown wheat to heat or drought stress was measured by quantifying efficiency of photosystem II (Fv/Fm), plant height, average seed weight (ASW), total seed weight (TSW), water-use efficiency (WUE) as well as time to 50% germination and percentage germination of second-generation seeds produced under heat stress, drought stress or well-watered conditions. The endophytic

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fungi tested increased wheat tolerance for drought and heat. They found out that Endophyte SMCD 2206 was the most beneficial, followed by SMCD 2210 and 2215. The second-generation seeds produced by drought-stressed wheat colonized by SMCD 2206, 2210, or 2215 had decreased WUE relative to those produced by endophyte-free, drought-stressed plants. Hubbard et al. (2013) reported that fungal endophytes can improve plant tolerance to abiotic stresses such as heat and drought. He hypothesized that six endophytic fungi, SMCD 2204, 2206, 2208, 2210, 2214, and 2215, would promote heat and drought tolerance in wheat during both seed germination and at later developmental stages as well as assessed mycomediated enhancement of seed germination (mycovitality) including seedling performance, in vitro in terms of percent germination, seedling fresh weight, energy of germination (EG), and hydrothermal time (HTT) of germination. He concluded that Endophytes SMCD 2206, 2210, and 2215 improved seedling heat or drought resistance, while SMCD 2204, 2208, and 2214 did not. He demonstrated that fungal endophytes SMCD 2206, 2210, and 2215 improve wheat tolerance for heat and drought both in vitro and in pot studies.

1.4.6

Applications of Endophytes in Soil Salinity Tolerance

Salinity affects 20% of agriculture lands worldwide and is a major cause of reductions in plant productivity and the degradation of land (Siddikee et al. 2010; Ramadoss et al. 2013). Soil salinity can be caused by the interaction of natural factors including geological processes, climate change, and water management. Salinity can also be induced by anthropogenic activity, e.g., the inappropriate use of fertilizers and improper irrigation practices (Bianco and Defez 2011; Paul and Lade 2014). Osmotic stress and associated imbalances in ions and nutrients adversely affect plant growth and function (Evelin et al. 2009). Ramaiah et al. (2020) demonstrated that a salt-tolerant endophyte isolated from salt-adapted Pokkali rice, a Fusarium sp., colonizes the salt-sensitive rice variety IR-64, promotes its growth under salt stress and confers salinity stress tolerance to its host. Piriformospora indica, has been reported to promote growth in a number of plant systems under abiotic stresses including salinity stress (Varma et al. 2012). In Arabidopsis, P. indica maintains the Na + and K+ homeostasis under salt stress (Arshad et al. 2017). Two bacterial endophytes, Bacillus subtilis and Mesorhizobium ciceri, confer salt tolerance to chickpea by decreasing H2O2 concentrations and increasing proline content (Egamberdieva et al. 2017). Pseudomonesa fluorescens and P. migulae ameliorate salinity stress in tomato plants by increasing the 1-aminocyclopropane-1-carboxylate deaminase activity, the key enzyme for ethylene biosynthesis. (Ali et al. 2014). Asaf et al. (2018) demonstrated Aspergillus flavus CHS1-mediated salinity tolerance in Glycine max. L. through the stimulation of the antioxidative system and endogenous hormone levels in the host. Bajaj et al. (2018) showed that colonization of soybean plants by P. indica resulted in the stimulation of genes associated with the phenylpropanoid and lignin pathways, both of which are known to play an

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important role in oxidative stress tolerance. Kearl et al. (2019) explained that the salttolerant halophyte rhizosphere bacteria stimulate the growth of Alfalfa in salty soil. Bacteria were isolated from the rhizosphere and as root endophytes of Salicornia rubra, Sarcocornia utahensis, and Allenrolfea occidentalis and a total of 41 independent isolates were identified by 16S rRNA gene sequencing analysis. Isolates were tested for maximum salt tolerance, and some were able to grow in the presence of up to 4 M NaCl. Halomonas, Bacillus, and Kushneria species were consistently isolated both from the soil and as endophytes from roots of all three plant species at all collection times. The most commonly identified bacteria were from several phyla commonly found in soil or extreme environments: Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, and Gamma- and Delta-Proteobacteria. Isolates were tested for the ability to stimulate growth of alfalfa under saline conditions. This screening led to the identification of one Halomonas and one Bacillus isolate that, when used to inoculate young alfalfa seedlings, stimulate plant growth in the presence of 1% NaCl, a level that significantly inhibits growth of uninoculated plants. The same bacteria used in the inoculation were recovered from surface sterilized alfalfa roots, indicating the ability of the inoculum to become established as an endophyte. They concluded that the results with these isolates have exciting promise for enhancing the growth of inoculated alfalfa in salty soil. Acknowledgments The authors are grateful to the Head of the Forestry Department and the Director, NERIST for the support. PK would like to thanks Early Career Research Award, SERB, Government of India (file no ECR/2017/001143) for their financial support to carry out this research work. Conflict of Interest There is no conflict of interest among the authors.

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Endophytic Bacteria: Mitigating Abiotic Stress from Inside Garima Malik and Rahul Arora

Abstract

Plants grow and develop under harsh environments, where they receive water, light, minerals, and other products in a dynamic cycle. Sometimes, the essentiality gets compromised when a plant’s local environment gets disturbed either due to scarcity or excessive availability of the abiotic factors. Plants have adapted a diverse variety of mechanisms to overcome these abiotic stresses. Apart from their own well-developed defence strategies, they sometimes require help from the organisms that live in close association with the plants. One such group of organisms are the endophytic bacteria that live inside the host plant and help them to survive harsh conditions such as salt stress, drought, flood, heavy metal stress, high and low temperatures, and variable light intensity. They also provide support to the plant to mitigate the risk caused by other biotic stress sources. They enter the plant or the host body from various entry points, both above and below the ground. They trigger the systemic response and interfere with the phytohormone signalling mechanisms by controlling the gene expression of various stress-associated genes. The endophytic bacteria have been a good and an alternative source to insecticides and pesticides to prevent a high loss of crops per hectare and thus are being used widely to address the food security challenge globally. Here, we discuss various classes of endophytic bacteria that live in

G. Malik Raghunath Girls’ Post Graduate College, C.C.S. University, Meerut, Uttar Pradesh, India R. Arora (*) The Francis Crick Institute, London, UK Division of Biosciences, Department of Genetics, Evolution and Environment, University College London, London, UK e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. K. Singh et al. (eds.), Bacterial Endophytes for Sustainable Agriculture and Environmental Management, https://doi.org/10.1007/978-981-16-4497-9_2

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association with the plants and help them to overcome the abiotic stresses by promoting plant growth and via other alternative mechanisms. Keywords

Abiotic stress · Salt stress · Heavy metal · Endophytes · Phytohormones · Plant defence · Metagenomics · NGS · Interactomics · Molecular defence

2.1

Introduction

Availability of food is essential for the survival of all living organisms. Food security happens when every person is capable of accessing sufficient food to fulfil their necessities for a healthy life in a sustainable way. However, food security is a complex issue to resolve as it faces a number of challenges throughout production and consumption. Over the past decades, food production outperformed food requirement due to extension in crop zone and irrigation that prompted the swift and sustained development in agricultural productivity and thereby better food security in many countries. However, future expectations highlight a slow-down in agricultural productivity and food shortage chiefly in zones across Africa and Asia which are having unending food security issues. Currently, more than one billion humans are facing starvation problem on a daily basis, affecting their health and life expectancy. The crisis of food insecurity is anticipated to worsen due to rapid population growth and additional rising challenges such as climate change along with various kinds of abiotic and biotic stresses. Gradual climate change presents multifaceted challenges such as droughts, floods, and will only make things difficult for future generations. It has been projected that we need to produce more food in the next 30 years than humans have ever produced in their history as growing population means more food demand. Catering to the food security needs of human population seems a colossal task on the basis of the expected shoot in human population with sustainable intensification of agriculture on the farmland that is accessible for use. As food production is reliant on different interconnected ecosystem services, it is vital that these services are maintained for optimal productivity of agriculture crops. Due to gradual climate change, crop plants are subjected to a vast range of environmental stresses that in turn hamper crop yield and productivity. Various abiotic stresses viz., salinity, drought, heat, cold, and heavy metals, pose a severe threat to agriculture as they adversely affect growth and crop yield worldwide (He et al. 2018). It is clear that we will need to use every technology available, alongside best practice farming to sustainably increase production. Also, there is a need to look beyond agriculture and invest in affordable and suitable farm technologies if the problem of food security is to be addressed in a sustainable manner. A major challenge is to understand how we can re-design the food system to be healthy, sustainable, and more resilient to climate change and abiotic and biotic stresses and help to meet the sustainable development goals.

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Plants face very many different stress conditions, both biotic and abiotic, which reduce the overall yield of important agricultural crops. Since plants are non-mobile unlike humans, they have to withstand these stresses, and thus over the years develop various defence mechanisms depending upon the stress encountered. Biotic stresses include attack to the plants by microbes, including bacteria, fungi, and virus and by pathogens such as nematodes and insects and by animals such as herbivores. The abiotic stress factors include salt, drought, excess water, UV, cold, high temperature, heavy metals, and chemical waste (Kumar and Verma 2018). These abiotic factors limit the growth and development of plants by interfering with their ion distribution system, which in turn restricts the yield. Due to constantly changing environmental conditions, it is not a surprise that in the near future the volume of freshwater availability would decrease severing the intensity of abiotic stresses and limiting the resources. Thus, there is an urgent need to push research in this avenue and look for the best methods to develop genetically modified varieties of plants that can withstand such harsh conditions. Since roots are the first plant parts to trigger a defence response, a soil rich in diversity of microbes that aids plant growth and provides them with nutrients in adequate quantities would help them sustain for long (Gull et al. 2019). Plants interact with a great diversity of microbes in the rhizosphere and the rhizoplane, but how does the soil get rich with these microbes? The answer is simple, when a plant grows, its roots absorb water and micronutrients from the soil. During the process of seed germination, growth, and development, a diverse variety of organic compounds such as sugars, amino acids, and other classes of organic acids are released in the soil. This makes soil a habitable place for many different species of bacteria and fungi that grow in the vicinity of plants and sometimes colonize the plant roots and shoot. This gives rise to endophytic class of microbes that secrete bioactive compounds which interact with other microbial products and the host plant machinery to mitigate different stresses faced by the host plant (Rosenblueth and Martínez-Romero 2006; Strobel et al. 2004; Thrall et al. 2007). The symbiotic relationship helps both the microbes and the host plant system to survive and thrive in harsh conditions without developing any pathogenic phenotype. Due to their unique association, a lot of research is being conducted to understand how the colonization of microbes inside the plant provides tolerance to the host against abiotic stresses. It has been suggested that microbial genome interacts with the host genome whereby upon encountering any stress, the microbial machinery synthesizes metabolic compounds such as phytohormones and siderophores that defend the plant by molecular signalling events against these stresses (Ahmad et al. 2011; Eid et al. 2019). Apart from this, these endophytic microbes also help the plant to grow by increasing the nutrient uptake, increasing phosphorus solubilization, increasing nitrogen fixation, synthesizing organic volatile compounds, and also simultaneously providing resistance against the pathogenic strains of microbes, thus fighting biotic stress. These endophytes not just help alleviate stress against salts, droughts, and heavy metals but also act as source of bioremediation, whereby they catalyze the process of breaking down of hazardous chemical substances in the soil and thus making it sustainable for plant growth (Eid

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et al. 2019; Kumar and Verma 2018). Many endophytes are members of common soil bacterial genera, such as Pseudomonas, Burkholderia, and Bacillus (Lodewyckx et al. 2002). These genera are well known for their diverse range of secondary metabolic products including antibiotics, anticancer compounds, volatile organic compounds, antifungal, antiviral, insecticidal, and immunosuppressant agents. This chapter reviews the dynamic role of endophytic microbes in abiotic stress management along with the use of endophytic inoculants to combat abiotic stresses in agricultural fields, which increases global crop production.

2.2

Diversity of Endophytic Bacteria and Associated Abiotic Stresses

The Plant Growth Promoting Bacteria (PGPB) induce tolerance in plants at both physical and chemical levels. This led to the coining of the term Induced Systemic Tolerance (IST) (Yang et al. 2009). The physical mechanism of stress tolerance mediated by the endophytes includes an overall reduction in the diameter of the roots, decreased root mass near the peripheral soil layers, and increase in the number of longer root hairs that could penetrate the soil through deep layers to reach ground water. These phenotypic modifications usually ensure that there is an increased water supply to the plant despite being susceptible to drought stress (Thrall et al. 2007). There are some other phenotypic modifications that have been reported in plants with endophytism, such as changes in the ratio of secondary metabolites due to alteration in stomatal opening frequency and fluctuations in osmotic levels, i.e., the ability of the plant cell to maintain turgor pressure by a gradual accumulation of solutes under water stress conditions. The accumulation of the solutes to maintain the osmotic balance has been attributed to the endophytic system (Malinowski et al. 2008; Theocharis et al. 2012). Further, the endophytic microbes have also been reported to make plants more efficient in low water availability conditions by helping them conserve the moisture (Morse et al. 2002). The endophytes have also been known to modulate the phytohormone concentrations such as that of ethylene and abscisic acid and an increased jasmonic acid production under salt stress (Khan and Doty 2011). These modifications in the phytohormone levels in turn affect the root morphology by increasing/decreasing their growth depending upon the stress encountered (Luo et al. 2009; Wittenmayer and Merbach 2005). Endophytes are also known to regulate the expression of enzymes during stress. One very common example is the regulation of the expression of the enzyme ACC deaminase. An increased production of ACC deaminase decreases the ethylene concentration by hydrolysing ACC, the ethylene precursor and thus promotes root growth (Saleem et al. 2007). Contrary to the use of ACC deaminase, if the plant under stress requires an increased ethylene production then the endophytes down-regulate the expression of this enzyme to trigger ethylenerelated stress signalling in the host plant. Apart from regulating phytohormones and enzymes, another mechanism to salt stress tolerance in plants also includes an increased uptake of two key nutrients: phosphorus and potassium. This increase in

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the uptake of nutrients has also been attributed to the endophytic associations of the host plant system (Mayak et al. 2004). In their study, Mayak et al. also reported that if tomato plants are subjected to salt stress and are inoculated with the bacteria Achromobacter piechaudii, this endophyte expresses ACC deaminase and reduces ethylene production in the plant which helps the plant to survive by alleviating the stress conditions (Mayak et al. 2004). Another class of endophytic bacteria that has grabbed attention of the researchers is Bacillus subtilis. This endophyte is known to synthesize organic compounds that induce systemic tolerance to the host plant under saline stress conditions (Ryu et al. 2004). Bacillus has also been reported to reduce the effect of osmotic stress in pepper plant (Jung et al. 2003). In a study conducted by Hamdia et al. in maize plant inoculated with Azospirillum bacteria under saline stress, it was reported that the bacterial species helps the plant to mitigate salt stress by altering the K+/Na+ ratio by selective influx and efflux of sodium, potassium, and calcium along with other changes such as increased chlorophyll content and a reduced proline content (Abd El-Samad Hamdia et al. 2004). Similarly, Azospirillum has been observed to alleviate salt stress-associated phenotype in barley; another study reports that upon inoculation of wheat seedlings with genetically engineered strain of Azospirillum lipoferum, the effects of salt stress were significantly reduced and similar results were also observed in chick pea plants (Bacilio et al. 2004; Siddiqui 2006; Zawoznik et al. 2011). Another endophytic bacteria, Piriformospora indica was also reported to provide tolerance to barley plant against salinity stress (Baltruschat et al. 2008). A whole diversity of microbes inhabit the rhizosphere but a few of them begin to occupy the internal parts of the plant, in the root tissue. As soon as they begin to colonize, they reach for the vascular system, mainly xylem. This leads to classification of such microbes into four different groups, namely associative microbes, neutralistic microbes, pathogenic microbes, and symbiotic microbes. The ones that seem to be of any advantage to the host plant are classified as endophytes (Hayat et al. 2010; Rosenblueth and Martínez-Romero 2004). Some plant growth promoting bacteria such as Acinetobacter lwoffii, Azospirillum brasilense, Bacillus pumilus, Chryseobacterium balustinum, Paenibacillus alvei, Pseudomonas fluorescens, Pseudomonas putida, and Serratia marcescens are known for their root colonization and active participation in alleviating abiotic stresses to protect agricultural crops (vegetables and fruits) and trees (Van Loon 2007). Apart from root colonization, other modes of bacterial entry into the host plant include lenticles, stomata, wounded sites in leaves or other parts, secondary roots, and through xylem vessels developing in a germinating seed. These endophytes have been reported to be present inside the seed endosperm, and upon germination they are released in the soil and then they enter the other parts of the developing plant. This ensures that the endophytes are transferred from one generation to the next. It can thus be stated here that plants have developed an intelligent system by co-evolving with the microbes to ensure their survival in different stress conditions. This coevolving strategy has also provided an opportunity for beneficial microbes to enter the host plant through localized multiple entry points and via other available routes throughout the plant body, depending upon whether the microbial community

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thrives in phyllosphere or rhizosphere (Johnston-Monje and Raizada 2011; Mano and Morisaki 2008; Strobel et al. 2004). Since plants and microbes have coevolved in an interdependent manner, plants also play a vital role in attracting beneficial bacterial species and aiding in their colonization. The organic compounds such as flavonoids produced by plants act as chemo-attractants for bacteria to invite them for colonization (Firdous et al. 2019). Plants that are capable of growing in low phosphorus challenged environments are known to synthesize malic acid to attract the microbes for endophytism (Piñeros et al. 2002). It is not just the plants that need endophytic microbes for better chances of survival in stress conditions, but microbes also need a habitable environment. Thus, microbes also release lipopolysaccharides that are identified by the plant metabolite products, such as isoflavanoids to establish an endophytic relationship between the two species (Chang et al. 2009; Surette et al. 2003). Microbes use different mechanisms to colonize the host plants, a few examples are the use of Major Outer Membrane Protein (MOMP) by Azospirillum brasilense to colonize germinating seeds of cereal plants; Pseudomonas is known to use type IV pili; Azoarcus uses type IV pili and the twitching motility, and P. flourescens uses a secretion system known as the SSIII system (Dulla et al. 2012; Lugtenberg and Kamilova 2009; Preston 2007; Reinhold-Hurek et al. 2006). This suggests that endophytes and the host plants have reached to a stage of mutualism during their course of co-dependent evolution where plants provide multiple habitable sites inside the plant body with multiple entry points and the endophytes use various systems to colonize them and in return provide tolerance against various abiotic stress conditions and a promise for a sustained life cycle.

2.3

Endophytes and the Molecular Defence Response

Endophytes play a major role in protecting plants from a variety of abiotic stresses. The bacterial species contribute either directly or indirectly to mitigate the stress and help plants to grow despite challenging conditions. The direct methods usually include the synthesis of various molecules by bacteria to contribute for molecular processes such as better uptake of micronutrients from the soil, better nitrogen fixation in case of leguminous plants, triggering the plant cells to synthesize phytohormones, such as ethylene and other stress hormones. Indirect methods on the other hand include the synthesis of extracellular compounds to fight against the pathogenic organisms and the production of hydrogen cyanide (HCN) in roots. Similarly, the vesicular-arbuscular mycorrhizal (VAM) fungi help the plant to sustain good health under different abiotic stress conditions. They remain attached to the plant with the help of very fine hyphae and help the plants by increasing the surface area for better absorption of nutrients from the soil. The microbes also interact with the plant machinery by either triggering the biosynthesis of metabolites or by inhibiting the process of their degradation. They also increase the expression

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level of defence-specific proteins such as pathogenesis-related proteins (Braud et al. 2009; Hayat et al. 2010; Meena et al. 2017). The contribution of a diverse group of microbes has been reported in mitigating different abiotic stresses (Table 2.1). The genera to which some of these endophytic bacteria belong include Azospirillum (Omar et al. 2009), Azotobacter (Sahoo et al. 2014), Bacillus (Sorty et al. 2016), Bradyrhizobium (Tittabutr et al. 2013), Burkholderia (Oliveira et al. 2009), Enterobacter (Grichko and Glick 2001), and Methylobacterium (Meena et al. 2012). The role of the endophytic fungi Trichoderma harzianum has also been reported in rice and Indian mustard. The rice plant showed excellent results upon inoculation with the T. harzianum against drought stress by the upregulation of the genes that code for aquaporin and dehydrin. Other physiological changes such as increased chlorophyll content and enhanced superoxide dismutase (SOD) activity against reactive oxygen species (ROS) were also reported alongside changes in the level of proline and malondialdehyde. It has also been reported that Trichoderma also imparts the ability to fight abiotic stress by upregulating the expression of the enzyme monodehydroascorbate reductase (MDHAR) by catalyzing the reaction of monodehydroascorbate reduction and thus regenerating ascorbic acid using electrons from the electron donor species such as NADH and NADPH (Ahmad et al. 2015; Brotman et al. 2013; Pandey et al. 2016; Park et al. 2016). Similarly, the bacterial species from the genera Acinetobacter and Pseudomonas have been reported to enhance the ability of the oat and barley plants from the poaceae family to thrive in salt stress conditions by upregulating the expression of the enzyme ACC (1-aminocyclopropane-1-carboxylate)-deaminase which reduced the production of ethylene and catalysed the conversion of ACC to α-Ketobutyrate and ammonia (Chang et al. 2014). Likewise, the Pseudomonas bacterial sp. produces the phytohormone Indoleacetic acid (IAA), and it has been reported to help grow the roots of the plant for better reach to the ground water in saline stress conditions (Meena et al. 2017). The mechanism of salt and other abiotic stresses mitigation varies depending upon the colonizing endophytic microbes. The molecular mechanism for salt stress includes the role of many molecular players, such as, phytohormones, metabolites and other molecular chaperones. In a study, it was reported that in the rice plant, the endophytic bacteria Bacillus amyloliquifaciens, strain SN13 alters the gene expression of 14 different genes, out of which BADH, EREBP, NADP-Me2, SERK1, and SOSI were reported to be upregulated whereas the genes GIG and SAPK4 were reported to be downregulated (Nautiyal et al. 2013). Similarly, in groundnut (Arachis hypogaea L.), the bacterial species Brachybacterium saurashtrense (strain JG-06) is known to alter the ratio of K+/Na+ and increase the uptake of Ca2+ and other nutrients such as phosphorus and nitrogen. The bacterial interaction with the plant system also leads to an increase in the concentration of auxin in roots as well as in shoots (Shukla et al. 2012). An exhaustive list of other microbial species conferring salt stress tolerance is provided in Table 2.1.

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Table 2.1 Microbe-mediated abiotic stress response Stress condition Drought

Microbial species A. lipoferum

Host plant Maize

Drought

Bacillus thuringiensis

Levandula dentata

Drought

P. libanensis and P. reactans

Brassica oxyrrhina

Drought

Azospirillum

Lettuce

Salt

Azospirillum and Pseudomonas

Wheat

Salt

Enterobacter and Pseudomonas

Maize

Salt

B. pumilus and P. pseudoalcaligenes

Rice

Salt

Azospirillum

Lettuce seeds

Mechanism of action Affect dry and wet weight of root and shoot; elevated accumulation of proline, free amino acids, and sugars Decrease in the activity of the enzymes ascorbate peroxidase and glutathione reductase; increased proline content Increase in pigment content and plant growth and a decrease in malondialdehyde and proline in leaves Increased content of ascorbic acid, increased chlorophyll and aerial biomass, increased antioxidant capacity, and a decreased browning intensity Accumulation of proline and soluble sugars and inorganic ions to maintain osmotic balance Increased uptake of nitrogen, phosphorus, and potassium; high ratio of Na-K ions; triple response is reduced Reduction in SOD activity and lipid peroxidation; reduction in ROS toxicity Reduced browning, increased antioxidant capacity and ascorbic acid content

Reference Bano et al. (2013)

Armada et al. (2014)

Kumar and Verma (2018)

Fasciglione et al. (2015)

Kumar and Verma (2018)

Nadeem et al. (2009)

Jha and Subramanian (2014) Fasciglione et al. (2015)

(continued)

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Table 2.1 (continued) Stress condition Salt

Microbial species P. putida

Host plant Canola and maize

Cd, Zn and Cu

Bacillus pumilus

Sedum

Cd, Pb and Zn

Enterobacter

Polygonum pubescens

Zn

P. brassicacearum and Rhizobium leguminosarum

Brassica juncia

Cd

Exophiala pisciphila

Zea mays

Chilling

B. phytofirmans

Grapevine

High temperature and drought

Bacillus and Paenibacillus sp.

Grapevine

Mechanism of action Modulation of ACC deaminase activity and alteration of the host plant differential protein expression Production of ACC deaminase, indoleacetic acid, and siderophores; increased phosphorus solubilization Improved phytoremediation; production of ACC deaminase, indoleacetic acid, and siderophore production Microbe-assisted phytoremediation, toxicity attenuation, and metal chelation Downregulation of ZIP, upregulation of PCS and MTP genes Increased expression of defence and trehalose related genes; increased accumulation of phenolics, malondialdehyde, and proline Increased anthocyanin content and elevated ABA metabolism

Reference Cheng et al. (2012)

Ma et al. (2015)

Jing et al. (2014)

Adediran et al. (2015)

Wang et al. (2016) Ait Barka et al. (2006)

Pacifico et al. (2019)

ZIP ZRT/IRT-like protein, PCS phytochelatin synthase, MTP metal tolerance protein, SOD superoxide dismutase, ROS reactive oxygen species

Apart from salt stress, many microbial communities interact with the plant systems to alleviate other stress conditions such as drought and heavy metal stress. A few examples of drought stress mitigation include bacterial species of Bacillus with its role highlighted in the maize (Zea mays) plant from the family Poaceae. The molecular mechanism involves the accumulation of free amino acids and proline and

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a few sugars. The decrease in the overall outflow of electrolytes has also been reported along with the alteration in the role of enzymes such as glutathione peroxidase and catalase, belonging to the class of antioxidant enzymes (Vardharajula et al. 2011). Another bacterial species, Pseudomonas putida, strain H-2-3, has been reported to modify the ratio of abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA) whereby ABA and SA levels are reduced, and the JA levels are elevated, thus suggesting an involvement of JA-mediated molecular defence signalling pathway as the line of response against drought in soybean. Kang et al., also reported a decrease in the levels of flavonoids and the enzyme superoxide dismutase (SOD) (Kang et al. 2014). Heavy metal stress is also a very concerning problem that inhibits plant growth and poses a major problem to global food security, as it contributes to soil pollution, making the land unsuitable for farming. Methods such as phytoremediation and bioremediation have long been used as techniques of choice of remedy against heavy metal soil pollution. Endophytic bacteria have been proved to play a vital role in overcoming such stress conditions while promoting plant growth. The molecular mechanism of their action involves enzymatic detoxification, alteration of efflux rates, metal complex formation, volatilization, and impermeability to a number of metals present in the soil (Meena et al. 2017). A few examples include the Bacillus thuringiensis strain GDB-1 from the plant Alnus firma where these microbes aid in the removal of metals such as Arsenic, Cadmium, Copper, Lead, Nickel, and Zinc. The mechanism involves various changes in the host plant such as increase in biomass, increase in nodule numbers, and an increased chlorophyll content. Along with these changes, the ratio of phytohormone productions is modulated, siderophores or iron-chelating compounds are formed that increase the influx of iron. Other changes include solubilization of phosphorus and the synthesis of ACC deaminase to promote plant growth (Babu et al. 2013). Yet another species of Bacillus, i.e., B. pumilus, strain E2S2, is known to promote plant growth in the plant Sedum by alleviating stress against Copper, Cadmium, and Zinc metals. The mechanism of action is similar to that of B. thuringiensis with an addition of synthesis of IAA and an increased metal uptake (Ma et al. 2015).

2.4

The Role of Multi-Omics: A Better Understanding of Molecular Interactions

In the post-genomic era, a lot of data has been generated for a better understanding of how endophytes interact within the host plant system to help it ease in its growth despite challenging abiotic factors. These abiotic factors, such as scarcity or excess of water, high temperature, saline and acidic conditions, and contamination of the environment with pollutants such as heavy metal, work in an intricate network,

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Fig. 2.1 Use of multi-omics techniques to discover new knowledge about the role of endophyte bacteria in alleviating different stresses faced by the host plant system

interacting with multiple systems at the same time. Thus, it is important to have a systems view to understand these complex interactions. Omic techniques, such as genomics, transcriptomics, proteomics, metabolomics, interactomics, phosphoproteomics, glycomics, and their meta counterparts have provided researchers with a completely new perspective to approach the same biological question, but with new insights (Figs. 2.1 and 2.2).

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Fig. 2.2 Interaction network of the protein NP2 in Arabidopsis thaliana. Source: STRING database. The circles represent nodes and the lines joining the nodes are edges representing interaction between the nodes. A few nodes are annotated with structural information. The nodes are labelled with their respective protein names

2.5

Genomics and Metagenomics

The next-generation sequencing has provided an edge to look for very interesting data objects that the genome stores. The coding region of the genome is highly conserved and so are the non-coding regions but there is a high degree of variability in the genomes of various species of endophytic bacteria and their host plant genomes. The core genome of all the endophytes have the same gene sets that might have played a crucial role in their co-evolution with their hosts, whereas the accessory genome would have genes specific to each species. Till date, 41 endophytic bacterial genomes have been sequenced and the number is increasing (De Maayer et al. 2014; Kaul et al. 2016; Medini et al. 2005). The genomic sequencing of endophytes has suggested that they contain genes that code for stress alleviating enzymes such as ACC deaminase; for phytohormones such as Indoleacetic acid (IAA) or Salicylic acid (SA); for mineral acquisition and stress tolerance; for their adhesion and colonization to the host, etc. (Firrincieli et al.

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2015; Kaul et al. 2016; Martínez-García et al. 2015). Moreover, single nucleotide polymorphism (SNP) genotyping has also aided in better understanding the variation at single nucleotide resolution. The use of biomarkers and mapping techniques has helped to understand the genome structure and organization in endophytes and the host plants (Meena et al. 2017). More investigation is required to better understand what other novel genes are present in the colonizing bacteria and how their interaction with the host machinery could help to get a better picture of these complex interactions. A suggestive approach could involve using comparative multigenomics, where the genome for one species could be compared with multiple other species to have a better idea of how divergent the genomes are and in what capacity does their organization help these specific bacterial types to promote plant growth in challenging stress conditions (Kaul et al. 2016; Meena et al. 2017). Metagenomics on the other hand helps to understand the community composition of the endophytes. The bacterial strains that cannot be cultured in a lab, no longer pose any limitation (Dinsdale et al. 2008). High throughput sequencing of the microbiome has been very helpful to decipher the gene content of the community and how the interactions between the microbes and the plants has evolved over time to overcome various abiotic stresses. An approach involving comparative metagenomics might be helpful to throw some light on the question of why different endophytic species colonize the same or different host plant depending upon the stress conditions (Akinsanya et al. 2015). A study involving the use of metagenomic approach in rice was reported with the outcomes that endophytes participate in various metabolic processes inside the plant roots and help the plant to grow despite the stress and the microbial community also helps in bioremediation (Sessitsch et al. 2012). Thus, in conclusion, more investigative studies involving a diversity of plants under different stresses in multiple environmental conditions could shed some light on how and why the microbiome genomes are under evolution to better suit the needs of its own community and for their host plant community as well.

2.6

Transcriptomics and Meta-transcriptomics

Techniques such as RNA-sequencing, RT-PCR, qRT-PCR, northern blotting, microarray and mi-RNA sequencing have greatly advanced our understanding of how expression studies provide better insight into the functioning of endophytes in stress mitigation. Other techniques such as ATAC-seq and CLIP-seq when integrated with orthogonal data suggests more about how various factors interact together to shape differential gene expression. The use of these techniques shed light on what set of endophytic genes are up and down regulated in response to a specific abiotic stress, which genes in the host plant in response to differential gene expression in endophytes alter their expression pattern, what proteins interact with these mRNAs, where do they bind, whether differential expression is spatially and temporally separated or it occurs globally and how microRNAs regulate the gene expression are a few questions that seek answers (Kaul et al. 2016; Meena et al. 2017). Metatranscriptomic studies help to understand the global expression pattern

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of a microbial community providing tolerance to the host plant. Comparative metatranscriptomic studies from soybean has been reported to suggest which endophytes colonize the plant, whether they are pathogenic or are free living and if others are in a symbiotic relationship or not (Molina et al. 2012). Defez et al. conducted an RNA-seq study on Sinorhizobium meliloti (a gram-negative bacteria) and they reported in their finding that the expression pattern of various genes leads to the overproduction of IAA and promotes plant growth (Defez et al. 2016). In another study conducted by Alavi et al., they reported a novel plant growth regulator, named spermidine in the rapeseed plant with Stenotrophomonas rhizophila as its endophytic bacterial counterpart (Alavi et al. 2013). Many miRNA expressions studies have also suggested their role in abiotic stress alleviation in plants such as Arabidopsis and Medicago. Apart from their role in stress signalling, these small RNA species also regulate developmental processes, transcription factors and other processes (Meena et al. 2017; Trindade et al. 2010). Therefore, transcriptomic studies integrated with genomic studies could suggest new insights to understand how interaction of endophytes with the host plant and how variable gene expression aid in this complex phenomenon to work in an orchestrated manner.

2.7

Proteomics and Meta-proteomics

The study of all the proteins in a sample at any given time is referred to as its proteome. Similarly, the entire protein content of a community at any given time existing in a specific environmental condition is known as the metaproteome (Wilkins et al. 1996). Single cell transcriptomics has already been achieved but single cell proteomics is a field rapidly developing. Protein extraction is followed by purification using 2D gel-electrophoresis which is further studied using Mass Spectrometry (MS) to identify the differentially expressed proteins in the meta-sample. A number of bioinformatics tools have been developed to get a better picture of these differentially expressed proteins, providing a platform for a better understanding of endophyte–plant interaction under different stress conditions (Kaul et al. 2016). A study conducted by Lery et al. with sugarcane as host plant reported the interaction between 78 differentially expressed proteins with Gluconacetobacter endophyte. Though little is known about its roles, but it has been suggested that this bacterial species helps in nitrogen fixation and supports plant growth (Lery et al. 2011). A number of studies have been conducted to understand what differentially expressed proteins in lab conditions with induced stresses help the plants to thrive in such challenging conditions but unfortunately, they do not mention much participation from their endophytic microbiomes. According to recent research avenues discovered in this domain, a lot of emphasis has been laid on understanding how halobacteria participate in alleviating the stress (Meena et al. 2017). Another class of bacteria, Pseudomonas is also under the spotlight of high throughput research. It has been identified for its role in alleviating stress in plants by secretion of plant growth promoting factors and by siderophore production (Meena et al. 2017). But more

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research is indeed required for understanding how these complex interactions work synergistically to curb the effects of various stresses. A limitation of proteomic studies is the sample preparation as during protein extraction, a lot of other non-protein substances also get extracted. Thus, we are still very far from reaching the full potential of exploiting proteomics and metaproteomics.

2.8

Interactomics and Metabolomics

Interactomics is the evaluation of molecular interactions in a system, such as protein–protein interactions, protein–metabolite interactions, and genetic interactions. An interaction network consists of nodes that represent each molecule with an associated annotation, including the structure of the molecule, the chemical activity or protein function, their known physical interactions and other similar information, and the edges that represent the interactions. In a network, these interactions come from experimental data, text mining, or database annotations. The following network depicts the interactions of the protein NP2 in Arabidopsis thaliana. The nodes are the proteins with which the NP2 protein has physical interactions, and the edges represent interactions predicted by experiments and text mining. So far, it has been possible to determine such interactions in the host plant machinery and in the endophytes but are not well characterized in the microbe– plant interactions. If in the near future these interactions can be deciphered then it will provide us with a voluminous amount of data to understand how these interactions work. Data from microbe–microbe and plant–microbe interactions could assist in understanding the role of co-expressed proteins and metabolites in both the microbial community and the plant to decipher how the association of these different organisms help each other to survive environmental stress. Metabolomics on the other hand, is an umbrella term that is used to study the metabolites, such as phenols (phenolomics), terpenes (terpinomics), and volatile organic compounds (VOCs—Volatomics), by characterizing them and investigating their participation in various metabolic pathways. Metabolome of an endophyte and that of a host plant are subject to changes due to the dynamic nature of the environment. A study conducted by Sorty et al. reported the production of Indole acetic acid (IAA) under saline stress by a number of microbes colonizing the plant Psoralea corylifolia L (Sorty et al. 2016). Likewise, the bacterial species of Bacillus have also been reported to increase phosphate solubilization in the fennel plants under salt stress (Mishra et al. 2016). Another class of bacteria, Nocardia is known to metabolize a number of organic molecules, such as ortho- and para-xylene, n-butylbenzene, and p-isopropyltoulene (Meena et al. 2017). Therefore, it is of utmost importance now to understand what metabolites are produced in the endophyte–plant system under different stress conditions and how they interact to develop a better understanding of the systems biology of the plant–microbe interactions.

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Integration of Multi-omics and Discovery of New Data

It is evident that it is important to integrate multi-omics data (genomics, transcriptomics, proteomics, interactomics, metabolomics) both from the endophytic microbiome and the host plant system to better understand how these multi-partite interactions give rise to a system that is able to survive and thrive in stressful environmental conditions. Bioinformatics expertise is required to identify novel genetic variants in the plant–microbe system and how they can be integrated with more data coming from kinomics, phosphoproteomics, proteogenomics, Riboomics, etc. New avenues can be discovered by applying techniques such as data mining to identify novel interactions in the system. These can further be integrated with Gene Ontology (GO) annotations, pathway annotations, and the reaction networks by using resources such as KEGG and Reactome databases. As the integration of multi-omics would be realized, the intricate biological questions of complex systems would become more clearer, and this will help us develop new ideas of creating genetically modified and high-yielding plants that could live in a symbiotic association with a great diversity of plants and can help to eradicate the global food shortage by thriving in non-habitable conditions as well.

2.10

Conclusion

Plants and endophytic bacteria live in a complex and an intricate manner where they complement each other to sustain in harsh environmental conditions. Great diversity of these microbes has co-evolved with plants to survive under various stresses that the whole host system is subjected to everyday. Intricate molecular defence strategies against these stresses have evolved over a course of time, and it is believed that they will continue to evolve. Data generation, validation, and its meaningful interpretation is required to understand systems biology of the host–microbe interactions. Therefore, this domain of research has great potential to offer in future and solve many new challenges faced by the agricultural domain due to unforeseeable factors that harm the crop fields.

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Diversity and Bioactive Potential of Endophytic Bacteria from High-Value Medicinal Plants Namita Ashish Singh and Rahul Jain

Abstract

Similar to human microbiome, plants are also colonized by a large number of tiny microorganisms. Plant endophytic bacteria colonize the internal tissues of plants without causing apparent diseases or showing any symptoms to their host. Endophytes that constitute the plant endobiome serve various functions from plant growth promotion and stress tolerance to the modulation of the plant’s chemistry. Medicinal plants are the inherent source for the production of a large number of bioactive phytochemicals, but with recent understanding on plant– microbe interactions, it has been observed that a significant number of these metabolites result from the contribution of associated microbial partners. Therefore, endophytes, particularly from medicinal plants whose microbiome remains largely unknown, have extended immense interest of scientific community in terms of bio-prospection for bioactive metabolites such as novel antimicrobial compounds, pharmacologically relevant drugs, and enzymes with novel properties. Majority of these bacterial endophytes producing bioactive metabolites belong to the Gram positive, high G + C, actinobacteria. For instance, endophytic Streptomyces spp. produce numerous novel antibiotics active against multi-drug-resistant bacteria. This chapter gives a comprehensive summary of the diversity of endophytic bacteria that colonize the internal tissues of various medicinal plants and their prospection for bioactive compounds. The chapter also describes the role of such compounds in agriculture, food, environment, and medicines. Possible future aspects of plant microbiome study in context to

N. A. Singh Department of Microbiology, Mohanlal Sukhadia University, Udaipur, Rajasthan, India R. Jain (*) Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. K. Singh et al. (eds.), Bacterial Endophytes for Sustainable Agriculture and Environmental Management, https://doi.org/10.1007/978-981-16-4497-9_3

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medicinal plants for better understanding the association of bacterial endophytes are also discussed. Keywords

Endophytes · Diversity · Bioactive compounds · Medicinal plants · Multi-omics

3.1

Introduction

Plant endophytes are now recognized as the integral component of biological diversity. Bacterial endophytes are widespread colonizers in the internal tissues including roots, leaves, and stem of all plants. Several crucial functions in the growth and development of a plant are mediated by the activities of endophytic microbes. High-value medicinal plants are no exception in being colonized by endophytic bacteria. The colonizers inside medicinal plants not only enhance plant growth but also stimulate biotransformation for the production of valuable metabolites (Hassan 2017; Song et al. 2017; Etalo et al. 2018). Therefore, analyzing the microbial diversity, composition, and function of medicinal plant-associated endophytes might be a sustainable resource for promoting growth of such plants and to enhance production of valuable secondary metabolites. Culture-dependent and metagenomics-based culture-independent methods have been employed for studying the plant-associated endophytes. Culture-dependent methods have advantage over culture-independent metagenomics approach, as the microorganisms can be isolated and examined for their beneficial effects on host plants. However, these methods are insignificant when dealing with the overall diversity and composition of colonizers in a plant tissue. This limitation arises from our ability to culture only 5%. Furthermore, authors reported maximum total species richness in leaf and minimum in stem. Similarly, Ma et al. (2013) elucidated phylogenetic diversity of bacterial endophytes with antagonistic characters from Panax notoginseng. Corresponding to most of the endophytic studies, Bacillus spp. (93.1%) from phyla Firmicutes was the most abundant genera. Thymus vulgaris and T. citriodorus (Family: Lamiaceae) are two related aromatic thyme species which differ in relation to composition of essential oil (Salehi et al. 2019). Study on endophytic microbiota of both thyme species revealed dominance of Gammaproteobacteria (Pseudomonas) in culturable diversity. Moreover, based on relative abundance, Pseudomonas was suggested responsible for

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most of the differences existing in essential oil composition between T. vulgaris and T. citriodorus as well as in leaf and roots (Checcucci et al. 2017). Plectranthus tenuiflorus (Family: Euphorbiaceae) known as ‘Shara’ grows naturally in highaltitude mountains of Taif province in Southwest of Saudi Arabia. It is one of the medically important plants used for earache and inflammation of middle ear, curing sore throat, etc. (Rahman et al. 2004). El-Deeb et al. (2013) isolated endophytic bacteria from different plant parts of P. tenuiflorus plant. Leaves were reported to possess maximum culturable bacteria followed by stem and root. A total of 28 endophytic isolates were assigned to 8 bacterial species including 4 Bacillus spp., and one each of Micrococcus luteus, Paenibacillus sp., Pseudomonas sp., and Acinetobacter calcoaceticus. These endophytic isolates also produced extracellular enzymes and showed antimicrobial activities. Similarly, a sum of 170 culturable endophytic bacteria belonging to 3 phyla were isolated from Ferula songorica, a medicinal plant of Apiaceae family (Liu et al. 2016). Traditionally, this plant is utilized in treating digestive disorders, rheumatism, headache, dizziness, toothache, etc. (Sun et al. 2013). The phylum Actinobacteria dominated as endophytes, followed by Proteobacteria and Firmicutes. Roots showed highest endophytic colonization than leaf and stem. Endophytic actinobacteria associated with medicinal plants have also been of research interest to the scientific community due to their numerous applications. For instance, Qin et al. (2012) revealed abundance and diversity of endophytic high G + C, gram positive actinobacteria associated with the medicinal plant Maytenus austroyunnanensis (Family: Celastraceae). Use of 10 different culture media and different culture methods resulted in isolation of 312 actinobacteria belonging to 21 different genera and >40 species. Streptomyces was the most abundant actinobacteria with at least 15 species. Majority of these endophytic actinobacteria were isolated from roots, followed by leaves and stems. Root being the major entry point for endophytes and enrichment of nutrients in rhizosphere, harbors maximum endophytic diversity as reported in various other studies too (Verma et al. 2009; Sun et al. 2013). The higher abundance of Streptomyces spp. in various medicinal plants and herbs has also been reported by Kim et al. (2012), Machavariani et al. (2014) and Passari et al. (2015). In contrast, higher abundance of actinobacteria other than Streptomyces spp. has also been reported. For example, Gynura cusimbua (Family: Asteraceae) is a medicinal plant with preventive effects for high blood pressure, coronary heart disease, Alzheimer’s disease, atherosclerosis, etc. Out of 63 clones of actinobacteria isolated from this plant, 59 strains belonged to Microbacterium, Arthrobacter, Micrococcus, Curtobacterium, Okibacterium, Quadrisphaera, and Kineococcus (Zhang et al. 2016). Furthermore, these reports on endophytic actinobacteria from medicinal plants also provide clues to explore these niches as source for potential novel actinobacterial species with new metabolic abilities (Li et al. 2017; Jiang et al. 2018). Conclusively, it is suggested that endophytic bacterial communities among different plants and tissues are influenced by the variations in origin of plant, cultivar, plant age, presence of active constituents such as essential oils in different plant tissues, environmental factors, and isolation strategies (Islam et al. 2010; Tan et al.

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2016; Checcucci et al. 2017; Khan Chowdhury et al. 2017). Bacterial adaptation against plants’ active constituents and metabolites specifically compounds with antimicrobial properties makes them a successor to localize into different plant tissues. Care should be taken when isolating bacteria from different plant tissues to maximize the recovery of cultivable bacteria as certain endophytic microbiota tend to be lost during tissue processing itself. Further, duration from sample collection to isolation should be minimized for effective isolation of bacterial endophytes.

3.2.2

Diversity Based on Culture-Independent Methods

With the progression of high-throughput next generation sequencing, culture independent metagenomic approach has become a choice to understand the plant microbial diversity. Metagenomics is the study of collective genome of microorganisms from an environmental sample, which provides information on the microbial diversity and ecology of a specific environment such as a plant. 16S amplicon sequencing as well as shotgun metagenome sequencing of plant tissue can be performed on different sequencing platforms such as Illumina, PacBio, and more recently Oxford Nanopore. Hypervariable regions (V1–V9) in the 16S rRNA gene are sequenced utilizing high-throughput gene sequencing. V3, V4, and V5 regions in 16S rRNA gene are reported to be the most suitable regions for metabarcoding, but metagenomics based on other domains including V2 and V7 are also reported (Bukin et al. 2019; Lucaciu et al. 2019). V4 domain, targeted by the primer pair 515F/806R, was recommended by the Earth Microbiome Project (Gilbert et al. 2014) and has been used in the plant-rhizosphere studies (Breidenbach et al. 2015). Environmental DNA sample from any plant tissue can be extracted to decipher the diversity, abundance, and community composition associated with it. However, several limitations of DNA extraction arise during plant microbiome studies due to the varying compositions of plants’ secondary metabolites, especially in medicinal plants, and difficulty in bacterial DNA recovery as large amount of DNA is extracted from the host plant. Therefore, an efficient method of DNA isolation with ability to recover maximum endophytic microbial DNA is a key prerequisite in such examinations (Corcoll et al. 2017). Due to these limitations, metagenomic studies on plants are scarce in comparison to other samples such as soil, sludge, water, and animal tissue. Nonetheless, attempts to understand plant microbiome have been increased utilizing 16S amplicon sequencing in last one decade. Culture-independent methods have advantages in terms of their efficacy to identify novel unculturable endophytic bacteria, which otherwise cannot be identified using traditional approaches (Raghavan et al. 2017). This provides us an opportunity to uncover yet to be isolated, identified, and characterized bacteria for their inherent beneficial functions and understand plant–microbe interactions. In Chinese herbal medicine, Coptidis rhizome is often used for detoxification, cure of various sicknesses such as high fever, diarrhoea, and heat burn and is also used as an active constituent of many proprietary Chinese pharmaceutical products (Wang et al. 2019a). Liu et al. (2020a) explored endophytic bacterial communities

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related to berberine content in different plant parts of wild-type and cultivated Coptis teeta (family: Ranunculaceae) by V3–V4 region analysis in 16S rRNA gene. Proteobacteria, Actinobacteria, and Bacteroidetes were the dominated phyla, and Mycobacterium, Salmonella, Nocardioides, Burkholderia-Paraburkholderia, and Rhizobium were the major genera in root, stem, and leaf tissues. In addition, berberine content in root was found positively correlated with Microbacterium. Purushotham et al. (2020) studied diversity and potential of endophytic bacteria in a primitive New Zealand medicinal plant Pseudowintera colorata (Family: Winteraceae). This medicinal plant possesses antimicrobial properties. Based on Illumina MiSeq analysis (V3–V4 region) it was conferred that tissue type strongly influenced the diversity and richness of endophytic bacteria in P. colorata. The relative abundance of Proteobacteria was high in all tissues (97.6%), followed by Actinobacteria (1.2%), Tenericutes (0.7%), Firmicutes (0.1%), Acidobacteria (0.1%), and Bacteroidetes (0.1%). At the genus level, Pseudomonas, Acinetobacter, Methylobacterium, Burkholderia, Actinomyces, and Frankia were some of the most common genera present. Further, Pseudomonas was identified as a member of core endomicrobiome due to occurrence of this genus in >75% of all P. colorata leaf, stem, and root samples. Moreover, authors also isolated and identified bioactive endophytic bacteria including Pseudomonas, Bacillus, Erwinia, and Pantoea. In another study, based on V1 hypervariable region of 16S rRNA genes, total bacterial diversity of Panax notginseng was reported to be grouped in 14 phyla with most abundant bacteria belonging to Proteobacteria (Dong et al. 2018). The relative abundances of Proteobacteria, Actinobacteria, Verrucomicrobia, Bacteroidetes, Acidobacteria, Firmicutes, Gemmatimonadetes, and Chloroflexi were 97.9%, 97.2%, 97.6%, 98.2%, and 97.7% in the samples from the flower, leaf, stem, root, and fibril, respectively. Fibril possessed the highest diversity of bacterial endophytes. Moreover, significantly higher abundances of Conexibacter, Gemmatimonas, Holophaga, Luteolibacter, Methylophilus, Prosthecobacter, and Solirubrobacter were observed in aboveground parts than in the underground parts, whereas the abundances of Bradyrhizobium, Novosphingobium, Phenylobacterium, Sphingobium, and Steroidobacter were noticeably lower in the aboveground parts. Illumina-based V5–V7 region analysis in Dendrobium classified bacterial diversity into 22 different phyla (Wang et al. 2019b). Proteobacteria (55.24%) followed by Actinobacteria (25.58%), Firmicutes (12.86%), and Bacteroidetes (5.46%) were the dominant phyla. It was reported that the overall composition of bacteria in each sample was similar but distribution of dominant genus in six different Dendrobium samples varied. Akinsanya et al. (2015b) performed V3–V4 region-based metagenomics analysis for studying endophytic diversity associated to A. vera plant. The analyses elucidated Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes as the dominant phyla. The roots possessed the most diverse bacterial community with 23% of bacteria absent in other tissues. Stem tissues showed dominance by Pseudomonas genus along with presence of genera (6%) such as Gluconacetobacter and Anoxybacillus which were not present in both the root and leaf tissues. Illuminabased endophytic diversity of tree Peony (Paeonia Sect. Moutan, family:

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Paeoniaceae) roots and leaves (Yang et al. 2017) and four Allium species (Family: Amaryllidaceae) (Huang 2019) utilizing V3–V4 region has also been reported. Peony tree harboring excellent ornamental and medicinal values as Chinese traditional plant showed five dominant phyla (Proteobacteria, Firmicutes, Bacteroidetes, Acidobacteria, and Actinobacteria) as endophytes. Prevalence of Pseudomonas and Enterobacter was higher in roots, whereas Succinivibrio and Acinetobacter dominated in leaves. Among four Allium species, Chinese leek showed dominance of Proteobacteria (42.68%) and Bacteroidetes (20.18%) phyla, while one unclassified (>70%) phyla dominated in the other three species. Similarly, Jin et al. (2014) also reported overall abundance of Proteobacteria (43.2%), Firmicutes (36.5%), and Actinobacteria (14.1%) as endophytes in Stellera chamaejasme (Family: Thymelaeaceae) on the basis of 16S rRNA gene clone library (499f/1492r primers). Bacteroidetes (2.1%), Chloroflexi (1.9%), and Cyanobacteria (1.7%) were found consistent but in lower abundance. In addition, when comparing different plant parts of S. chamaejasme, Proteobacteria were dominant in the roots, and Firmicutes were dominant in leaf and stems. Diversity of actinobacteria in a medicinal plant Maytenus ustroyunnanensis (Family: Celastraceae) in Xishuangbanna tropical rainforest was explored by Qin et al. (2012) by both culture-dependent and culture-independent methods. The medicinal plant is used for the extraction of maytansinoids antibiotic. The study included endophytic enrichment technique for constructing clone libraries of 16S rRNA gene. Following this approach, 84 distinct operational taxonomic units (OTUs) distributed among Actinomycetales and Acidimicrobiales orders, with a number of rare actinobacteria genera, were identified. Analysis at the genus level revealed prevalence of Brachybacterium, Streptomyces, and Friedmanniella, followed by Zhihengliuella, Microbacterium, and Catenuloplanes genera in the order Actinomycetales. Based on V3–V4 sequencing, Oberhofer et al. (2019) explored the diversity of actinobacteria in the endosphere of the native alpine medicinal herb Leontopodium nivale Subspecies alpinum (Family: Asteraceae) which is traditionally used against numerous illnesses. Authors identified 336 unique Actinobacterial OTUs across the 4 tissues examined including 292 in the root, 213 in the rhizome, 29 in the leaves, and 11 in the inflorescence. A descending diversity from roots to rhizomes, leaves, and inflorescences was observed. Moreover, this study identified some uncultured Actinobacteria specifically in belowground tissues, which provides opportunity for a targeted isolation strategy. In another study, Melia toosendan (Chinaberry; Family: Meliaceae) has been explored for its endophytic actinobacterial diversity utilizing culture-dependent and -independent methods (Zhao et al. 2018). Culture-based methods resulted in the identification of most isolates as Streptomyces spp., whereas amplicon sequencing (Actinobacteria-specific primers Com2xF and Ac1186R for amplifying 270 bp region of 16S rRNA gene) resulted in the identification of the most abundant OTU as Rhodococcus while Tomitella as the most diverse genus. With the advancements in sequencing technology and its decreased cost, researchers are attempting to profile overall microbial community present in a specific niche utilizing high-throughput shotgun sequencing. Whole genome

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metagenome is a recent approach which can not only study community composition and diversity of microbiota but can also provide information on the novel genes which are yet to be discovered from unculturable bacteria and/or identify new functional genes relevant in plant–microbe interactions (de Souza et al. 2019; Regalado et al. 2019).

3.3

Applications of Endophytic bacteria

Bioactive compounds produced from endophytic bacteria have wide applications in agriculture, environment, food, pharmaceutics, and medicine. Endophytic colonizers have also been reported to produce certain metabolites of plant origin. Therefore, they provide tremendous opportunity for active exploration of novel bioactive compounds.

3.3.1

Pharmaceutical and Medical Applications

Endophytes of the ethnomedicinal plants are known as a good source of bioactive compounds (Nongkhlaw and Joshi 2015). These bioactive compounds are characterized into alkaloids, terpenoids, peptides, antibiotics, flavonoids, quinones, and phenols; (Fig. 3.2) and are listed in Table 3.1. These compounds have anticancer, antimicrobial, anti-inflammatory, and antioxidant activities (Korkina 2007).

Fig. 3.2 Bioactive compounds synthesized by endophytic bacteria

Bacillus cereus, Bacillus thuringiensis, Bacillus licheniformis Aranicola proteolyticus, Serratia liquefaciens Microbiospora sp. LGMB259

7

Streptomyces sp. TPA0556

Streptomyces sp. GT2002/1503

10

11

B. Terpenes and terpenoids 9 Streptomyces sp. TPA0456

8

6

5

Streptomyces sp. SUK10 Pseudomonas sp. Enterobacter sp. Lysinibacillus sp. Bacillus cereus Micromonospora violae

3 4

S. no. Endophytic bacteria/actinobacteria A. Alkaloids 1 Streptomyces sp. TP-A0595 2 Streptomyces sp. SUC1

Aucuba japonica Bruguiera gymnorrhiza

Aucuba japonica

Vochysia divergens

Pinellia ternate

Viola philippica

Miquelia dentata Bedd.

Allium tuberosum Ficus benjamina Shorea ovalis Pinellia ternate

Host plant

Antiviral

Antibacterial Anti-Candida Antimicrobial

Anti-bacterial

Anticancer Antiinflammmatory

Anticancer

Anti-cancer

Lansai A-D

Antifungal and anticancer Antiprotozoal Antitumor and antiviral

Xiamycin

Demethylnovobi-ocins

Diversity and Bioactive Potential of Endophytic Bacteria from High-Value. . . (continued)

Ding et al. (2010)

Igarashi (2004)

Sasaki et al. (2001)

Savi et al. (2015)

β-Carbolines and 3-indole compounds

Cedarmycin A and B

Liu et al. (2015)

Zhang et al. (2014)

Singh et al. (2013)

Tuntiwachwuttikul et al. (2008) Zin et al. (2017) Liu et al. (2010)

Sasaki et al. (2002)

References

7-Isoprenyl indole-3-carboxylic acid and 3-acetonylidene-7-prenylindolin-2one Guanosine and Inosine

Camptothecine

Diketopiperazine gancidin W Guanosine and inosine

6-Prenylindole

Active component

Antifungal

Therapeutic application

Table 3.1 Bioactive compounds produced by endophytic bacteria/actinobacteria and their therapeutic applications

3 55

Endophytic bacteria/actinobacteria Streptomyces sp. KIB 015

Streptomyces sp. BT01

Streptomyces sp. TC022

Verrucosispora maris AB-18-032

Micromonospora sp. PC1052

18

19

20

E. Quinones 21 Streptomyces sp. DSM 1175 22 Streptomyces Q21, Streptomyces MaB-QuH-8

Streptomyces sp. NRRL 30562

17

D. Peptides and their derivatives 15 Streptomyces sp. 16 Streptomyces sp. R-5

14

C. Flavanoids 13 Streptomyces sp. Tc052

S. no. 12

Table 3.1 (continued)

Alnus glutinosa Plants of Celestracae family

Monstera sp. Rhododendron sp. Kennedia nigricans Alpinia galangal Sonchus oleraceus Puereria candollei

Alpinia galangal Boesenbergia rotunda (L.)

Host plant Panax notoginseng

Antibacterial Antimycobacterial, antibacterial

Antimalarial, antibacterial Antifungal, antitumor Antibacterial, antitumor Antioxidant, antibacterial

Antimicrobial Antimicrobial

Antibacterial

Antioxidants

Therapeutic application Antimicrobial Anti-inflammatory

Alnumycin Celastramycins A and B

S-adenosyl-N-acetylhomocysteine

Proximicin

Actinomycin D

Munumbicins E-4 and E-5

Coronamycin Actinomycin X2

Kaempferol, Isoscutellarin, Umbelliferone and Cichoriin 7-Methoxy-3, 30 ,40 ,6-tetrahydroxyflavone and 20 ,7-Dihydroxy40 ,50 Dimethoxyisoflavone, Fisetin, Naringenin, 30 -Hydroxydaidzein, Xenognosin

Active component Labdanmycins A and B

Bieber et al. (1998) Pullen et al. (2002)

Boonsnongcheep et al. (2017)

Ezra et al. (2004) Shimizu et al. (2004) Castillo et al. (2006) Taechowisan et al. (2006) Roh et al. (2011)

Taechowisan et al. (2009) Taechowisan et al. (2014)

References Xiong et al. (2018)

56 N. A. Singh and R. Jain

Micromonospora lupini

24

F. Phenols 25 Dactylosporangium sp. strain SANK 61299

Streptomyces sp. CS

23

Cucubalus sp.

Maytenus hookeri Lupinus angustifolius Antifungal

Antitumor

Antitumor

Streptol

Lupinacidin C

Naphtomycin A

Okazaki (2003)

Igarashi et al. (2011)

Lu and Shen (2007)

3 Diversity and Bioactive Potential of Endophytic Bacteria from High-Value. . . 57

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For example, endophytic Paenibacillus spp. produce exo-polysaccharides having antioxidant and anti-tumor properties, while mutanase enzymes reduce tooth decay (Grady et al. 2016).

3.3.1.1 Alkaloids Alkaloids are the low molecular weight, organic compounds containing basic nitrogen atoms. Mostly alkaloids are derived from amines by the decarboxylation of various amino acids, namely histidine, lysine, ornithine, tryptophan, and tyrosine. Endophytic Bacillus cereus, Aranicola proteolyticus, Serratia liquefaciens, Bacillus thuringiensis, and Bacillus licheniformis isolated from Pinellia ternate produce alkaloids (guanosine and inosine) in fermentation broth (Liu et al. 2015). 3.3.1.2 Terpenoids Terpenes and terpenoids are derived biosynthetically from isoprene units and are the main constituents of the essential oils of many medicinal plants. Terpenoids have been widely used for fragrances in perfumery and medicines. Terpenoids are also produced from microbes, apart from plants (Tholl 2015; Zhou et al. 2015). Terpenes and terpenoids have been produced by several endophytic bacteria. For instance, Micrococcus sp. isolated from Catharanthus roseus has been reported to enhance in-planta content of vindoline, ajmalicine, and serpentine (Tiwari et al. 2013). Streptomyces spp. is the major producer of vast volatile terpenes used in various applications (Singh et al. 2017). 3.3.1.3 Flavonoids Flavonoids are polyphenolic compounds, widely found in fruits, vegetables, and some beverages. Flavonoids have antioxidant effects associated with various diseases such as cancer, Alzheimer’s disease, and atherosclerosis (Panche et al. 2016). 3.3.1.4 Peptides and Their Derivatives Microbes play a significant role in the production of peptide antibiotics. Endophytic actinobacteria produce some novel therapeutic peptides with antimicrobial, antioxidant, and antitumor activities. 3.3.1.5 Quinones Quinones are a group of secondary metabolites derived from the oxidation of hydroquinones and produced by various plants, fungi, and bacteria. Quinones show antibacterial, antioxidantal, neurological, antitumor, and anti-HIV activities (Villamil et al. 2004). 3.3.1.6 Phenols Polyphenolic compounds are helpful in improving human health due to their antioxidant, anti-carcinogenic, and antimicrobial activities. Polyphenols lower the risks of various chronic diseases, including cancer, cardiovascular diseases, and chronic inflammation. These phenolic compounds may be synthesized by the seven-step shikimate pathway (Valdes et al. 2015; Carvalho et al. 2016).

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3.3.1.7 Antibiotics Antibiotics are low molecular weight natural compounds which are active against other microorganisms. Antibiotics are produced by endophytic microorganisms; actinobacteria are known for the production of antibiotics. In actinobacteria, 80% of the total antibiotics is produced by Streptomyces sp. (Sathiyaseelan and Stella 2011). Antibiotics and drugs produced by some endophytic bacteria are listed in Table 3.2.

Table 3.2 Antibiotics and drugs produced by endophytic bacteria/actinobacteria S. no. 1

Endophytic bacteria/ actinobacteria Bacillus subtilis 168

Therapeutic application Antifungal

2

B. subtilis

3

B. thuringiensis

Antibacterial, Antiinflammatory Insecticidal

4 5 6

B. subtilis, B. amyloliquefaciens B. subtilis B. subtilis

Antifungal, hemolytic Antibacterial Antibacterial

Subtilin Bacteriocins

7 8

Pseudonocardia sp. Streptomyces sp.

Antimalarial Antimalarial

Artemisinin Coronamycin

9

S. caespitosus S. lavendulae Streptomyces sp. Hedaya 48 Streptomyces sp. NRRL 30562

Chemotherapeutic agent

Mytomycin C

Anti-dermatophyte

Saadamycin Munumbicins A, B, C and D

Monensin

10 11

Compounds Bacilysocin Amicoumacin b-exotoxin Bacillomycin

12

Streptomyces sp.

13

S. cinnamonensis

Antimicrobial, antimalarial, antitumor Treatment of breast cancers and tumors Prevent coccidiosis

14

S. albus DSM 40763

Immunotherapy

Strepturidin

15

S. scabies

Thaxtomin A

16

Streptomyces sp. MK932-CF8

Cellulose synthesis inhibitor Anti-prostate cancer

Doxorubicin

Androprostamines

References Tamehiro et al. (2002) Pinchuk et al. (2002) Espinasse et al. (2002) Aranda et al. (2005) Stein (2005) Sansinenea and Ortiz (2011) Li et al. (2012) Ezra et al. (2004) Danshiitsoodol et al. (2006) Gendy and Bondkly (2010) Castillo et al. (2002) Brayfield (2013) Lowicki and Nski (2013) Pesic et al. (2014) Francis et al. (2015) Yamazaki et al. (2015)

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Plant Growth Promotion Abilities

Endophytic bacteria are involved in plant growth promotion by different mechanisms including phosphate solubilization, indole acetic acid (IAA) production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, hydrogen cyanide (HCN), siderophore production, and nitrogen fixation listed in Table 3.3. Ullah et al. (2017) isolated bacterial endophytes from two medicinal plants namely Withania coagulans Dunal and Olea ferruginea Royal. Many of these bacteria were able to solubilize phosphate, produced IAA, ammonia as well as HCN, synthesized extracellular enzymes, and showed antagonistic activities against phytopathogenic fungi. Degrading corn root-associated Paenibacillus polymyxa CR1 can directly influence plant growth by producing IAA and other auxin phytohormones (Weselowski et al. 2016). The bacterium showed phosphate solubilization activity and can fix atmospheric nitrogen. The bacterium also displayed biocontrol activity against phytopathogens by triggering induced systemic resistance and producing a variety of biocidal substances. Shan et al. (2018) has reported plant growth promoting activities of endophytic actinomycetes from tea plants (Camellia sinensis; Family: Theaceae). IAA is the broadly known growth regulator from endophytic actinobacteria (Khamna et al. 2010). Bacterial IAA increases root surface area and subsequently provides the soil nutrients to plant. IAA production also relaxes the cell walls and increases the release of exudates simultaneously providing extra nutrients to support the growth of other helping bacteria of rhizosphere. Therefore, endophytic bacterial IAA plays a Table 3.3 Plant growth promotion activities of endophytic actinobacteria isolated from medicinal plants

S. no. 1.

2

3

4

Endophytic bacteria/ actinobacteria Enterobacter cloacae, E. dissolvens, E. hormaechei, Cronobacter sakazakii Streptomyces sp. AzR-051, AzR-049, AzR-010 Brevibacterium sp. S10S2, Microbacterium sp. S4S17 Bacillus sp. 3A8, Kocuria sp. R7S1, Janibacter sp. R4S4

Host Withania coagulans Dunal and Olea ferruginea royal Azadirachta indica A. Juss.

Tissue Root, stem, and leaves

Ferula sinkiangensis

Stem

Ferula sinkiangensis

Root

Root

Plant growth promotion activities IAA, HCN, Siderophore, ammonia production, hydrolytic enzymes, phosphate solubilization

References Ullah et al. (2017)

IAA and siderophore production and suppression of Alternaria alternata IAA and siderophore production, nitrogen fixation

Verma et al. (2011)

IAA and siderophore production, nitrogen fixation

Liu et al. (2017a, b)

Liu et al. (2017a, b)

3

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crucial role in plant–microbe interactions i.e. phytostimulation (Spaepen and Vanderleyden 2011). According to Passari et al. (2017) the majority of endophytic IAA producer actinomycetes from the traditional medicinal plant Rhynchotoechum ellipticum (Family: Gesneraceae) belong to genus Streptomyces. The maximum IAA production was observed in Streptomyces olivaceus strain BPSAC77 (52.3μg/mL). Pseudomonas, Bacillus, Erwinia, and Pantoea isolated from Pseudowintera colorata possessed inhibitory activity against phytopathogens and also promoted P. colorata seedling growth as compared to control (Purushotham et al. 2020). The endophyte Bacillus subtilis, isolated from Speranskia tuberculata (Family: Euphorbiaceae) was found antagonistic to the pathogen B. cinerea under in-vitro conditions (Wang et al. 2009). New strains of Burkholderia pyrrocinia JK-SH007 and B. cepacia were identified as effective biocontrol agents against poplar canker (Ren et al. 2011). Conclusively, endophytic microorganisms do not just act as effective biocontrol agents suppressing various phytopathogens but also provide nutrients required for plants’ optimal growth. However, identification of a consortium of such microbes rather than an individual microorganism remains to be a pre-requisite for achieving maximal plant growth.

3.4

-Omics in Plant–Microbe Interactions Study

Throughout the life cycle of plant, whether medicinal or an agricultural crop, every plant interacts with numerous microorganisms in its environment. In particular, medicinal plants, which have been categorized as endangered or threatened, need special attention to identify the associated microbial communities and their functions for making strategies to conserve these plants and propagate in large numbers. Omics approach has been increasingly used in plant microbiome studies (Großkinsky et al. 2018; Plett and Martin 2018; Liu et al. 2020a, b), which includes genomics, to study genes and their functions, transcriptomics, to quantify gene expression by measuring mRNA transcript level, proteomics, to quantify proteins and their expression, and metabolomics to study cellular metabolites. The high-throughput integration of multi -omics provide a way forward in elucidating critical endo-microbiome gene functions and gene products which can be used for improving plant growth. For example, integrating transcriptomics with proteomics or metabolomics can provide insights in to coordinated gene expression and their protein products or biosynthetic gene clusters producing secondary metabolites or bioactive compounds (Levy et al. 2018). Metabolomics is a powerful tool to study the cellular metabolites and can be integrated with metagenomics for studying the functions of small molecule metabolites associated with a specific microbial community in a plant. Combinatorial -omics approach has resulted in identification of gene responding to changes in plant metabolism due to drought (Xu et al. 2018), genes and protein involved in legume–rhizobia symbiosis (Delmotte et al. 2010), etc. However, there are several challenges that limit the use of such high-throughput techniques in plant-microbiome study. This may include specific sample preparation, limited public reference database, contamination from

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host plant limiting in-planta studies, limited technical expertise, and cost. Nevertheless, integration and analysis of the -omics data while overcoming such limitations can lead to great outcomes that can be significantly implemented in the fields.

3.5

Conclusion and Prospects

Considering the great potential of microbes in mediating plant health, it is confirmed that plant-associated microbes cannot be ignored and need active lookout. With the growing global population, a demand for the medicinal plants for their use as traditional medicine as well as for their bioactive compounds is increasing. Besides, overexploitation of medicinal plants for their commercial use has pushed several medicinal plants into the International Union for Conservation of Nature (IUCN) red list of threatened species. Utilization of plant microbiome (endo- and rhizosphere) and plant–microbe interactions could prove to be a useful strategy for the efficient conservation of medicinal plants as well as in enhancing high-valued bioactive compounds production. Novel culturomics strategies are needed to maximally characterize the plant microbiota for unleashing the full potential of plant microbiome. Further, omics strategies should be integrated into plant microbiome research to unravel the secrets of plant–microbe interactions. Acknowledgements The editors of “Bacterial Endophytes: Advances in Sustainable Agricultural and Environmental Management” are gratefully acknowledged for extending the invitation for sharing our views on one of the most promising research areas of plant–microbe interactions.

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Zhang X, Gao Z, Zhang M et al (2016) Analysis of endophytic actinobacteria species diversity in the stem of Gynura cusimbua by 16S rRNA gene clone library. Microbiology 85:379–385. https://doi.org/10.1134/S0026261716030176 Zhao K, Li J, Shen M (2018) Actinobacteria associated with Chinaberry tree are diverse and show antimicrobial activity. Sci Rep 8:11103. https://doi.org/10.1038/s41598-018-29442-2 Zhou JY, Yuan J, Li X et al (2015) Endophytic bacterium triggered reactive oxygen species directly increase oxygenous sesquiterpenoid content and diversity in Atractylodes lancea. Appl Environ Microbiol 82:1577–1585. https://doi.org/10.1128/AEM.03434-15 Zin NM, Baba MS, Zainal-Abidin AH (2017) Gancidin, W, a potential low-toxicity antimalarial agent isolated from an endophytic Streptomyces SUK10. Drug Des Devel Ther 11:351–363. https://doi.org/10.2147/DDDT.S121283

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Plant Growth Promoting Rhizobacteria (PGPR)-Assisted Phytoremediation of Contaminated Soils Garima Malik, Samira Chugh, Sunila Hooda, and Ritu Chaturvedi

Abstract

The unprecedented augment in the concentration of diverse contaminants in the environment has grim impacts on the ecological balance of our ecosystem. Soil, being a major sink, holds up the maximum load of environmental contaminants. Heavy metals and petroleum hydrocarbons are the most common pollutants present in the soil. Plant growth promoting rhizobacteria (PGPR)-assisted phytoremediation is one of the competent methods for removal of pollutants, which has proven its efficiency in reclamation of contaminated soils. PGPR are bacteria that reside in close association with plant roots and facilitate growth and development of plants by influencing their physiological and metabolic activity. Rhizobacteria are known to amplify the effectiveness of phytoremediation by modulating contaminants transportability and accessibility to the plant via acidification, chelating agents, solubilization of phosphate, and redox changes. This chapter aims to explore the role of rhizomicrobiome in the phytoremediation of heavy metal- and petroleum-contaminated soils, the successful commercialization of PGPR, and the insights into the recent advances in PGPR research.

Garima Malik, Samira Chugh, Sunila Hooda and Ritu Chaturvedi contributed equally with all other contributors. G. Malik (*) Raghunath Girls’ Post Graduate College, C.C.S. University, Meerut, Uttar Pradesh, India S. Chugh Gargi College, University of Delhi, New Delhi, India S. Hooda Ram Lal Anand College, University of Delhi, New Delhi, India R. Chaturvedi St. John’s College, Agra, Uttar Pradesh, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. K. Singh et al. (eds.), Bacterial Endophytes for Sustainable Agriculture and Environmental Management, https://doi.org/10.1007/978-981-16-4497-9_4

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Keywords

Abiotic stress · Heavy metals · Plant growth promoting rhizobacteria · Petroleum · Phytoremediation

4.1

Introduction

The persistence of heavy metals and petroleum hydrocarbons is one of the serious environmental concerns demanding attention of researchers throughout the world. The plants which can accumulate metal or tolerate metals stress have been used for phytoremediation in metal-polluted soils. Phytoremediation is the use of green plants for removal of both inorganic and organic pollutants from air, water, and soil (McCutcheon and Jorgensen 2008). The name phytoremediation is obtained from the Greek word phyto, i.e. plant, and the Latin word remedium, i.e. cure. Its usage in scientific literature for the first time has been traced to paper written by Cunningham and Berti in 1993 (Novo et al. 2018). Speight (2020) rightly states that the description of phytoremediation can be redefined to include the utilization of green plants and the allied microorganisms, along with appropriate soil amendments and agronomic methods to either contain, eliminate, or render lethal environmental pollutants undamaging. Many phytoremediation projects have been carried out worldwide to mitigate contaminants like pesticides, metals, crude oil, and explosives. The phytoremediation practice utilizes specific plants with roots that can absorb contaminants over time. Many plants such as hemp, mustard, pigweed, and alpine pennycress have the ability to hyper-accumulate contaminants from polluted sites (Speight 2020). Phytoremediation has immense potential as a natural, low cost, in situ approach driven by solar energy to moderately treat polluted sites spreading over large areas. However, the plants have to be cautiously selected depending on the type contaminants (Schwitzguébel 2017). The added advantage of phytoremediation over other technologies is that various kinds of nutrients, organic materials, and oxygen are supplemented to the soil through metabolic processes of plant and microbes. This enhances the value and consistency of remediated sites, stabilizes soil, and checks wind and water erosion as well (Schwitzguébel 2017). It can be concluded that this is an esthetically pleasing technology which helps in reducing erosion, increasing biodiversity, and fixing atmospheric carbon dioxide (Cunningham and Berti 1993). Phytoremediation techniques should, however, avoid the use of food crops for cleanup. The use of ornamental plants reduces the possibility of metals passing into the food chain along with the extra benefit of improving the environment’s esthetics and producing extra earnings, together with added job opportunities from cut-flower trading and/or travel industry (Nakbanpote et al. 2016). A survey of recent literature brings to light the numerous advantages of plant growth–promoting rhizobacteria (PGPR) to environment, agriculture, landscaping,

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etc. PGPRs are also known to have the potential to enhance phytoremediation processes (Jing et al. 2007). The application of PGPR for improving metal tolerance of plants is increasingly being utilized these days. Plant roots have restricted capacity to absorb metals from soil, chiefly because metals are not very soluble in the soil solution. Phytoremediation of metal-contaminated soil depends on the rate of uptake of the metal by plants. Also the phytoavailability of metal depends on soil properties and the associated PGPRs. In this chapter we attempt to outline the benefits of PGPR in phytoremediation by enhancing mitigation of petroleum and heavy metal pollutants in the environment. It also focuses on the recent developments that have taken place in decoding the genetics and genomics of PGPR.

4.2

Rhizomicrobiome

The evolution and colonization of terrestrial plants has apparently been antedated by microbiome relationships (Berg et al. 2014). A land plant does not exist individually in nature; rather a consortium of bacteria is generally associated with plants and constitutes a phytomicrobiome (Smith et al. 2017). The phytomicrobiome is of crucial importance in determining the existence of plants, or rather the holobiont. Certain plant–microbe associations (e.g., Cycas, Azolla) are so inseparable that they are known to be symbiotic ubiquitously. Members of phytomicrobiome even ascertain the survival efficiency of plants under conditions of stress. Though microbes are associated with all the major plant structures, microbes associated with rhizosphere constituting a rhizomicrobiome are most elaborated and populous (Backer et al. 2018). Venturi and Keel (2016) define the rhizosphere as a complex zone around the roots of plants with a large population of microorganisms including bacteria, protists, invertebrates, nematodes, and fungi. Bacterial communities in the rhizosphere are called the rhizobacteria. Rhizosphere has much greater amount of bacteria than the bulk soil due to the presence of root exudates such as amino acids and sugars, called rhizodeposition, which provides energy and nutrients for development (Novo et al. 2018). Rhizodeposition may comprise nearly 15% of plant total nitrogen and 10% of photosynthetically fixed carbon (Venturi and Keel 2016). The complex composition and well-guarded regulation of rhizomicrobiome has assisted land plants against various stresses in due course of evolution (Lundberg et al. 2012; Smith et al. 2015; Zhang et al. 2017). Plant roots secrete exudates of various compositions and signal compounds in order to recruit preferential microbes (Chaparro et al. 2012; Smith et al. 2017). Apart from the considerably controlled regulation by plants, microbes do exhibit facets of self regulation (by virtue of quorum sensing in favorable conditions) depending upon the ecological conditions, which are reciprocated by plants as a mechanism for further regulation (Leach et al. 2017; Ortiz-Castro et al. 2009). This degree of regulation is directly dependent upon the affinity between roots and microbes, i.e., it is much higher for endophytes and rhizospheric bacteria (Backer et al. 2018). The co-evolution of plants and microbes has facilitated certain free-living bacteria such as PGPRs to become endophytes (Bulgarelli et al. 2013). Members of the rhizomicrobiome play pivotal roles in

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enhancement of plant growth by aiding in nutrient acquisition and assimilation, improving soil texture and modulating the secretion of extracellular molecules that also influence plant stress responses (Backer et al. 2018).

4.2.1

Plant Growth Promoting Rhizobacteria

Rhizobacteria can be characterized as neutral, beneficial, and harmful depending on their outcome on plant growth and development (Huang et al. 2014). Many of them have been aptly called Plant Growth Promoting Rhizobacteria (PGPRs) (Kloepper and Schroth 1978). A bacterium is called a PGPR when it can induce a positive impact on the plant after inoculation. Around, 2–5% of rhizosphere bacteria qualify as PGPR (Goswami et al. 2016). Most PGPRs belong to genera Acinetobacter, Agrobacterium, Arthobacter, Azospirillum, Azotobacter, Bacillus, Bradyrhizobium, Burkholderia, Frankia, Pseudomonads, Rhizobium, Serratia, and Thiobacillus (Glick 1995; Vessey 2003). They are known to aid plant growth by assisting in acquisition of minerals such as nitrogen and phosphorus. They also promote plant growth and development by controlling the plant hormonal balance, eliciting immune responses, mobilizing nutrients, and protecting the plant against pathogens (Glick 2012). The plant-beneficial rhizobacteria can also help to lessen the reliance on harmful fertilizers (Ahemad and Kibret 2014). Somers et al. (2004) have categorized PGPR according to their activities as: 1. Biofertilizers: ones expanding the accessibility of nutrients to the plant. 2. Phytostimulators: ones that promote plant growth and development by releasing plant growth regulators. 3. Rhizoremediators: ones that break down organic contaminants. 4. Biopesticides: ones that control diseases by producing antibiotics and antifungal metabolites. Thus we can say that PGPR can directly and indirectly influence plant growth. The direct mechanism involves the synthesis and modulation of phytohormones or the acceleration of resource accumulation including nitrogen fixation, phosphorus bioavailability, and iron sequestration. Indirect mechanism includes biocontrol which is the reduction of the effects of phytopathogens by production of antibiotics and antifungal compounds (Glick 2012; Novo et al. 2018).

4.3

Role of PGPRs in Phytoremediation

The use of plants and allied microbes for elimination of metal pollutants and soil reclamation has both ecological and economic benefits. In general, plant-associated microbes utilize one of these mechanisms to alleviate metal stress to plants (a) bioaccumulation, (b) bioavailability by transformation of metals into soluble form, (c) production of extracellular polymeric substances (EPSs) for binding, and

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(d) production of iron siderophores. Therefore, a strategy utilizing the combinatorial effect of metal-tolerant plant species along with metal-resistant plant–associated microorganisms will be more efficient. Such a novel in situ approach for bioremediation is known as rhizoremediation and the microorganisms are called heavy metaltolerant-plant growth promoting (HMT-PGP) microbes (Mishra et al. 2017). It exploits the combined capacities of the roots of plants and allied microbial communities of rhizosphere to tackle heavy metal contamination in soils (Ullah et al. 2015). The knowledge that plants assisted microbes could prove more beneficial in enhancing metal tolerance in plants has opened up new avenues. PGPRs provide metal tolerance by either using one or multiple mechanisms such as bioaccumulation, bioavailability, and production of binding and chelating compounds, and also they enhance plant growth in such conditions by protecting against various types of abiotic and biotic stress. For instance, PGPRs produce a phytohormone indole acetic acid (IAA) which can augment the uptake of metals in the roots of plant (Khan et al. 2009; Tak et al. 2013). These microorganisms are found in abundance in plant rhizosphere, they help to diminish metal buildup in plant tissues and in addition assist in reducing metal bioavailability in soil through a variety of mechanisms. PGPRs release siderophores, organic acids, and plant growth regulators which increase the rate of phytoremediation (Tak et al. 2013). Kloepper et al. (1980) reported that some strains of the Pseudomonas fluorescens-putida act as PGPR by producing extracellular siderophores which are microbial iron transport agents. They probably deprive native microflora of iron and make it less available to them and significantly improve the yield of potato, radish, and sugar beet.

4.3.1

Role of Rhizobacteria in Phytoremediation of Contaminated Soil

4.3.1.1 Metal-Contaminated Soil The mining and extraction of mineral resources is important for development but frequently causes great harm to neighboring ecosystems (Novo et al. 2018). Industrialization and modern life style have led to drastic pollution of biosphere. Different types of inorganic (heavy metals) and organic (hydrocarbons, volatile organic compounds, and solvents) contaminants are being incessantly released into the environment by mining and industrial activities (Manoj et al. 2020). Soils from mining areas are nutrient deficient and have reduced organic matter, pH, and cohesion and elevated concentration of metals (Novo et al. 2018). Mining waste leaches into aquifers and contaminates agricultural lands and accumulates in plants planted for food or livestock feed; hence can enter food chain (Mendez and Maier 2008). It causes damage to water and soil flora and has lethal impacts on human health because of the mutagenicity, cytotoxicity, and carcinogenicity. Release of untreated industrial waste containing precarious heavy metals such as mercury, arsenic, and cadmium into the water bodies and soil is another source of heavy metal pollution of surrounding soil and water (Nordberg et al. 2009). Aluminum (Al), Cadmium (Cd), Chromium (Cr), Copper (Cu), Lead (Pb), Mercury (Hg), and

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Zinc (Zn) are the most widespread polluting toxic heavy metals. These heavy metals have been declared as “priority pollutants” by United States Environmental Protection Agency because of their mutagenic and carcinogenic nature. Presence of heavy metals in soil is noxious to most plants as heavy metals ions are absorbed by roots and translocated to shoot; leading to reduction in metabolism and growth of plants (Jing et al. 2007). Soils contaminated with high metal concentration led to reduction in activity of soil microbes and thereby soil fertility (Jing et al. 2007). For example, Cd is recurrently accumulated by chief agricultural crops and its high concentration affects nutrient uptake and inhibits root and shoot growth. Such crop plants with Cd affect the health of animals and humans and negatively affect biodiversity. Cd pollution affects the activity of soil microbial communities, and the contaminated soil may eventually become unusable for crop production (Ma et al. 2011a). Therefore, research on remediation of heavy metals contaminated soils is now the topmost priority for scientists. Few methods like thermal treatment, acid leaching, excavation and land fill, and electro-reclamation were explored for cleanup of contaminants, but they are timeand cost-consuming (Zubair et al. 2016). Currently, phytoremediation is being accepted as in situ eco-friendly technology (Abou-Shanab et al. 2019). Phytoremediation depends on the fact that numerous plant species have the capability to accrue large quantities of metals in their vegetative as well as reproductive organs. Depending upon the metal accumulating skills and tolerance, plants can be metal sensitive (excluders), having poor metal uptake and transport (indicators) or those with higher uptake efficiency (hyperaccumulators) (Khan et al. 2009). Hyperaccumulator plants have the capacity to endure high level of noxious heavy metal concentration (Ma et al. 2011a). The potential for use of a particular plant for phytoremediation depends on the BCF i.e. bioconcentration factor and the TF i.e. translocation factor. The BCF specify the capability of a plant to take up the contaminant and its accumulation in its tissues. The TF specify the capacity of the plant to transfer contaminants from the root to its aboveground parts (Novo et al. 2018). The efficiency of such plants to uptake and accumulate heavy metals also depends on edaphic factors like soil, temperature, redox potential, cation exchange capacity of the soil particles (CEC), metal bioavailability, pH, aeration, and amount of organic matter and water (Eliana Andrea et al. 2019). Also, plants chosen for the purpose of phytoremediation ought to be fast growing with high biomass production, widespread root system, ability to accumulate the pollutants, and preferably hardy, native species (Manoj et al. 2020). However, high levels of pollutants are also toxic to the plants used for reclamation of affected soil, and phytoremediation by plants alone is a very slow process. Still, this process can be accelerated by the synergistic action of plant and microbes. Plant-Microbe association improves plant development by enabling the sequestration of noxious heavy metals especially by phytostabilization and phytoextraction (Ma et al. 2011a). Rhizobacteria improve adaptation of host plants to altering environment by altering plant cell metabolism, so they can withstand exposure to high concentrations of metals (Welbaum et al. 2010).

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The synergistic interaction between rhizobacteria and plants is now being investigated by many workers because of its potential to enhance plant growth, metal uptake, and tolerance during environmental stresses (Ma et al. 2011a). Rhizobacteria not only improve fertility of polluted soil but also enhance the growth and development of plants by exuding plant growth hormones (Zubair et al. 2016). Many plants like Alyssum lesbiacum and Arabidopsis halleri have been documented for phytoremediation as hyperaccumulators of Nickel (Ni) and Zn (Cluis 2004; McNair et al. 2000). Ni and Zn are known as most accumulated metals by different hyperaccumulator species (Pandey and Bajpai 2019). Khan and Bano (2018) have also emphasized the role of PGPRs in remediation of heavy metals by affecting heavy metal portability and accessibility to the plant through acidification, phosphate solubilization, and release of chelating agents. PGPRs used for phytoremediation of metal-contaminated soil in various laboratory and field experiments have been compiled in Table 4.1.

4.3.1.2 Petroleum-Contaminated Soil Petroleum hydrocarbons symbolize the biggest group of organic pollutants (HawrotPaw and Nowak 2012). Phytoremediation of petroleum is proving to be a low-cost and sustainable approach for sustainable waste management technology. This strategy can be useful in petroleum-contaminated soils where other techniques have been unsuccessful. Attempts of remediation of soils polluted with petroleum products with various herbaceous plants including Cynodon dactylon, Digitaria sanguinalis, Cyperus rotundus, Chloris babata, and Pasparlum vaginatum have also been reported (Borowik et al. 2019). However, PGPR-associated plants are much better in withstanding the pressure of growing in the crude oil contaminated soils as compared to the plants without allied PGPR (Gurska et al. 2009). Afzal et al. (2012) also stated the importance of plants and associated microorganisms to remediate petroleum hydrocarbons-contaminated soils. Gurska et al. (2009) have reported the successful establishment of a system using PGPR to enhance phytoremediation of soil contaminated with total petroleum hydrocarbons. Addition of PGPRs to soil supports an active rhizosphere, minimizes plant stress in contaminated soils, causes an increase in root biomass, and promotes degradation of oil contaminants by the plants. They also noted a noteworthy boost in fresh weight and length of shoots in experimental PGPR-associated plants. Afzal et al. (2012) reported that not only the strains used for inoculum purpose but also the inoculation strategy (seed imbibement and soil inoculation) employed determines bacterial colonization, plant growth advancement, and degradation of hydrocarbon. When the soil contaminated with diesel, where Italian ryegrass was planted, was inoculated with amalgamation of three alkane-degrading strains namely Pantoea species ITSI10, Pantoea species BTRH79, and Pseudomonas species MixRI75, maximum hydrocarbon degradation was achieved as compared to soil where single strain was used. Also soil inoculation method gave better results than seed imbibement method. PGPRs utilized for phytoremediation of petroleumcontaminated soil in various laboratory and field experiments have been compiled in Table 4.2.

Oryza sativa Triticum aestivum Glycine max

Brassica napus

Cicer arietinum

Sulla conoraria

Eruca sativa

Ocimum gratissimum Medicago lupulina Medicago sativa Alyssum serpyllifolium Lupinus luteus

Enterobacter sp. Pseudomonas moraviensis

Bradyrhizobium sp. Per 3.61

Bacteroidetes bacterium, Variovorax sp.

Trichoderma sp.

Rhizobium sullae, Pseudomonas sp.

Pseudomonas putida (ATCC 39213)

Arthrobacter sp. TISTR 2220

Bradyrhizobium sp. 750, Pseudomonas sp., Ochrobactrum cytisi

Pseudomonas sp. A3R3

Sinorhizobium meliloti

Sinorhizobium meliloti CCNWSX0020

Host plant Lycopersicon esculentum Oryza sativa

PGPR Pseudomonas aeruginosa, Burkholderia gladioli Bacillus subtilis

Cu, Cd, Pb

Ni

Cd

Cu

Cd

Cd

Cu, Zn, Pb

As

Cd, Zn

Cd Cd, Cr, Cu Mn, and Ni As

Cd

Contaminant Cd

In situ (Field scale)

Pot study, lab scale Pot study

In vitro

Field study

Greenhouse study In situ (field scale) Pot study

Pot study, lab scale Pot study

In vitro Field study

Type of study/scale In vitro

Table 4.1 List of plant growth promoting rhizobacteria (PGPR)-assisted phytoremediation studies

Increase in phytostabilization

Increase in Ni accumulation

Increase in cadmium accumulation and translocation Increase in plant growth and tolerance to Cu Increase in Cd phytoextraction

Increase in Cd uptake

biotransformation of As and ameliorates stress Increase in Zn phytostabilization

Increase in accumulation

Decrease in Cd uptake Decrease in accumulation and translocation Decrease in translocation factor

Decrease in Cd uptake

Effect of PGPR Decrease in Cd uptake

Dary et al. (2010)

Ghnaya et al. (2015) Ma et al. (2011b)

Bianucci et al. (2018) Dąbrowska et al. (2017) Tripathi et al. (2017) Saadani et al. (2016) Kamran et al. (2015) Prapagdee and Khonsue (2015) Kong et al. (2015)

References Khanna et al. (2019) Treesubsuntorn et al. (2018) Mitra et al. (2018) Hassan et al. (2017)

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Table 4.2 List of some PGPRs that assist in phytoremediation of petroleum-contaminated soil PGPR Klebsiella D5A Pseudomonas strains, UW3 and UW4

Pseudomonas sp. AJ15

Bacillus circulans, Enterobacter intermedius and Staphylococcus carnosus Serratia liquefaciens, Pseudomonas aeruginosa, Bradyrhizobium japonicum and Flavobacterium sp. Pseudomonas aeruginosa strains AS 03 and NA 108

Plant Festuca arundinacea L. Festuca arundinacea, var. Inferno, Secale cereale, Hordeum vulgare Withania somnifera

Type of study Pot experiment after isolation of strain Field study, seeds treated with inoculum

References Liu et al. (2014) Gurska et al. (2009)

Seed priming with biosurfactant

Zea mays

In vitro studies, seeds treated with inoculum

Vicia faba

In vitro studies on nodule

Das and Kumar (2016) Ajuzieogu et al. (2015) Radwan et al. (2007)

Tea (TV1 type)

Pot experiments in greenhouse using 1 year old tea plants TV1 type In vitro studies using both solid and liquid medium Pot experiments

Azospirillum brasilense strain SR80

Triticum aestivum L. Saratovskaya 29

Proteobacteria

Cajanus cajan

γ-proteobacteria and Bacteroidetes

Festuca arundinacea L.

4.4

In vitro studies

Roy et al. (2013)

Muratova et al. (2005) Allamin et al. (2020) Hou et al. (2015)

From the Lab to the Field and Commercialization

Certain PGPR mechanisms i.e., nitrogen fixation, phytohormone synthesis, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, siderophore production, antibiosis, and phosphate solubilization are the basis of laboratory screening assays designed to develop new PGPR inocula. Under laboratory conditions, these mechanisms are difficult to screen for because of complexity of the mechanisms along with gaps in understanding. Henceforth, results obtained in laboratory conditions are not always replicated under field conditions and vice versa. Consequently, promising strains are rejected due to underperformance at classical laboratory screening scale (Cardinale et al. 2015). PGPR formulations propound to green alternatives over conventional agrochemicals as they promote plant growth, aid in soil fertility, and suppress

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phytopathogens without contaminating the environment (Arora et al. 2016). The development of bioformulations includes (Backer et al. 2018): (a) Isolation of PGPR from rhizospheric soil. (b) Laboratory screening of traits. (c) Field trial under different conditions (crop varieties, soil types, seasons, locations) and management practices (use of agrochemicals, etc.) (d) Assessment of synergistic effects of possible PGPR combinations. (e) Confirmation of safety against ecotoxicology. (f) Refining PGPR formulation and knowledge about its texture and storage conditions. (g) Product registration and approval by regulatory agency of country. (h) Commercialization. All these steps are time-consuming, laborious, and costly to perform. To facilitate this process, collaborations among industries, research institutes, and government organizations can play a vital role. Development of bioformulations is a business of intellectual property. Though living creatures and natural products can no longer be patented, formulations and their applications are patentable (Matthews and Cuchiara 2014). Patenting holds a prominent place between discovery and commercialization of promising PGPR in the field of environmental management. Variovorax paradoxus JHP31 strain (EP2578675A1) was patented by Koga and Masuda (2015) for assisting Cd phytostabilization in the plants of Brassicaceae, Chenopodiaceae, Compositae, Gramineae, Leguminosae, Liliaceae, Polygonaceae, and Solanaceae families. PGPRs such as Achromobacter piechaudii, Agrobacterium tumefaciens, Delftia acidovorans, and Stenotrophomonas maltophilia were patented (Banerjee and Yesmin 2011) for their ability to oxidize elemental sulfur and in turn enhancing plant growth. Some other PGPRs that have been patented are Microbacterium arabinogalactanolyticum, Microbacterium liquefaciens, and Sphingomonas macrogoltabidus for assisting phytoextraction ability of Alyssum murale (US7214516B2) (Angle et al. 2007). Highly potent microbes possessing long shelf-life and good colonization rates present a major challenge to commercialization. Colonization rates are largely affected by inoculation and field conditions. PGPR inoculated without a suitable carrier or in amount not enough to compete with native soil microbes are the major challenges to successful rhizosphere colonization (Backer et al. 2018). Additionally, fumigation of soils with broad-spectrum biocidal fumigants during cultivation of high-value crops alters the soil ecology by affecting microflora and their interactions with plants in aiding nutrient acquisition and mobilization (Dangi et al. 2017). Many underlying issues should be tended to for substantial commercialization of PGPR strains such as the following:

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(a) Identification of PGPR responsively suitable for particular soil conditions and overcome environmental constraints. (b) Choosing ideal rhizoinoculation techniques depending upon cultivation conditions (i.e., greenhouse vs. field) and training farmers to apply them efficiently. (c) Selection of desirable traits-possessing strains. (d) Uniformity among regulatory agencies of different countries regarding safety and use of PGPB strains. (e) Knowledge of potential interactions among PGPR and native microflora (other bacteria, algae, and fungi) and the advantages of using them over others. With view of all above points, PGPR strains that have been commercialized have been enlisted in Table 4.3 (Glick 2012). Progressing from research lab and greenhouse analysis to field assessments and commercialization involves development of new processes to inoculate, formulate, store, and ship these strains. Necessary instructions will need to be imparted to the users of these formulations.

4.5

Genetics and Genomics of Heavy-Metal Resistance in PGPRs

Many plant-associated microorganisms mainly bacteria and fungi are well-known to display plant-growth advancing qualities under heavy metal stress by means of various direct and indirect mechanisms e.g. Pseudomonas sp., Bacillus sp., Arthrobacter, Streptromyces, Methylobacterium, and filamentous fungi such as Trichoderma, Aspergillus, and Fusarium. There have been many genetic studies to evaluate if the heavy metal-resistance and plant growth promoter-producing bacteria found in soils would support phytoremediation. Much of the studies were done in symbiotic rhizobia and has been reviewed by Fagorzi et al. (2018). In Sinorhizobium meliloti CCNWSX0020 genetic mechanisms responsible for Cu resistance were elucidated through transposon mutagenesis combined with RTPCR (Li et al. 2014). The transcriptional analysis of Rhizobium etli revealed the increase in the levels of defense-related genes namely PvWRKY33, PvERF6, and PvPAL2 as well as ABA-synthesis-related gene PvAAO3 following infection with the pathogen (Díaz-Valle et al. 2019). Genetic screening of a cosmid genomic library of Mesorhizobium metallidurans for Cd or Zn endurance revealed the presence of a gene encoding PIB-type ATPase homologous to CadA (Maynaud et al. 2014). The mechanism of arsenite [As(III)] resistance via methylation and successive volatization was characterized by Qin et al. (2006) and the enzyme for this function was encoded by the As(III) S-adenosylmethionine methyltransferase (arsM) genes. Rhizobium leguminosarum bv trifolii, which lacks an endogenous arsM gene, was genetically engineered by using an algal As(III) methyltransferase gene (CrarsM) for arsenic bioremediation and it was able to successfully methylate arsenic reducing toxicity. In Mesorhizobium amorphae genetic mechanism of Cu resistance was investigated by transposon mutagenesis, and CopA was found to be the major

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Table 4.3 List of commercialized PGPR and their effects PGPR Agrobacterium tumafaciens strain K-84 (formerly A. radiobacter) Azospirillum brasilense

Azospirillum lipoferum Azotobacter chroococcum

Bacillus firmus

Bacillus licheniformis

Bacillus megaterium Bacillus mucilaginous

Bacillus pumilus

Bacillus subtilis

Application Biocontrol of crown gall disease

Intended crop Commercial and ornamental plants

References Bhattacharyya and Jha (2012)

Nitrogen fixation, promotes plant growth via synthesis of phytohormones, provides resistance against biotic and abiotic stress Promotes growth, ameliorates drought stress Potential biofertilizer, nitrogen fixation, P and K solubilizer, promotes plant growth via synthesis of phytohormones Phosphate solubilization, nitrogen fixation, promotes plant growth via synthesis of phytohormones, provides protection against nematodes Potential biofertilizer, phosphate solubilization, nitrogen fixation produces auxins, siderophores, and antifungal cellulases, induces tolerance to both biotic and abiotic stress Phosphate solubilization, produces auxins, promotes plant growth Phosphate and potassium solubilization, nitrogen fixation

Turf grass and forage crops

Fukami et al. (2018)

Corn, wheat, rice, vegetables, and turf grass Wheat, barley, oats, rice, sunflowers, maize, line, beetroot, tobacco, tea, coffee, and coconuts

Bashan and de Bashan (2005) Wani et al. (2013)

Maize, Cotton, Tomato

Mendis et al. (2018)

Vegetable and grain crops

Mahdi et al. (2020), Lim and Kim (2013)

Wheat, maize, rice, and cotton

Tabassum et al. (2017)

Sorghum, wheat

Bhattacharyya et al. (2016), Wu et al. (2005) Tabassum et al. (2017)

Phosphate solubilization, produces auxins, induces systemic resistance against wilt, molds, mildews, blights, rusts Phosphate solubilization, biocontrol agent against soil-borne pathogens such as Fusarium and Rhizoctonia

Millets, Soybean, oak trees, and green house crops

cotton, peanut, soya bean, corn, vegetables, and small grain crops

Nakkeeran et al. (2005)

(continued)

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Table 4.3 (continued) PGPR Burkholderia cepacia

Delfitia acidovorans Paenobacillus polymyxa

Application Phosphate solubilization, antifungal in nature (provides protection against Pythium, Fusarium) S-oxidizing PGPR, promotes growth Nitrogen fixation, promotes growth

Pantoea agglomerans

Nitrogen fixation, synthesize auxins

French beans

Pseudomonas aureofaciens

Biocontrol against Pseudomonas tolassi (Dollar spot, Anthracnose) Biocontrol against Pythium spp., Rhizoctonia solani, Fusarium oxysporum Biocontrol agent against major diseases Biocontrol agent against Botrytis cinerea, Penicillium spp., Geotrichum candidum Nitrogen fixation, induction of plant stress resistance, synthesis of auxins, siderophore production Biocontrol against fungal phytopathogens Resistance against soilborne diseases (mildews)

Mushrooms Turf and other crops

Banerjee and Yesmin (2002) Chauhan and Bagyaraj (2015), Anand et al. (2013) Chauhan and Bagyaraj (2015) Tabassum et al. (2017)

Vegetables and ornamental plants

Tabassum et al. (2017)

Edible, oil, cash, and ornamental crops Pome fruit, citrus, cherries, and potato

Ganeshan and Kumar (2005) Bhattacharyya and Jha (2012)

Legumes

Vejan et al. (2016)

Field, vegetables and ornamental plants Fruits and vegetables

Bhattacharyya and Jha (2012) Tabassum et al. (2017)

Pseudomonas chlororaphis

Pseudomonas fluorescens Pseudomonas syringae

Rhizobium spp.

Streptomyces griseoviridis K61 Streptomyces lydicus

Intended crop Alfalfa, Barley, Beans, Clover, Cotton, Maize, Peas, Sorghum, Vegetable crops, and Wheat Canola French beans, lodgepole pine

References Zhao et al. (2014)

determinant (Hao et al. 2015). Sinorhizobium melilotinia was shown express a P1B-5-ATPase in the nodule and its expression is activated by the presence of Ni2+and Fe2+ions (Zielazinski et al. 2013). Genomic analysis of the role of the plant-beneficial function contributing genes (PBFC genes) was probed utilizing the genomes of 25 PGPR species, and it showed favored associations among certain genes engaged in phytobeneficial qualities (Bruto et al. 2014). Currently, use of a novel phytobacterial strategy that uses genetically engineered plant growth promoting bacteria along with plants seems to be promising approach to mitigate heavy metal stress in plants (Gupta and Singh 2017; Ullah et al. 2015;

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Ashraf et al. 2017, Tiwari and Lata 2018). Many genes belonging to metal uptake and its regulation, metabolic enzymes, metal chelators, and metal homeostasis can be used as potential target genes for such manipulation. Undoubtedly, these genetically modified microorganisms have better remediation prospective, yet their effect on biomes needs to be studied in detail. Pseudomonas aeruginosa strain Psew-MT, which was genetically modified by expressing metallothioneins to capture Cd2+, showed tolerance along with plant growth-advancing properties (Huang et al. 2016). Similarly, enhanced Cd, Hg, and Silver (Ag) resistance and accumulation were shown by genetically modified Pseudomonas putida KT244 (Yong et al. 2014). Rhizobium–legume associations have been studied for various reasons in the past and they provide an excellent strategy which can be exploited in reclamation of heavy metal-polluted soils (Pajuelo et al. 2011; Ahemad 2012). For example, a genetically engineered Ensifer medicae MA11 strain having copAB gene from Pseudomonas fluorescens was analyzed for enhanced Cu resistance and reducing toxic effect of Cu in Medicago truncatula (Perez-Palacios et al. 2017; Delgadillos et al. 2015). Four different reports are available for the use of transgenic Meshorhizobium huakuii subsp. rengei strain B3 for Cd bioremediation in association with different plants (Ike et al. 2007, 2008; Sriprang et al. 2002, 2003). Wu et al. (2006) reported the use of genetically engineered Pseudomonas putida strain 06909 for enhanced Cd tolerance in alliance with the host plant Helianthus annuus. In another study, Weyens et al. (2013) analyzed the phytoremediation prospective of willow and its genetically engineered allied bacteria in Cd- and toluenecontaminated soils. In an independent study, the impact of genetically engineered Burkholderia pyrrocinia JK-SH007E1 on microbial communities of soil in the poplar rhizosphere during long-term use as biological control was analyzed (He et al. 2018). Whole genome sequence analysis has also been used to characterize the genetic basis of the PGPR and plant interactions. P. fluorescens Pf-5 is a remarkable organism widely recognized for its use in PGPR for its rhizosphere competence and production of broad range of secondary metabolites and antibiotics. The genome of Pseudomonas fluorescens Pf-5 was sequenced to identify the genetic features and molecular determinants responsible for biocontrol (Paulsen et al. 2005). The genome sequence Pseudomonas psychrotolerans CS51 was determined to understand the plant growth-promoting characteristics under multiple heavy metal stress (Cd, Cu, and Zn), and the existence of genes accountable for cobalt-Cd-Zn resistance, transportation of Ni, and Cu homeostasis was confirmed in the P. psychrotolerans CS51 genome. Genomes of other PGPR strains including the Serratia fonticola strain AU-P3, and Bacillus sp. strain JS, Sinorhizobium meliloti CCNWSX0020 have been sequenced, which is serving to comprehend the correlation among genes and PGPR activities (Devi et al. 2013; Song et al. 2012; Li et al. 2012). There are few studies that have focused on the role of bacterial consortium in PGPR-mediated beneficial effects (Zolla et al. 2013). A synthetic microbial consortium containing seven 2,4-DNT-degrading microbes affiliated to Bacillus, Burkholderia, Pseudomonas, Ralstonia, and Variovorax species was found to augment root length of Arabidopsis under 2,4-DNT stress (Thijs et al. 2014). In another

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study, phytoremediation potential of Lupinus luteus was improved when it was inoculated with a PGPR conglomerate inclusive of Bradyrhizobium sp. and two metal resistant bacteria including Ochrobactrum cytisi and Pseudomonas sp. (Dary et al. 2010).

4.5.1

Genetically Engineered PGPRs

There have been numerous attempts to understand the molecular features that define PGPR. But, it has remained largely unsuccessful due the ability of PGPR to occupy different habits, to display alternative/selective ecological niches. Moreover, the genes that are implicated in plant-beneficial functions are also involved in the essential primary metabolism like phosphate solubilization, nif (nitrogen fixation), and phl (phloroglucinol synthesis) or in the secondary metabolic functions like pqq (pyrroloquinoline quinone synthesis). So the role of PGPR in producing plant beneficial properties needs to be experimentally verified under controlled conditions and that too in isolation for each species. That has made the process of identification of plant-beneficial traits and their corresponding genes in PGPR a relatively difficult task. In the last few decades, there have been various reports on introduction of specific genes accountable for the expression of certain enzymes from microbial species lineally into crop plants, but very few studies have been reported on genetic manipulation in the PGPR for enhancing plant productivity under environmental stress or metal stress (Ullah et al. 2015; Saxena et al. 2019). The transgenic techniques are used to either overexpress or knock down genes playing a crucial function in metal detoxification and tolerance to metal stress like genes encoding metal binding, transport, and chelation (Dhankher et al. 2011; Ullah et al. 2015; Sarwar et al. 2017; Saxena et al. 2019). The enzyme ACC deaminase, encoded by AcdS gene, is common in bacterial and fungal species in soil. It breaks down ACC, precursor of the plant hormone ethylene, to α-ketobutyrate and ammonium. ACC deaminase enzyme has been recognized in soil bacteria and has been anticipated to play an important function in microbe–plant association by decreasing the harmful impacts of biotic and abiotic stress to plants. The activity of ACC deaminase is one of the most widespread qualities among PGPRs (Glick 2014). ACC deaminase microbes aid allied plants in phytoremediation by biotransformation of poisonous components, rhizodegradation facilitated by root exudates, as well as detoxification of heavy metals that let host plants to sustain under unfavorable conditions. The bacterial AcdS gene has been utilized to generate transgenic plants to improve their tolerance to abiotic and biotic stress. Many genetically modified plants with foreign AcdS gene have been generated to lessen the harmful ethylene levels in plants as reviewed by Saleem et al. (2007). When Mesorhizobium ciceri was exogenously transformed with acdS gene, it showed improved plant performance under salinity stress by enhancing nodulation suggesting the significant function of ACC deaminase in assisting symbiotic interaction under salinity stress (Conforte et al. 2010; Nascimento et al. 2012;

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Brıgido et al. 2013). However, there is limited information on performance of these transgenic plants under farm conditions due to the environmental risks associated with them. Thus biotechnological interventions can prove to be promising alternatives for enhancing agricultural productivity through PGPR-mediated beneficial effects. However, since the genetically engineered plant-associated microbes are mainly distributed in the rhizosphere, a detailed experimental validation is required for their use in field conditions. Currently, efforts are underway to understand the beneficial effects of root microbiome on plant productivity and stress endurance. Many differentially regulated genes were recognized in PGPR-treated roots of rice plants through microarray technique (Agarwal et al. 2019). Transgenic plants overexpressing OsASR6 (ABA STRESS RIPENING 6) showed a significant result on the growth of plant and root architecture, which could be the main reason for the positive impact of PGPRs in rice.

4.6

Conclusion

PGPRs play significant roles in assisting plant growth on soils polluted with diverse contaminants and in detoxification of soils. Microbial diversity and their interactions play an essential role in facilitating plant-based degradation of toxins. Therefore, microbiome analysis or a detailed rhiozbiome analysis in PGPR–plant interactions may provide more useful insights. The potential of “omics” technologies (such as genomics, transcriptomics, proteomics, metabolomics, and metagenomics) has to be utilized to get a clear holistic view of the role of various genetic, molecular, and regulatory mechanisms in microbe-assisted phytoremediation. For effectual utilization of genetically engineered PGPR for phytoremediation; well-structured, costefficient, and time-efficient tools for a trustworthy forecast of their effectiveness on contaminated sites and their repercussion on biomes required to be discoursed prior to commercialization. Also, assurance needs to be provided regarding safety upon large-scale release of strains, since public acceptance also comes into count.

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Endophytic Bacteria: Role in Phosphorous Solubilization Neha

Abstract

For complete growth and development of plants phosphorous (P) is the second key nutrient after nitrogen. Predominantly two major forms of phosphorous exist in soil: organic P and inorganic P, which are however mostly in insoluble forms. This unavailability of P is the result of fixation and precipitation, which causes P inadequacy and limits the growth of plants. To reassure the nutritional demand of crop, P is generally incorporated in soil in the form of chemical P fertilizer. However, the use of mineral P fertilizer has very long-term implications in the environment such as eutrophication, soil fertility depletion, and aggregation of harmful chemicals. So, it is important to generate alternative sustainable and economical method to fulfil the P requirements. In this regard, phosphate solubilizing microbes including P-solubilizing bacterial endophytes provided an unconventional and eco-friendly biotechnological solution to accomplish the phosphorous demands of crops. The bacterial endophytes are used as bio-inoculants and facilitate the growth of plants in many ways other than Psolubilization. This work emphasized on the plant colonizing ability of endophytic bacteria, their functional diversity and process involved in phosphorous solubilization or mineralization mechanism for their possible use to attain sustainable agriculture system. Keywords

Soil P · Bacterial endophytes · P-solubilization/mineralization · Bio-inoculants · Sustainable agriculture

Neha (*) Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. K. Singh et al. (eds.), Bacterial Endophytes for Sustainable Agriculture and Environmental Management, https://doi.org/10.1007/978-981-16-4497-9_5

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Introduction

Phosphorous is the major vital macronutrient for the growth and development of plants. It plays a key role in numerous plant metabolic processes including energy transfer, biosynthesis of macromolecules, photosynthesis, and respiration (Fernández et al. 2007). In soil, the total P content is approximately around 0.05% (w/w); however, merely 0.1% of the total P is available to plants (Scheffer and Schachtschabel 1992; Otieno et al. 2015), as in acidic soil it is fixed as insoluble iron phosphates and in alkaline soil in the form of calcium phosphates. This insoluble form of phosphorous is not available and is not absorbed by plants and results in the elevated use of mineral phosphatic fertilizer to crops (Sharma et al. 2013) that cause environmental pollution like eutrophication. The increasing price of chemical P fertilizers, their adverse effect on environment and low efficiency of plant to use P from soil have highlighted the interest in the study of microbial solubilization of P in soil. The eco-friendly agriculture practices and sustainable evolution of food sector is anticipated to move towards enhancing the productivity without compromising the needs of forthcoming generations. Extensive productivity is expected to be achieved through developing new biotechnological methods and employing high crop yield strategies. In this view, the organisms with phosphate solubilizing ability, generally termed as P-solubilizing microbes, may offer feasible replacement to chemical phosphorous fertilizers. Among the various P-solubilizing microbes, bacterial endophytes are assessed as one of the principal group to escalate the bioavailability of soil insoluble P for plant biological growth and development (Zhu et al. 2011). Since the development of the rhizosphere concept in 1904 by Hiltner, many research studies have established that the rhizosphere soil environment is a hotspot of microbial activities, abundance and diversity because of the presence of root exudates and rhizodeposits (Hiltner 1904; Hartmann et al. 2008). The bacterial colonization in healthy plants has become an interest because of their capability for manipulation to enhance crop productivity (Turner et al. 1993). The group of microbes, either bacteria or fungi, that colonize within plant tissues symbiotically without harming the host plant are called as endophytes. The term endophytes was first introduced by De Bary (1866), which indicates the organisms that grow internally in plant tissues. Nowadays they are more appropriately explained, in respect to their various groups either bacterial or fungal associations, obligate or facultative with the host plant (Cabral et al. 1993; Hallmann et al. 1997; Rosenblueth and Martínez-Romero 2006). A large array of bacterial endophytes have been reported that are able to grow and survive on roots and in soil as well. The plantassociated bacteria that reside internally in plants are known as bacterial endophytes, which precisely regulate the host plant cells transmitting responses as a result of association (Hardoim et al. 2008) without any negative effect on host plant (Reinhold-Hurek and Hurek 2011). Bacterial endophytes can provide various beneficial aspects to host plants preferably plant growth promotion, defence from pathogens and under varied environmental situations endophytic bacteria are capable of communicating and interacting with the host plant more effectively in comparison to rhizosphere bacterial population (Ali et al. 2012;

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Coutinho et al. 2015). In accordance to their life strategies, the endophytic bacteria possibly can be classified into three groups: obligate, facultative and passive endophytes. Obligate endophytes purely depend on host plant for their growth and viability and transmit to other plants through specific vectors. Whereas facultative endophytes complete their life cycle outside the host plants (Hardoim et al. 2008) and the third group (passive endophytes) colonizes the host plant tissue via several open injuries. The passive endophytes are less efficient, as for colonization it is necessary for the host to have cellular machinery (Verma et al. 2004; Rosenblueth and Martínez-Romero 2006). For the first time, endophytic bacteria was probably isolated by Mundt and Hinkle (1976) from plants, and till date, in 16 phyla over 200 bacterial genera are identified as endophytes. These genera of endophytic bacteria comprise both culturable and unculturable groups. The most extensively studied bacterial endophytes found abundantly across several phyla involving Proteobacteria, Firmicutes, Actinobacteria and Bacteriodetes (Hardoim et al. 2015; Bulgarelli et al. 2012; Wemheuer et al. 2017) and covers the members of Pseudomonas, Enterobacter (Taghavi et al. 2009, 2010), Bacillus (Deng et al. 2011), Burkholderia (Weilharter et al. 2011) and Stenotrophomonas (Ryan et al. 2009). The culturable methods for isolation of bacterial endophytes have been broadly reviewed (Hallmann et al. 1997; Reinhold-Hurek and Hurek 1998). Brígido et al. (2019) isolated, identified and characterized culturable endophytic bacteria inhabiting the roots of chickpea (Cicer arietinum L.) grown in different types of soils. They found that the most common endophytic bacteria were Enterobacter and Pseudomonas, which produced indole acetic acid (IAA) siderophores and facilitate dissolution of P. In another study, 55 isolates were isolated from sap, leaves and roots of maize crop that are able to solubilize tricalcium phosphate by producing organic acid (Abreu et al. 2017). In a similar study, 22 bacterial endophytes were isolated from rhizosphere and roots of wheat (Triticum aestivum L.) plants and these isolates solubilized P from tricalcium phosphate and liberated IAA (Emami et al. 2020). Endophytic bacteria play a crucial role in plant growth promotion by possessing favourable effect on host plant. These bacterial endophytes can stimulate plant growth in several ways such as increasing rate of germination of seeds, root and shoot biomass, chlorophyll content, and abiotic stress tolerance. (Wahla and Shukla 2017). They also enhance the growth of plants through nitrogen fixation, phytohormone production, and phosphorous solubilization (Iniguez et al. 2004). These bacteria play a significant role in the biocontrol of phytopathogens in the plant root zones by the production of antifungal/antibacterial compounds, siderophore production and elicitation of systemic acquired resistances (Rosenblueth and Martínez-Romero 2006). This chapter highlights the mechanism of phosphorous solubilization by endophytic bacteria as they are efficient in solubilizing the soil insoluble P and make it available to plants. This capability to transform insoluble P to available orthophosphate form is a very important aspect of plant growth promoting bacteria for enhancing yields (Rodríguez et al. 2006). Hence, it is crucial to have in-depth understanding of plant, soil and microbial phosphorous cycle to develop sustainable agriculture system.

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Colonization of Bacterial Endophytes

Endophytic bacteria might have an interest over rhizosphere bacteria as it has characteristics feature of living inside the plants tissues and expresses opportunity to inevitably be in contact with cells of plant and consequently exert the direct favourable effect. Certainly rhizospheric bacteria may also have the ability to penetrate and colonize the plant root (Santoyo et al. 2016). This microhabitat has been extensively reported as one of the key source for colonization of endophytes (Hallmann et al. 1997). Actually, the diversity of endophytic bacteria can be accounted as a member of rhizospheric bacterial population (Marquez-Santacruz et al. 2010). The rhizospheric environment is very competitive for microbes to inhabit and acquire nutrients (Raaijmakers et al. 2002). Rhizospheric colonization has been linked to root exudation mechanism (Lugtenberg and Dekkers 1999). The endophytes utilize various mechanisms to enter inside the plant tissues, especially in roots. Primary and lateral cracks in the root and various tissue wounds are the most conventional routes of entry of bacterial endophytes into plant tissues (Sprent and De Faria 1989; Sørensen and Sessitsch 2007). Numerous nutrients providing plant metabolite for root-inhabiting bacteria like organic acids, amino acids and various other compounds are liberated in the rhizospheric region (Walker et al. 2003; Compant et al. 2010). Root wounds ooze plant metabolites and as a result they chemo-attract the bacteria (Hallmann et al. 1997). Root exudates and other nutrients captivate detrimental rhizobacteria and also beneficial bacteria, fungi and many other soil entities (Walker et al. 2003). Therefore, plant growth promoting bacteria (PGPB) or bacterial endophytes must be really very competitive to colonize successfully to the root zone. There are some other areas which allow endophytes to enter plant tissue such as stomata, young stems (Roos and Hattingh 1983), lenticels (Scott et al. 1996) as well as growing radicals (Gagnet et al. 1987). Rhizospheric region is studied as a hot spot for phosphate solubilizing bacteria (PSB) indicating that PSB rapidly grow in both rhizospheric and root endospheric region (Hui et al. 2011). The occurrence of high amount of PSB in rhizosphere is because of the availability of high intensity of nutrients, mainly root exudates, which support the growth and metabolism of bacteria (Sharma et al. 2007). In phosphorous-limited soils the population of phosphate solubilizers are high and they help in the solubilization of insoluble phosphorous in the vicinity of the roots in available form (Aranda et al. 2011; Zhou et al. 2011) by producing organic acids and enzymes (Otieno et al. 2015; Illmer and Schinner 1995).

5.3

Role of Bacterial Endophytes in Phosphorous Solubilization

The concentration of P in soil is very low around 1 ppm or less (Goldstein 1994). As it is well established that P in soil exists in two forms, the organic form of P is obtained from humus and other organic matter comprising dead decayed plant, animals and microbes, which accounts as a significant pool for nearly 20–80% of

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total soil P (Richardson 1994). A second main part is insoluble inorganic phosphate or mineral phosphorous. In soil they are represented as primary minerals such as dicalcium and tricalcium phosphate, hydroxyapatite, rock phosphate and oxyapatite (Goldstein 1986; Rodriguez and Fraga 1999). In majority of agricultural soils huge reserves of P are accumulated as a result of consistent application of chemical P fertilizers (Richardson 1994). However, large fraction of soluble chemical P fertilizer is quickly immobilized shortly after its implementation and as a result become inaccessible to plants (Dey 1988). The mineralization and solubilization of P are key processes to enhance its availability in the soil, and these activities can be efficiently conducted by endophytic bacteria. There are a vast majority of bacterial endophytic strains that efficiently serve as phosphate solubilizers like Bacillus, Pseudomonas and endosymbiotic Rhizobia (Igual et al. 2001). Many other bacteria have been isolated like Klebsiella, Rhizobium, Erwinia, Micrococcus, Pseudomonas, Bacillus and Mesorhizobium, which are associated with phosphate solubilization (Villegas and Fortin 2002). In soil the occurrence of P fixation and precipitation is generally dependent on soil type and pH. Thus, in acidic soil, P is fixed by free oxides and in alkaline soil it is fixed by calcium (Goldstein 1986, 1994; Jones et al. 1991).

5.4

Mechanism of Soil P-Solubilization

There are numerous mechanisms through which the solubilization of P takes place like lowering of pH, production of organic acid, secretion of extracellular enzyme like phosphatase, phytase. These processes is carried out by P-solubilizing microbes (Fig. 5.1) residing in various soil ecosystems (Rodriguez and Fraga 1999; Sharma et al. 2013; Khan et al. 2013). Both organic and mineral forms of complex phosphorous compound are solubilized and mineralized by soil endophytic bacteria making P available to plants (Wani et al. 2007a; Richardson and Simpson 2011). The following processes are employed by endophytic bacteria for solubilization of phosphorous: 1. 2. 3. 4.

Organic P mineralization by release of extracellular enzymes. Mineral P solubilization by production of organic acids. By proton liberation. By secretion of siderophores and exopolysaccharides.

5.4.1

Mechanism of Organic P-Solubilization

It is evident that soil includes a wide array of organic substances which possibly act as a prime source of P for growth and development of plant. In soil, organic P constitutes about 4–90% of total P (Khan et al. 2009). The organic form of P is mineralized to make it in available form i.e. first it must be hydrolyzed to inorganic P (Rodriguez and Fraga 1999). There are diverse microbes, especially the endophytic bacteria, which possess the potential to transform insoluble organic P into available

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Fig. 5.1 Schematic presentation of soil P solubilization/mineralization by bacterial endophytes

form of P. This process of mineralization of P is carried out by extracellular enzymes, most importantly phosphatases (Tarafdar and Claassen 1988; Khan et al. 2014; Rodriguez and Fraga 1999), phytases (Nannipieri et al. 2011; Maogual et al. 2014) C–P lyase (Wahla and Shukla 2017), phosphonatases (Nannipieri et al. 2011).

5.4.1.1 Phosphatases Phosphatases have been comprehensively explored in soil (Tabatabai 1994; Nannipieri et al. 2011). These enzymes catalyse the hydrolysis of both ester and anhydride bonds of phosphoric acid (Schmidt and Laskowski Sr 1961). Various kind of phosphatases are found in soil like phosphomonoesterases, phosphodiesterases, triphosphoric monoester hydrolases and enzymes functioning on phosphorylcontaining anhydrides and on P-N bonds (Nannipieri et al. 2011). Phosphomonoesterases comprising phytases are the studied enzymes for organic P

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mineralization by microbes (Jones and Oburger 2011). Further, based on the pH optima, phosphomonoesterases are categorized further into acid and alkaline phosphatases which catalyse the hydrolysis of monoester bonds of mononucleotides and sugar phosphates (Jorquera et al. 2008). Usually acid phosphatase prevails in the acidic soils and alkaline phosphatases are abundantly available in neutral to alkaline soil (Juma and Tabatabai 1977, 1998; Renella et al. 2006). Microorganisms capable of generating both acid and alkaline phosphatases (Nannipieri et al. 2011) and plant root produce only acid phosphatase (Hinsinger et al. 2018). The enzymes acid and alkaline phosphatases are exo-enzymes (liberated exterior to the cell), which are non-specific in nature and utilize organic form of P like a substrate and transform it into available inorganic P (Beech et al. 2001). There are a number of factors that regulate the phosphatase activities like availability of soil P and organic matter content (Štursová and Baldrian 2011). It is reported that the application of mineral P in soil inhibits the activity of phosphomonoesterases (Nannipieri et al. 2011). Phosphatases of microbial origin have elevated affinity for organic P in contrast to phosphatase originated from roots of plants (Chen et al. 2003; Walia et al. 2017). But the interaction between phosphorous solubilizing microbes in soil, phosphatase activity and mineralization of organic P is roughly understood till date (Chen et al. 2003).

5.4.1.2 Phytases Phytases hydrolyses P from phytate degradation and is the key source of inositol. It is the stored form of P in seed and pollen and is dominant form of organic P in soil (Richardson 1994). All the six phosphate groups of inositol hexaphosphate are hydrolysed by phytase (Nannipieri et al. 2011). Soil microbes regulate the phytate mineralization in soil. In a study it is revealed that the vicinity of rhizospheric phosphate-solubilizing microbes render chance to plants to draw P straight from phytate (Richardson and Simpson 2011). 5.4.1.3 C-P Lyases and Phosphonatases These are the enzymes that take part in the breakdown of C-P bond in organophosphonates (Rodríguez et al. 2006).

5.4.2

Inorganic P Solubilization

There are various bacterial species reported which solubilize insoluble inorganic phosphate compounds notably tricalcium phosphate, dicalcium phosphate, rock phosphate and hydroxyapatite (Rodriguez and Fraga 1999). Numerous bacteria have the ability to solubilize mineral phosphate compounds e.g. Pseudomonas, Bacillus, Rhizobium, Burkholderia, Micrococcus, Achromobacter, Flavobacterium, Agrobacterium and Erwinia. Long et al. (2008) isolated 77 bacterial endophytes from Solanum nigrum grown in two different native habitats and reported that among the isolated strain; six were capable of solubilizing inorganic P. In another study from ginseng plant, Thamizhvendan et al. (2010) screened 18 endophytic

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isolates out of which nine isolates have P-solubilizing capability. The estimation of P solubilization potential of microbes has been achieved using serial dilution plate screening technique. Gerretsen (1948) suggested that microbes could solubilize unavailable form of phosphorous in soil and make it accessible to plants. From then, various methods and culture media have been proposed such as Pikovskaya (Pikovskaya 1948), Bromophenol blue dye method (Gupta et al. 1994), and National Botanical Research Institute P (NBRIP) medium (Nautiyal 1999). By using this method the P-solubilizing ability is detected by the formation of clear halo zone around the microbial colonies, in the culture media comprising mineral P mainly tricalcium phosphate as the only source of P. Although plate screening method is the most reliable technique for isolation and primary characterization of phosphate solubilizing microbes (Illmer and Schinner 1992). The sub-culturing of bacterial cultures is carried out to analyze the potential of P solubilization. When the potent PSB are chosen the P released by the PSB is quantitatively evaluated and the most potential phosphate solubilizers are further mass produced and evaluated under pot/field conditions with varying crops (Zaidi et al. 2009). Phosphate-solubilizing microbes solubilize mineral P by organic acid production (Table 5.1). Organic acids (OA) are the metabolic products released by microbes by the process of fermentation of organic carbon or oxidative respiration (Trolove et al. 2003). These OA originate in the periplasmic space of bacteria following direct oxidation pathway (Zhao et al. 2014; Alori et al. 2017). As a consequence of OA production the acidification of the microbial cell and its vicinity occurs (Goldstein 1994). Organic acid released in the vicinity of P-solubilizing microorganisms results in the decrease in pH to make P available in solution (Zaidi et al. 2009) and simultaneously results in the release of P ions out of mineral P by substituting H+ for Ca2+ (Goldstein 1994).OA are capable of chelating cations like Al, Ca and Fe associated with P (Omar 1997; Sharma et al. 2013). There are numerous organic acids produced namely oxalic acid, 2-ketogluconic acid, succinic, gluconic, citric, lactic, malic, malonic, fumaric and tartaric acid (Ahmed and Shahab 2011). Among all these OA produced, gluconic acid is reported to be the predominant OA involved in solubilization of mineral P (Rodriguez and Fraga 1999). In several studies it is reported that gluconic acid (GA) is the main organic acid produced by PSBs such as Burkholderia cepacia (Rodriguez and Fraga 1999), Pseudomonas sp. (Illmer and Schinner 1992) and Pseudomonas cepacia (Goldstein 1994). Another important organic acid produced by PSB is 2-ketogluconic acid which is synthesized by Rhizobium leguminosarum (Halder et al. 1990), Rhizobium meliloti (Halder and Chakrabartty 1993), Bacillus firmus (Banik and Dey 1982), Enterobacter intermedium (Hoon et al. 2003) and Bacillus subtilis (Banik and Dey 1983). Since production of organic acids has been considered as the key process in phosphorous solubilization, some other mechanism have been also taken into consideration like the microbial production of chelating compounds (Sperberg 1958; Duff and Webley 1959) and production of some inorganic acids like HCl, nitric acid and carbonic acid for solubilizing P (Hopkins and Whiting 1916). However, in a study it is revealed that HCl has less ability to solubilize P from hydroxyapatite in comparison to that of organic acid at equal pH (Kim et al. 1997). Nitromonas and Thiobacillus species are also found to release P

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Table 5.1 List of organic acid produced by P-solubilizing endophytic bacteria P-Solubilizing Bacteria Bacillus megatarium, Bacillus subtilis, Pseudomonas Arthrobacter sp.

Oxalic acid, malonic acid

Micrococcus sp.

Oxalic acid

Bacillus polymyxa, Bacillus licheniformis Pseudomonas cepacia

Oxalic acid, citric acid Gluconic acid, 2-Ketogluconic acid

Enterobacter intermedium

2-Ketogluconic acid

Bacillus megatarium

Propionic acid

Serratia marscescens

Citric acid

Pseudomonas fluorescens

Citric acid, malic acid, tartaric acid, gluconic acid Gluconic acid, malic acid

Enterobacter sp. FS-11 Bacillus methylotrophicus CKAM

Organic acids Lactic acid, malic acid

Burkholderia cepacia

Gluconic acid, 2-Ketogluconic acid, formic acid Gluconic acid

Pseudomonas fluorescens

Gluconic acid

Burkholderia gladioli

Oxalic acid, acetic acid, butyric acid, lactic acid Gluconic acid, acetic acid

Bacillus sp.

References Taha et al. (1969) Banik and Dey (1982) Banik and Dey (1982) Gupta et al. (1994) Bar-Yosef et al. (1999) Hoon et al. (2003) Chen et al. (2006) Chen et al. (2006) Fankem et al. (2006) Shahid et al. (2012) Mehta et al. (2014) Zhao et al. (2014) Otieno et al. (2015) Istina et al. (2015) de Abreu et al. (2017)

compounds by production of nitric acid as well as sulphuric acid (Sharma et al. 2013). It has however found that the effectiveness of these processes in the contribution of P solubilization appears to be insignificant (Rudolfs 1922).

5.4.2.1 Role of Proton Liberation P- Solubilization Proton excretion from the cell is one of the main features of phosphate solubilization (Krishnaraj et al. 1998). Parks et al. (1990) suggested that the release of H+ from NH4+ assimilation may be the other alternative process of P solubilization. In a study Illmer and Schinner (1995) reported that Pseudomonas sp. solubilized the P without producing organic acids as detected by HPLC analysis. They reported the release of protons accompanying NH4+ assimilation as one of the possible reason for P solubilization in absence of organic acid production. Different species of microbes possess different mechanism of proton release. The form of C i.e. glucose versus fructose had significant effect on proton release than the N (NH4+ versus NO3
)

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supply (Park et al. 2009). In a study, the acidification of the cactus seedlings rhizosphere after inoculation with endophytic bacteria Azospirillum brasiliense in presence or absence of NH4+ and NO3
, the effect of inoculating this plant growth promoting bacteria was assumed to have effect on one or more metabolic processes of the plant which enhances efflux of proton and release of organic acid from roots which ultimately results in rhizosphere acidification (Carrillo et al. 2002).

5.4.2.2 Role of Siderophores in Mineral P- Solubilization Siderophores are low molecular weight, iron-chelating compounds which form complexes with iron from mineral and make it soluble Fe3+ complexes under iron starvation condition by microbes and transport it to the cell. Siderophores production by P-solubilizing bacteria is a potent mechanism to ameliorate plant growth in iron limiting condition (Wani et al. 2007b; Ahmad et al. 2008). Several studies have revealed the excretion of siderophores from P-solubilizing microbes (CaballeroMellado et al. 2007; Hamdali et al. 2008; Ahmad et al. 2008; Singh et al. 2008; Selvakumar et al. 2008; Jiang et al. 2008). As studies revealed that the mineral dissolution is dominant over ligand exchange via organic acid anions as a phosphate solubilizing process, it is obvious to consider the function of production of siderophores in increasing P-solubilization (Parker et al. 2005). 5.4.2.3 Role of Exopolysaccharides in Phosphate Solubilization Exopolysaccharides (EPS) are polymeric material comprised of sugar residues secreted by microbes into their vicinity. EPS vary in their structure and composition. They can be homopolysaccharides or heteropolysaccharides. Moreover EPS may also include a wide variety of organic and inorganic substituents (Sutherland 2001). Yi et al. (2008) assessed the role of EPS in the solubilization of tricalcium phosphate by microorganisms. He studied for bacterial strain i.e. Enterobacter sp. EnHy-401, Arthrobacter sp. ArHy-505, Azotobacter sp. AzHy-510 and Enterobacter sp. EnHy402 which have potential to solubilize TCP (tricalcium phosphate) to examine the possible role of EPS in P-solubilization. All these four strain have capacity to produce EPS and solubilize TCP, but despite that further studies are important to unravel the association between production of microbial EPS and P-solubilization.

5.4.3

Plant Growth Promoting Attributes of P-Solubilizing Endophytic bacteria

Phosphate solubilizing endophytic bacteria enhances the overall performances of plants by exhibiting multifunctional properties (Khan et al. 2013). They are not only potent P-solubilizers but also promote growth and development of plants by producing phytohormones like indole acetic acid (Wani et al. 2007b; Ahmad et al. 2008), siderophores (Wani et al. 2007c), cyanide and antibiotics (Wani et al. 2008). These bacteria also have capability to produce essential enzyme 1-aminocyclopropane-1carboxylate (ACC) deaminase (Madhaiyan et al. 2007) and can reduce the metal toxicity in stressed soils. In a study, endophytic bacteria was isolated from the cacti

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Table 5.2 Plant growth promoting compounds produced by P-solubilizing bacteria Endophytic bacteria Pseudomonas putida

Plant growth promoting traits HCN, Siderophore

Pseudomonas fluorescens

IAA, Siderophore

Pseudomonas, Bacillus

Siderophore, IAA

Bacillus sp.

IAA, Siderophore

Burkholderia

ACC deaminase, IAA, Siderophore ACC deaminase, IAA, Siderophore ACC deaminase, Siderophore ACC deaminase, IAA, Siderophore Siderophore, IAA

Enterobacter sp. Bacillus sp. Bacillus sp. Bacillus methylotrophicus CKAM Pseudomonas sp., Paenibacillus, Bacillus sp., Enterobacter

IAA

References Tripathi et al. (2005) Gupta et al. (2005) Rajkumar et al. (2006) Wani et al. (2007c) Jiang et al. (2008) Kumar et al. (2008) Farajzadeh et al. (2012) Kumar et al. (2012) Mehta et al. (2014) Emami et al. (2020)

rhizoplane growing on bare lava rocks which not only solubilize P but in addition also stimulated growth the wild cactus species (Puente et al. 2004a, 2004b, 2009). Some other physiological traits of PSB include the liberation of ecologically critical cyanide (Wani et al. 2007b). The plant growth promoting substances generated by these P-solubilizing bacteria are given in Table 5.2.

5.4.4

Genetics of Phosphate Solubilization

5.4.4.1 Genetics of Inorganic Phosphate Solubilization The organic acid production is considered as a principal mechanism for mineral P solubilization (Rodriguez and Fraga 1999). In gram negative bacteria, glucose oxidation into gluconic acid is the principal mechanism for solubilization of mineral P i.e. MPS (Goldstein 1996). Biosynthesis of gluconic acid (GA) is catalysed by glucose dehydrogenase enzyme and pyrroloquinoline quinine (PQQ) as the cofactor by following direct oxidation pathway. PQQ is linked to the family of quinone and it performs as a cofactor for various bacterial dehydrogenases e.g. glucose and methanol dehydrogenase. A P-solubilizing gene was cloned from the Erwinia herbicola. When this gene was expressed in E.coli HB101 produces GA and is shown to solubilize hydroxyapatite (Goldstein and Liu 1987). The sequence analysis of this gene revealed its possible participation in the synthesis of enzyme PQQ synthase (Liu et al. 1992). This enzyme catalyses PQQ synthase, which is the essential

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cofactor in the formation of holoenzyme glucose dehydrogenase-PQQ, which helps in the synthesis of GA from glucose. PQQ synthesis genes from Acinetobacter calcoaceticus (Goosen et al. 1989) and Klebsiella pneumoniae (Meulenberg et al. 1992) have been cloned and 5 pqq genes were recognized and sequenced from A. calcoaceticus (Goosen et al. 1989). In a similar way another gene involved in PS and GA production, gabY, was cloned from Pseudomonas cepacia (Babu-Khan et al. 1995). This gene does not show visible homology along with the formerly cloned PQQ synthetase gene but showed similarity with histidine permeases membrane bound proteins. When gabY gene is present, the production of GA takes place only when E.coli expresses a functional glucose dehydrogenase (gcd) gene (Rodríguez et al. 2006). In Pseudomonas cepacia this gabY gene could possibly play an alternative role in expression/regulation of the direct oxidation pathway, hence behaving as a functional MPS gene in vivo. A fragment of DNA isolated from Serratia marcescens induces gluconic acid (GA) synthesis in E.coli but exhibited no homology to pqq or gcd genes (Krishnaraj and Goldstein 2001). Many other MPS genes isolated are not associated with pqq and gcd biosynthetic gene. A DNA fragment isolated from Enterobacter agglomerans exhibited MPS activity in E. coli JM109; however, the pH of the medium was not changed (Kim et al. 1997), which suggested that acid production is not a single method for bacterial P-solubilization (Illmer and Schinner 1995). The knowledge of molecular basis of P-solubilization trait is limited, and to bridge this knowledge gap the complete study of genetic basis of MPS is important.

5.4.4.2 Genetics of Organic P Mineralization Since organic form of P can be mineralized to available form by group of enzymes: phosphatases, phytases, C-P lyases and phosphonatases (Rodríguez et al. 2006). Several genes involved in organic P mineralization have been isolated and characterized. The key mechanism involved in production of phosphatase is regulated by concentration of inorganic phosphorous i.e. Pi repressible phosphatases. As a part of phosphorous starvation mechanism, enhanced activity of phosphatases occurs as a result of phosphate deficiency. This process of regulation of phosphatase has been best received in phoA alkaline phosphatase gene isolated from E.coli (Rosenberg 1987). The genes regulated by inorganic phosphate (Pi) and activated by Pho B represent PHO regulon (Santos-Beneit 2015). Other bacteria like Pseudomonas fluroscens MF3 exhibit alkaline phosphatase activity in phosphorous-deficient condition (Gügi et al. 1991). Bacterial acid phosphatases are comprised of three gene families entitled as molecular class A, B and C (Thaller et al. 1995a). The acpA gene expressed acid phosphatase and shows optimal activity at pH 6 having broad range of substrate specificity (Reilly et al. 1996). Genes isolated from Morganella morganii encoding class A (Pho C) and class B (Nap A) acid phosphatase are very promising; moreover, they show broad substrate specificity at pH 6 and temperature 30  C (Thaller et al. 1994; Thaller et al. 1995b). A gene has been isolated from rhizobacteria Burkholderia cepacia exhibiting phosphatase activity (Rodríguez et al. 2000). This gene was reported to code for protein present in outer membrane and increases the activity in P starving conditions and possibly

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participates in phosphorous transportation within cell from Rhizobium meliloti. Deng et al. (1998, 2001) cloned two non-specific periplasmic acid phosphatase gene napD and napE. A phosphatase gene (napA) from soil bacteria Morganella morganii was inserted into an endophytic bacterium Burkholderia cepacia IS-16 utilizing broadhost range pRK293 vector (Fraga et al. 2001). This recombinant strain shows improved phosphatase activity. Moreover, many other phosphatse encoding genes have been isolated from E.coli including ushA (Burns and Beacham 1986) agp (Pradel and Boquet 1988; Pradel et al. 1990) and cpdB (Beacham and Garrett 1980). The rhizospheric colonizing bacteria of the plant have significant impact on the host physiology (Antoun and Kloepper 2001). The comprehensive study of phosphate solubilization mechnism is still in infancy stage. Moreover, molecular level study to understand the mechanism involved in P solubilization mechanism by PSB is also ambiguous (Rodríguez et al. 2006). Although, various genes have been identified and cloned till date to characterize their role in inorganic and organic P solubilization (Sharma et al. 2013). Genetic manipulation of these genes is carried out by cloning and their expression in desired rhizobacterial strains to get improved phosphorous solubilizing capability for agricultural purpose as inoculants (Sharma et al. 2013). Several investigators have been worked on both phosphatase and MPS gene for their cloning and further characterization (Fraga et al. 2001; Krishnaraj and Goldstein 2001). Goldstein and Liu (1987) cloned a gene responsible for phosphate solubilization from Erwinia herbicola. The expression of mps genes in E.coli from Renella aquatilis showed enhanced gluconic acid production and solubilization of hydroxyapatite (Kim et al. 1998). Another rhizospheric bacteria Pseudomonas produces gluconic acid following oxidative glucose mechanism and overexpression of GDH gene, and PQQ biosynthesis enhances their P-solubilizing ability. In other study, expression of citrate synthase gene of bacteria in tobacco roots revealed increased exudation of organic acid and P availability. This unravels the possible role of organic acid synthesis gene in P assimilation. Genetic engineering or gene manipulation is a major conclusive method but there are some difficulties in gene insertion like dissimilarity of metabolic apparatus and regulating mechanism which should be addressed. In spite of problems and difficulties there is significant progress in acquiring genetically engineered microbes for improved agricultural purpose (Armarger 2002). However, further studies are required for better understanding of the different aspects of P-solubilizing bacteria for their better use in sustainable agriculture.

5.4.5

Bacterial Endophytes as Crop Bio-Inoculant

The conventional agricultural system is dependent on agrochemicals comprising phosphate fertilizers to achieve enhanced crop yields. These chemical fertilizers are not completely utilized by the crop plant and persist in the soil and disturb the rhizospheric microbial community (Ai et al. 2012). The extensive application of chemical fertilizers causes hazardous effect on environment sustainability. Therefore, due to high cost and hazardous impact of chemical fertilizer on environment,

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(López-Bellido et al. 2013) it become crucial to find cost-effective alternatives such as bio-inoculants which could be economical and environment friendly for sustainable agriculture (Adesemoye and Kloepper 2009). P-solubilizing microbes are considered as successful approach for providing proper nourishment (Martins et al. 2004) and used as soil inoculants to amplify growth and yield of crop plants (Otieno et al. 2015). Soil microbial communities play a significant role in solubilizing and mineralizing inorganic and organic form of phosphorous to make it accessible to plants (Adhya et al. 2015). P-solubilizing bacterial inoculants can be produced by the following steps: 1. Soil sample collection. 2. Serial dilution of soil samples. 3. Inoculation of serially diluted samples on desired media with sources of insoluble phosphorous. 4. Isolation, screening and selection of P-solubilizing bacteria producing clear halo zone around the colonies (halo zones indicate P solubilization). 5. Bioassay of phosphorous solubilizing ability of isolated bacterial strains. 6. Identification and characterization of PSB. 7. Plant-growth promoting activities were assessed. 8. Selection of appropriate carrier and development of bio-inoculants (microphos). 9. Field / pot trials of microphos. 10. Standardization. 11. Commercially prepared for agricultural implementation. Hence, using P-solubilizing bacteria as bio-inoculants will reportedly enhance the uptake of P through plants (Chen et al. 2006). The microphos including P-solubilizing bacteria can be utilized in a distinct way i.e. as seed treatment, seedling root dip and soil application. Several studies revealed that P-solubilizing species of Rhizobium, Bradyrhizobium and Azotobacter in leguminous and non-leguminous plant enhance the P-content and growth of the plant.

5.4.6

Conclusions

Phosphorous is of paramount importance for plant nutrition after nitrogen. The adverse environmental effect, depleting rock phosphate and increasing price of chemical phosphate fertilizer have compelled to find sustainable method of agriculture to accomplish the increasing demand of food for the ever-increasing human population. Therefore, it is very important to make substitutes for chemical P fertilizers that are cost-effective. In this perspective, the research on P-solubilizing endophytic bacteria gained interest and used as economically efficient bio-inoculants or biofertilizers. Phosphorous solubilizing or mineralizing bacteria play a significant role in maintaining sustainable agriculture. However, limited knowledge and understanding of P-solubilizing bacteria has been achieved till date and require detailed study of interactions of microbes in the rhizosphere and their P- solubilizing

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potential from different fractions of soil. The phosphate solubilizing bacteria unfold new opportunities for extensive research to identify and characterize more phosphate-solubilizing endophytic bacteria with pronounced efficiency and as a result it can be used as biofertilizers in the field conditions. This can be achieved by extensive research on genetic engineering of specific P- solubilizing bacteria for strain improvement to get target results, and this technology should be transferred to farmers for better and eco-friendly agricultural practices.

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Endophytes of Medicinal Plants: Diversity and Bioactivity Sandeep Kumar Singh, Vipin Kumar Singh, Dharmendra Kumar, Dinesh Prasad Gond, and Ajay Kumar

Abstract

Plants are home to a diverse range of microbial communities. These microbes are present either on the epiphytic regions or inside the plant tissue as endophytes and play an integral role in growth promotion, phytopathogen control, and the management of various biotic and abiotic stresses. In this chapter, we summarized the diversity pattern of endophytic microorganisms associated with medicinal plants and their potential role in metabolite synthesis within the host plant. Medicinal plants harbor a plethora of bioactive compounds that are directly or indirectly used for the treatments of various human ailments. The endophytic diversity pattern of medicinal plants will provide insight into their potential and beneficial use, especially for the modulation of bioactive compounds. Keywords

Endophyte · Bioactive compounds · Diversity medicinal plants · Plant growth promotion

S. K. Singh · V. K. Singh · D. Kumar Centre for Advanced Study in Botany, Banaras Hindu University, Varanasi, Uttar Pradesh, India D. P. Gond Department of Endocrinology and Metabolism, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India A. Kumar (*) Department of Postharvest Science, Agriculture Research Organization (ARO), The Volcani Centre, Rishon LeZion, Israel # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. K. Singh et al. (eds.), Bacterial Endophytes for Sustainable Agriculture and Environmental Management, https://doi.org/10.1007/978-981-16-4497-9_6

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6.1

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Introduction

Microbes are an integral part of plants and interact in diverse ways such as growth promotion, phytopathogen management, and the management of various biotic and abiotic stresses (Kumar et al. 2018; Singh and Jha 2017; Singh et al. 2019). In fact, every plant is home to some specific microbial communities that vary from organs to tissue. Plants secrete various exudates or chemicals via plant roots or the rhizosphere, which are generally composed of amino acids, lipids, phenols flavonoids, and polysaccharides. These exudates act as chemoattractants or signal molecules for the microbial population, including bacteria, fungi, and actinomycetes, to achieve effective colonization (Kumar et al. 2015, 2016a). Microbes enter plant tissues and reside as endophytes without causing apparent diseases. These endophytes act as an integral part of the host plant and play a significant role in regulating various physiological processes, growth promotion, phytohormone synthesis, and elevating the defense response of the host plant (Kumar et al. 2016b). Currently, endophytes are a fascinating research topic due to their broad range of applications, including sustainable agriculture as a plant growth regulator, in phytopathogen management (Singh et al. 2017a, 2020), abiotic stress management (Eid et al. 2019; Singh et al. 2018a), bioremediation of environmental contaminants (Gupta et al. 2020), and bioactive compound synthesis (Singh et al. 2017b). Endophytes have been isolated in almost all the plant species, equaling nearly 30,000, but only 6–7% of them have been explored (Strobel and Daisy 2003; Zhang et al. 2018; Ling et al. 2014; Hawksworth 2001). Most of the plant species harbor at least one microorganism as endophytes. However, specific environmental conditions irrespective of plant variety and age immensely affect the endophytic diversity (Strobel 2001). Recent omics and approaches, especially the metagenomics studies, enable exploring the endophytic microbiota from diverse habitats. The endophytic microbial composition and diversity depends upon various factors, including genotypes of the host plant, plant organs, sampling time, practices, and the surrounding environmental conditions (Kumar et al. 2020). Generally, the population count of endophytic microbials is comparatively lower than the epiphytic microbiota (Liu et al. 2014). It has been reported that the endophytic microbial population (104–108 per gram of root tissues) is lower than the rhizosphere or the bulk soil (106–109 bacterial cells g
1 soil; Bulgarelli et al. 2013). Indeed, the endophytic microbial population also varies in different organs of the same plant. The concentration of bacterial microbiota in the root zone is comparatively higher than the upper stem, leaf, flower, and fruits (Zinniel et al. 2002: Compant et al. 2011; Hardoim et al. 2015).

6.2

Entry and Colonization of Endophytes

The entry of endophytes inside the tissue is a complex phenomenon that comprises the attachment, entry, or colonization of the microbes into the host plant. The endophytic microbes generally preferred natural openings such as stomata, lenticels,

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wounds, germinating radicles, site of root branch emergence, and root hairs at the any stage of the life cycle. During entry, endophytic strains preferred to cross through the cortex zone of endodermis or the emergence site of root tissue to reach the cortex or vascular tissue of the root (Gupta et al. 2019; Kumar et al. 2020). The wounds present on the plant surface or tissue, generally formed by several biotic factors, including insect or nematodes attack, or the abiotic factors, such as temperature fluctuation, root pruning, and tillage, also act as entry sites for endophytes (Quadt-Hallmann et al. 1997). However, some of the studies also reported that endophytic strains secrete cell wall degrading enzymes such as cellulose and pectinase that help endophytic microbes enter host tissue (Benhamou and Chet 1996; Quadt-Hallmann and Kloepper 1996).

6.3

Movement and Localization of Endophytes

The microbial strains associated with the host plants colonize the host tissue either via vertically or horizontally. The term vertically is used for the transmission of microbes via the seed or the offspring and horizontally via the environment or mixed methods. Generally, vertical transmission is preferred by the microbial strains that show a symbiotic, obligate relationship with the host plants to complete their life cycle within the host tissue and are unable to survive outside the host plant (Gupta et al. 2019). However, after entry inside the host tissue, endophytic strains either localize at the host tissue or move systemically with the help of vascular tissue or the apoplast (James et al. 1994). It has also been mentioned that sometimes endodermis act as a physical barrier in the movement of endophytes from the cortex to vascular system (Kloepper and Beauchamp 1992). The colonization pattern of the endophytic bacterial strains are strain specific and depend upon the genotypes of the host plant. Most often, the endophytes begin colonization in intercellular spaces (Hurek et al. 1994). Hurek et al. (1994) studied the colonization route of the endophytic strain Azoarcussp in the Keller grass and found that the strains proliferated in the intercellular space and moved through the stele and reached the parenchyma cells. Levanony et al. (1989) reported that the strain Azospirillum brasilense was present only in intercellular spaces of the wheat and grasses root cortex and not in the endodermal layer of the vascular system.

6.4

Medicinal Plants

Ancient medical systems such as Ayurveda have acknowledged several plants for their medicinal properties against a range of human diseases, including jaundice, fever, etc., and these methods of healthcare management have been widely practiced in the Indian subcontinents. Even to date, a large number of the global population living in rural and remote areas remain dependent on folk and traditional medicines (Singh et al. 2018b). Recently, these medicinal plants have been explored for their medicinal or pharmaceutical properties. Currently, numerous pharmaceutical drugs

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have been reported in Allopathic or Ayurveda practices of treatments that are derived from plant or plant secondary metabolites (Taylor 2000; Newman and Cragg 2007). These natural plant products or metabolites are broadly utilized in the pharmaceutical field as analgesic, antibiotics, antipyretics, diuretics, cancer treatments, and even HIV treatments (Makkar et al. 2009; Vaishnav and Demain 2011). According to the World Health Organization (WHO) any plant or plant organs that contain substances of pharmaceutical or therapeutic use or are precursors of pharmaceutical products are defined as medicinal plants. According to a report, approx. 80% of the global population directly or indirectly depends upon medicinal plants or plant metabolites for the treatment of various human ailments (Balick et al. 1996). Plants secrete various phytochemicals, alkaloids, and metabolites in the form of side products of the biochemical cycle. Based on their role, phytochemicals are classified into primary and secondary metabolites. However, the major metabolites used in the medical industry are plant secondary metabolites in the form of alkaloids, polyphenols, glycolipids, tannins, terpenoids, and sequiterpenoids, and after isolation, purification, or structural modification, these compounds are formulated as drugs or medicines (Shukla et al. 2014; Leicach and Chludil 2014; Singh et al. 2018b). For decades, researchers and pharmaceutical companies have been heavily exploiting the natural metabolites of plants in the form of raw materials for a broad range of pharmaceutical products or direct use as herbal medicines.

6.5

Endophytic Diversity in Medicinal Plants

The diversity and variation in the endophytic microbial composition depends on and varies with the seasons, genotypes, developmental stages, and tissue types of the host plants (Strobel and Daisy 2003; Liu et al. 2014). Endophytic microbes, including both bacteria and fungi, have been isolated and reported in numerous plant species (Zhao et al. 2010a, b; Miller et al. 2012; Kaul et al. 2012; Liu et al. 2014; Jasim et al. 2014; Kumar et al. 2016b). Medicinal properties have been reported in various herbs, rhizotomous herbs, shrubs, and trees, and their products have been directly or indirectly utilized in the treatment of various human ailments. There are numerous reports available regarding microbial diversity in medicinal plants. Kumar et al. (2016b) reported six cultivable endophytic bacterial strains—Bacillus cereus, Bacillus thuringiensis, Bacillus sp., Bacillus pumilis, Pseudomonasputida, and Clavibacter michiganensis—from the rhizome of Curcuma longa L. and all these endophytic strains have shown plant growth promoting attributes. Some of the fungal endophytes, Eurotium sp., have been reported by Jalgaonwala and Mahajan (2014) and 44 fungal strains have been reported by Bustanussalam et al. (2015) in the turmeric plant. Curcuma longa L., commonly known as turmeric, is a rhizotomous herb and has been broadly utilized in the Indian subcontinent since antiquity as an antipyretic, analgesic, antibacterial, antiviral, antimycoidal, anticancerous, anti-HIV, and is nowadays used to boost the immune system against COVID-19. Curcumin is one of the main constituents of turmeric and is frequently utilized in the treatment of various human diseases (Kumar et al. 2016a, 2017; Rao

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et al. 1995; Srimal 1997; Mukerjee and Vishwanatha 2009; Panahi et al. 2014). Ayob and Simarani (2016) isolated endophytic filamentous fungi Colletotrichum sp., Macrophomina phaseolina, Nigrospora sphaerica, and Fusarium solani from Catharanthus roseus. The herb C. roseus is an ornamental plant, and its alkaloids vincristine and vinblastine have been very widely used for their anticancer properties. Akinsanya et al. (2015) reported 29 endophytic bacterial strains from the root, stem, and leaf of Aloe Vera, which were classified into 13 different genera Aeromonas Bacillus, Cedecea, Cronobacter Enterobacter, Chryseobacterium, Pantoea, Shigella Sphingobacterium, Providencia, Klebsiella, Macrococcus, and Pseudomonas. Some of the bacterial strains of these genera were actively secreting bioactive compounds. Similarly, Chandrakar and Gupta (2019) isolated endophytic Streptomyces parvulus actinomycetes from the roots of an Aloe Vera strain having novel antimicrobial properties against some of the multidrug resistance strains. Kumar et al. (2015) isolated Bacillus subtilis, Agrobacterium tumefaciens, Bacillus sp., Pseudomonas putida, and Pseudomonas sp. endophytic bacterial strains from the Cassia tora L., and these bacterial strains have shown plant growth promoting activities. Ginger (Zingiber officinale) is a household spice and traditional medicine having antioxidant, antimicrobial, and analgesic properties that is commonly used in the Indian subcontinents (Aggarwal and Shishodia 2004). Zhang et al. (2018) reported 57 bacterial strains from the ginger rhizome, which belong to the genera Acinetobacter, Agrobacterium Bacillus, Enterobacter, Serratia, Pseudomonas, Stenotrophomonas Tetrathiobacter, and Ochrobactrum; in addition, Agrobacterium Bacillus, Enterobacter, Pseudomonas, Stenotrophomonas, and Serratia, as endophytic strains were reported by Jasim et al. (2014). Rustamova et al. (2020) isolated endophytic bacterial strains Micrococcus endophyticus, Bacillus megaterium, Pseudomonas chlororaphis, P. kilonensis, Stenotrophomonas pavanii, B. endophyticus, S. maltophilia, Pantoea ananatis, B. atrophaeus, and M. favus from Vernonia anthelmintica and found these bacterial strains showed biological properties such as antimicrobial, anti-vitiligo, and antidiabetic similar to the plants. This indicates that properties of the endophytes may be modulated by the plant physiological properties. Details of the diversity pattern of endophytic microorganisms in medicinal plans are described in Table 6.1.

6.6

Endophytic Microbes in Metabolites Production

Microbes, especially the endophytes, synthesize and play an important role in co-production of similar metabolites to those of the host (Aly et al. 2013; Kusari et al. 2013). These metabolites have been broadly utilized against the treatment of various human diseases. The co-evolution and growth of endophytic microorganisms share a symbiotic relationship between the host plants, which directly or indirectly modulates the growth and metabolite synthesis of the plants (Jia et al. 2016; Chaturvedi et al. 2014; Sadananda et al. 2011). Indeed, these endophytic microorganisms modulate metabolite synthesis either partially or

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Table 6.1 Diversity of endophytic microorganism in different medicinal plants Host plant Caralluma acutangula, Rhazya stricta, and Moringa peregrina Asclepias sinaica

Curcuma longa L.

Teucrium polium L.

Catharanthus roseus

Saraca indica

Collected from Oman’s sultanate (57 400 07.0000 E; 23 040 22.0000 N)

Endophytes Phoma sp., Alternaria sp., Cladosporium sp., Bipolaris sp.

Mechanism Crop growth and reduction of oxidative stress

References Khan et al. (2017)

Saint Katherine, South Sinai of Egypt, Ain Shakaya (28.543386 N, 33.933071 E) Botanical Gardens of BHU, Varanasi, India (80 36 E, 20 180 N; 80.71 m height above sea-level)

Alternaria alternata Aa_27, Penicillium chrysogenum Pc_25

Improve plant growth

Fouda et al. (2015)

Bacillus pumilis, P. putida, B. cereus, Clavibacter michiganensis, B. thuringiensis

Kumar et al. (2016b)

Wadi AlZwatin, Saint Katherine Protectorate, Egypt (28.539290 to 28.53919 N, 33.930784 to 33.92044 E). Peat soil in Alor Setar in Kedah, North of Peninsular Malaysia (6.1167 N, 100.3667 E) Jabalpur Dumna Forestry Area, India

B. subtilis, Penicillium chrysogenum, P. crustosum, B. cereus

Secretion of growth hormones, siderophore and growth inhibition of K. pneumoniae, E. coli; A. alternata, F. solani Improve plant growth

Colletotrichum sp., Macrophomina phaseolina, Fusarium solani, and Nigrospora sphaerica

N.A.

Ayob and Simarani (2016)

Phomopsis sp., Phialophora sp., Phyllosticta sp., Aspergillus terreus Xylaria sp., Phomopsis sp. P. putida, A. tumefaciens, B. subtilis, Pseudomonas sp.

Antibacterial activity

Sandhu et al. (2014)

N.A.

Nath et al. (2012) Kumar et al. (2015)

E. officinalis

Meghalaya, India

Cassia tora L.

Campus BHU, India (80 360 E, 20 180 N, height above sea-level 80.71 m)

PGP attributes

El-Din Hassan (2017)

(continued)

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Table 6.1 (continued) Host plant Plectranthus tenuiflorus

Collected from Taif Province in northwestern Saudi Arabia, 99 4400 and 79 450 E

Tinospora cordifolia, Phyllanthus amarus, Azadiracta indica, Carica papaya

Biligirangana Hill, the region of the eastern Ghats Chamarajanagar, Karnataka ranges from 77 010 to 77 050 east and 12 090 to 11 400 north The Ayurvedic and Siddha, Uzhavoor, Kottayam Study Centre, Navajyothisree Karunakara Guru NBAIM, Maunath Bhanjan (25 520 5300 N; 83 330 3200 E); Uttar Pradesh Nursery field for Sungai Buloh Selangor, Malaysia (3.235283 N, 101.568342 E) CSIR-NBRI (Banthara unit) experimental areas, Lucknow, India (80 5900 E; 26 5500 N,) Shimla, NBPGR Regional Station India (77.1734 E
31.1048 N)

Zingiber officinale

Ocimum tenuiflorum L.

Aloe vera

Withania somnifera

Artemisia annua L.

Piper longum

Thrisshur, Kerala NBPGR India

Endophytes B. licheniformis, Paenibacillus sp., B. megaterium, Pseudomonas sp., Micrococcus luteus Leptosphaerulina arachidicola, Lycopersici stemphylium, Phomopsis azadirachtae, Epicoccum nigrum

Mechanism Antimicrobial and extracellular enzyme activity

References El-Deeb et al. (2013)

Bioactive compound

Sushma et al. (2018)

Stenotrophomonas sp., Pseudomonas sp., Staphylococcus sp., Bacillus sp.

Production of growth promoting attributes

Jasim et al. (2014)

B. safensis, B. tequilensis, B. haynesii, B. siamensis, B. paralicheniformis, B. altitudinis Macrococcus caseolyticus, B. anthracis, P. hibiscicola

Biocontrol activity against blight disease

Sahu et al. (2020)

Production of bioactive compounds

Akinsanya et al. (2015)

P. fluorescens, B. amyloliquefaciens

Growth enhancer, or secondary metabolites production

Mishra et al. (2018)

Cochliobolus lunatus, Curvularia pallescens, Colletotrichum gloeosporioides, Acremonium persicum Enterobacter sp., Alishewanella sp.,

Increase biomass and artemisinin content

Hussain et al. (2017)

Produces HCN and IAA, growth promotion and

Mintoo et al. (2019) (continued)

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Table 6.1 (continued) Host plant

Collected from

Endophytes

Mechanism

Bacillus casamancensis

biocontrol activity

References

completely. There are several reports available that showed the efficacy of endophytic microbial strains in synthesizing similar metabolites with the host plants (Nair and Padmavathy 2014; Kaul et al. 2013). Zhao et al. (2010a, b) reported on several endophytic strains that synthesized vincristine & vinblastine, anticancer metabolites. Strobel and Daisy (2003) studied the strain Rhinocladiella sp., which synthesized the compounds cytochalasins, having anticancer and antibiotic properties. Similarly, Cui et al. (2012) reported on the compound Ginkgolide B synthesized by the endophytic strain Fusarium oxysporum used in the treatment of cardiovascular disease. Parthasarathy and Sathiyabama (2014) studied the compound Gymnemagenin synthesized by the endophytic strain Penicillium oxalicum that has anti-diabetic properties. Similarly, Yin et al. (2009) reported that Gentiopicrin synthesized by endophytic strains has antimalarial properties. In addition to these, the host and endophytic microbes modulate some of the important alkaloids that are used in various other aspects, including contamination degradation, flavoring agents, etc. Zabetakis (1997) reported that furanoids, preset in strawberries, having an active role in flavoring, are also present in their endophytes. Van Elst et al. (2013) studied pavettamine present in Fadogia, Pavetta, and Vangueria, and reported that it was present only in the endosymbioant bacterial strain Burkholderia spp., which might be due to endophytic mediated synthesis in the host plant. Similarly, Li et al. (2012) reported about modulation in the artemisinin of Artemisia after inoculation of Pseudonocardia.

6.7

Conclusion and Future Prospects

In the present scenario, endophytic microbiota are broadly used in sustainable agriculture for growth promotion, phytopathogen control, or for the management of various abiotic stresses. However, endophytic microbiota have recently gained increased attention due to bioactive compounds or secondary metabolites and their broad range of applications in the pharmaceutical industry. Endophytic microbes produce metabolites in association with the host plant. Currently, a myriad of herbal or medicinal plants are being extensively studied for medical applications because of their cost effectiveness and fewer side effects. Therefore, there is an urgent need to explore the associated endophytic microbes of medicinal plants and their products for beneficial uses in the pharmaceutical or nutraceutical industries.

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Shukla ST, Habbu PV, Kulkarni VH, Jagadish KS, Pandey AR, Sutariya VN (2014) Endophytic microbes: a novel source for biologically/pharmacologically active secondary metabolites. Asian J Pharmacol Toxicol 2(3):1–6 Singh RP, Jha PN (2017) The PGPR Stenotrophomonas maltophilia SBP-9 augments resistance against biotic and abiotic stress in wheat plants. Front Microbiol 8:1945 Singh VK, Singh AK, Kumar A (2017a) Disease management of tomato through PGPB: current trends and future perspective. 3 Biotech 7(4):255 Singh M, Kumar A, Singh R, Pandey KD (2017b) Endophytic bacteria: a new source of bioactive compounds. 3 Biotech 7(5):315 Singh VK, Singh AK, Singh PP, Kumar A (2018a) Interaction of plant growth promoting bacteria with tomato under abiotic stress: a review. Agric Ecosyst Environ 267:129–140 Singh AK, Singh SK, Singh PP, Srivastava AK, Pandey KD, Kumar A, Yadav H (2018b) Biotechnological aspects of plants metabolites in the treatment of ulcer: a new prospective. Biotechnol Rep 18:e00256 Singh M, Singh D, Gupta A, Pandey KD, Singh PK, Kumar A (2019) Plant growth promoting rhizobacteria: application in biofertilizers and biocontrol of phytopathogens. In: PGPR amelioration in sustainable agriculture. Woodhead Publishing, Cambridge, pp 41–66 Singh M, Srivastava M, Kumar A, Singh AK, Pandey KD (2020) Endophytic bacteria in plant disease management. In: Microbial endophytes. Woodhead Publishing, Cambridge, pp 61–89 Srimal R (1997) Turmeric: a brief review of medicinal properties. Fitoterapia 68:483–493 Strobel G (2001) Stegolerium kukenani gen, et sp nov., an endophytic taxol producing fungus from the Roraima and Kukenan tepuis of Venezuela. Mycotaxon 78:353–361 Strobel G, Daisy B (2003) Bioprospecting for microbial endophytes and their natural products. Microbiol Mol Biol Rev 67(4):491–502 Sushma KS, Jayashankar M, Vinu AK, Saeed MA (2018) Identification of endophytic fungi from the medicinal plants of Biligirirangana hill, Karnataka. J Appl Nat Sci 10(4):1156–1161 Taylor L (2000) Plant based drugs and medicines. Rain Tree Nutrition, Carson City, NV, pp 1–5 Vaishnav P, Demain AL (2011) Unexpected applications of secondary metabolites. Biotechnol Adv 29(2):223–229 Van Elst D, Van Wyk B, Schultz A, Prinsen E (2013) Production of toxic pavettamine and pavettamine conjugates in the gousiekte-causing Fadogia homblei plant and its relation to the bacterial endosymbiont. Phytochemistry 85:92–98 Yin H, Zhao Q, Sun FM, An T (2009) Gentiopicrin-producing endophytic fungus isolated from Gentiana macrophylla. Phytomedicine 16(8):793–797 Zabetakis I (1997) Enhancement of flavour biosynthesis from strawberry (Fragaria x ananassa) callus cultures by Methylobacterium species. Plant Cell Tiss Org Cult 50(3):179–183 Zhang Y, Kang X, Liu H, Liu Y, Li Y, Yu X, Zhao K, Gu Y, Xu K, Chen C, Chen Q (2018) Endophytes isolated from ginger rhizome exhibit growth promoting potential for Zea mays. Arch Agron Soil Sci 64(9):1302–1314 Zhao J, Zhou L, Wang J, Shan T, Zhong L, Liu X, Gao X (2010a) Endophytic fungi for producing bioactive compounds originally from their host plants. Curr Res Technol Educ Trop Appl Microbiol Microbial Biotechnol 1:567–576 Zhao L, Deng Z, Yang W, Cao Y, Wang E, Wei G (2010b) Diverse rhizobia associated with Sophora alopecuroides grown in different regions of loess plateau in China. Syst Appl Microbiol 33(8):468–477 Zinniel DK, Lambrecht P, Harris NB, Feng Z, Kuczmarski D, Higley P, Ishimaru CA, Arunakumari A, Barletta RG, Vidaver AK (2002) Isolation and characterization of endophytic colonizing bacteria from agronomic crops and prairie plants. Appl Environ Microbiol 68 (5):2198–2208

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Biotechnological Applications of Bacterial Endophytes Mohit Mishra, Sudheer Pamidimarri, V. Balasubramanian, Sneha Kumari, Shalini Pandey, Bhairav Vaibhav, and Sushma Chauhan

Abstract

Endophytes are microorganisms such as fungi and bacteria that can exist within healthy plant tissues and cells by making colonies within them. Endophytes coexisting with plant systems are involved in a broad range of associations such as symbiotic, communalistic, mutualistic, and trophobiotic. Their mutualistic relationship and synergistic interaction assist in producing valuable chemicals, hormones, nitrogen rich nutrients, and secondary metabolites. In addition, endophytic microbes are able to enhance and promote the growth rate of plants under biotic and abiotic stresses. Endophytic association assists the host plant in counteracting these biotic and abiotic stresses and acquiring tolerance. A promising area of study involves the endophyte symbiotic relationships and synergetic interactions. This will help to understand the product of the mutual relationship and can help in exploring these aspects to develop strategies for future genetic improvement of crop plants to promote stress tolerance. Because of such characteristic features, currently, endophytes are gaining increased attention for numerous biotechnological applications. Additionally, endophytes in association with host systems are unique systems showing promising applications in phytoremediation. On the other hand, sustainable production of biomass is also possible from the non-edible plants. Furthermore, it is also believed that a few groups of endophytic microorganisms can synthesize nanoparticles naturally (in vivo) in association with hosts, expanding their applications in nanobiotechnology. In this chapter, a comprehensive discussion of the

M. Mishra · S. Pamidimarri · V. Balasubramanian · S. Kumari · S. Pandey · S. Chauhan (*) Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, India e-mail: [email protected] B. Vaibhav Vidya Jyoti Institute of Higher Education, Dera Bassi SAS Nagar, Punjab, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. K. Singh et al. (eds.), Bacterial Endophytes for Sustainable Agriculture and Environmental Management, https://doi.org/10.1007/978-981-16-4497-9_7

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endophytic association and its applications in several biotechnology fields were presented. Keywords

Endophytes · Phytoremediation · Biofuels · Hormones and nanoparticles · Synergetic interaction · Symbiotic

7.1

Introduction

Endophytes are microorganisms associated with various tissues of plants that live without interfering with the host plant’s basic physiology. Unlike pathogenic microbes, endophytes exhibit a harmless association with plants. The term endophyte was introduced by de Barry in 1866, and related research has been highly active in the past few decades because of the many synergistic benefits endophytes provide to plant systems. Bacteria, fungi, and actinomycetes are the most common microorganisms associated with plants. The reported studies found that endophytic microorganisms protect themselves from adverse conditions by establishing close association with their host plants and are insulated from extreme climate (Arora and Ramawat 2017). However, due to resource limitations and the cost of supporting, some endophytes opportunistically turn from symbiotic association to commensalism or parasitic association. The endophytic symbiotic relationship is generally the association of two or more different species together. This symbiotic association is considered as a positive endophytic relation, but if it turns negative for one or the other, then it leads to communalism. Similarly, commensalism also benefits the host plant after the interaction takes place between the host plant and endophytic microorganism; likewise, the commensalism occurs when microbial groups become modified in response to environmental changes (Ibáñez et al. 2017). The most common endophytic microorganisms are commensals, which have undiscovered metabolic functions in plants. For this reason, endophytic research has become one of the most studied areas in the field of plant interactions with microorganisms (Yan et al. 2019). Most of the endophytic research activity is centered on positive association (mutualism), and later studies have focused on the negative activity of microbial associations (antagonism) included in the pathology. These properties have been tested in a single species of plant and within the closely related group of plant genotype, but rarely over the wide taxonomical spectrum of plants. However, establishing the suitable environmental conditions where the association of endophytes–plant interaction are analyzed remains underdeveloped, and in many species, they have been hardly investigated (Ibáñez et al. 2017). Therefore, bacterial and fungal endophyte communities are being investigated separately to study the more detailed mechanism of their mode of association with the host and their separate response regarding biotic and abiotic stresses (Ren et al. 2011). In this chapter, discussions were presented on various aspects of endophytic association with plants and its synergic mutual benefits.

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History of Endophytes

An endophyte was first described by the German botanist Heinrich Friedrich Link in 1809 and named an endophytae. In the nineteenth century, it was believed that the nutritious and naturally growing plants were sterile and hence free of microorganisms (Compant et al. 2012). Galippe et al. reported that various microorganism were seen in the interior of vegetable plants, and those microorganism were mostly bacteria and fungi that originated from the soil ecosystem and migrated into the plants, where they formed a beneficial relationship with the host plant. The confirmation of the occurrence of beneficial microorganisms in plants occurred in the late nineteenth century and early twentieth century; thus, the existence of the beneficial microorganisms known as endophytes was known (Blanc et al. 2012). For the past two decades, researchers have been investigating the endophytic association with host plants for various kinds of microbial endophytes, such as bacteria, fungi, archaea, and unicellular eukaryotes, including amoebae and algae. Nowadays, exploration of endophytic application occurs in several fields of research. In 1888, the Dutch microbiologist Martinus Willem Bejerinck successfully isolated the root nodule bacteria, which were efficient for atmospheric nitrogen fixation, from Leguminosae plants nodules, and they were later named rhizobium leguminosarum (Martin and Schwab 2013). In addition, Hermann Hellriegel and Hermann Wilfarth showed that the leuminous plants serve as important symbiotic hosts and are capable of nitrogen fixation by rhizobia. Another mutualistic symbiotic (relationship between root of plant and underground bacteria or fungi) relationship between the plant and microorganism was reported by Albert Bernhard Frank, and he introduced the term mycorrhiza. More recently, in 1999, Orland Petrini stated that all organisms occupying plant organs can at some time in their life cycle colonize with internal plant tissue without having a harmful effect on the host plant (Petrini 1991). Over the past decade, endophyte studies have shown several applications in various fields, including agriculture, pharmaceuticals, and enzyme industries.

7.3

Endophytes and Their Mode of Association with Host

The endophytic association of plants with bacterial/fungi has been actively investigated over the past couple of decades by many researchers; however, the associations are diverse, and hence remain ambiguous. Nevertheless, the association between endophytes and plants takes place by two major events: (1) The endophytes generally reside in the rhizosphere and enter inside the host via various routes, which include root wounds, surface of leaves via stomatal opening, injured stem, etc. (Ren et al. 2011); (2) The chloroplast and mitochondria play an important role in the endophytic origination and are believed to have comparable genetic support, which assists the mutual association with the host plant. Through the mutual association, endophytes could be able to influence the physiological and molecular responses toward environmental changes and biotic stresses. It has been well established that some endophytes provide drought resistance, which was reported by Arora et al.

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(2017). Similarly, through association with endophytes, popular species such as Paspalum vaginatum (turf grass) and forage legume also showed enhanced drought resistance (Arora and Ramawat 2017). Studies have also been conducted on the association of various endophytic species, and their relative efficacy of resistance toward biotic and abiotic factors varies. The bacterial species invade the corresponding host species and colonize in the intracellular spaces because of the presence of intracellular fluids with rich minerals and sugars (Liu et al. 2017). Even though there is competition for bacterial association with roots of the host plant, a huge diversity is seen. In addition, fungal association in the plant roots is also very frequent, and reports have indicated that 90% of terrestrial flowering roots show mycorrhizal association. Smith and Read (2008) reported that these associations of fungi with plants aided the host plants to absorb essential mineral nutrients along with water. Also, in return, the endophytic fungi received photosynthetic carbon through the host (Khare et al. 2018). In addition to this, the direct association of plants with mycorrhizal fungi can help in better performance in host physiology and results in good agronomical outcome (Kogel et al. 2006; White Jr and Torres 2010). Also, endophytic association is reported to induce flowering in host plants. In 1995, Musselman and Press studied the mode of flowering in plants, and they found that endophytic association induced the flowering in the plants (Mus et al. 2016; Puri et al. 2018). These studies were conducted over 4000 species, including trees, herb, and shrubs from tropical forests. In the later part of this section, the discussion will be presented on the association of various endophytes with the host plant.

7.3.1

Symbiotic Association of Fungi with Host

Fungal species are the most diverse species that form mutual associations with plants. Upon association, endophytic fungi can provide the host plant with various benefits, some of which have industrial potential. A noteworthy example of this context is the association of actinomycetes endophytes with host plants that induces the host plant species to produce several value-added chemical substances, which have potential medical properties (Pinski et al. 2019). Endophytic fungi live noninvasively within the plant part, and this association is said to be synergetic with the host plant because they accept nutrition and protection from the host plant, whereas the host plant receives help from endophytes by being protected from pathogens, herbivores, and different abiotic stresses (Latch 1993; Compant et al. 2005). Most endophytes are derived from the surrounding environment, and a variety of them are often transferred through seed or vegetation. The association of fungi imparts many beneficial effects in host plants. The association of endophytes promotes various vegetation aspects. They can stimulate root growth through the secretion of auxin within the plants, which is the usual mechanism of indole-3-acetic acid secretion. Indole-3-acetic acid along with auxin promotes plant growth, which further stimulates the cell growth, and influences the reaction toward light, gravity, and tissue distinction (Fisher et al.

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1992). Fungi endophytes are well documented to improve the drought resistance of plants, and it has been recognized in various species of fungi. Endophytic fungi can occur in almost all habitats and occur mostly in agricultural grasses. Furthermore, those endophytes provide drought resistance, protection from seed predators and water stress. In turn the endophytes receive nutrition and other mineral support from the host plant. According to a previously conducted survey of grassland, 25 species out of 41 species of grasses were found to be associated with Neotyphodium endophytes. This mutualistic association is known to improve the drought resistance potential in the population of wild grass (White Jr and Torres 2010). Morse et al. found that in Festuca arizonica vasey endophytic association was beneficial because the host was able to survive under the condition of low water availability (Morse et al. 2002). Rudgers and Swafford (2009) reported that the association of fungal endophytes declined under the condition of drought stress in Elymus virginicuse while benefiting the host, but no benefits have been reported for endophytic infection in Festuca rubra and F. pratensis (White Jr and Torres 2010). In the case of fungal species, Colletotrichum was reported to be associated in two ways: it can be either parasitic or mutualistic based on the host genotype, and this species of fungus can establish different lifestyles while colonizing in the distinct host. For example, C. magna species are known to be a virulent pathogen in cucurbits, but it also exerts an endophytic mutualistic lifestyle on various other species such as tomato, wheat, potatoes, etc. (Puri et al. 2018).

7.3.2

Symbiotic Association of Endophytic Bacteria with Host

Endophytic bacteria have a remarkable role in symbiotic relationships with their host. They are generally found in intercellular spaces and xylem vessels of the host plant. Endophytic and host interactions are established on mutual exploitation (Compant et al. 2010). Their consortium is often induced or elective and causes no damage to the host plants. They show a complex interchange with their host that incorporates hostility and mutualism (Glassner et al. 2018). Endophytic bacteria generally occupy the aboveground plant tissues, which differentiate them from the more well-known mycorrhizal symbionts such as mycorrhizae endophytic fungi. They are by concept interrelated synergistically with their host plants mainly by expanding host resistance to herbivores. Endophytic bacteria can suppress the proliferation of nematodes, which can benefit other crops in rotation with a host plant. Endophytic bacteria can solubilize phosphate and contribute to phosphate assimilation reported in the soybean plant. Also, there are a few endophytic bacteria that have the capability to stimulate and develop immunity in the host plant (Vinagre et al. 2019). Endophytic bacteria have been well studied regarding plant growth promotion, phosphate-solubilizing ability, developing the immune system, secondary metabolite production, and production of several antibiotics in the host. These features frequently prompt scientists to focus on these fields to gain complete information and knowledge about their association. After an in vitro study carried out by culturing different species of endophytes from various sources, scientists were

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able to understand to some extent their mutualistic relationships. Endophytes have a plant defense mechanism, pathogen antagonistic substances for colonization, and availability of nutrition. Salmonella species is one of the bacterial species that have been reported as an endophyte in alfalfa sprouts, and they have a mutualistic relationship with each other. It seems that bacteria have adapted for symbiotic living in a host and are selected naturally. Endophytic bacteria produce adenine ribosides, which are responsible for the stimulation of expansion. Endophytes found in potato plants are reported to show antagonistic activity against the fungus Curtobacterium flaccum, which results in better persistence to pathogenic infection. Endophytic actinobacteria are also stated to be effective antagonists against fungus in wheat (Herrera et al. 2002). It has also been described that bacterial endophytes enhance plant nitrogen usage efficiency by the respective mechanism, which include root growth enhancement, improving the plant metabolism to boost nitrogen, development of additional root hyphae for nutrient absorption, nitrogen fixation, changing soil directly, or modifying root transudes (Fisher et al. 1992). Recently, it has been proposed that synergistics relationship between endophytes and host plants are not being established in either ecological or evolutionary time or through geographically, and it varies from synergetic and antagonistic, depending on genetic strains, phylogeny, and other interrelating species (Fisher et al. 1992). Endophytes alter root development through a unique mechanism, and most of them seem to receive or block plant hormones, which comprise auxin, gibberellin, cytokinin, and ethylene. Root exudate is rich in biomolecules that are magnetized or acknowledged by genial microbes comprising endophytes. They are also rich in water and nutrients, which attracts all the groups of microbes. Flavonoids are classified as chemoattractant, which is one such metabolite that is secreted from a variety of plants and plays a major role in endophytic interaction with root hairs (Wang and Dai 2010). Wang et al. (2017) suggested that endophytes play a crucial role in the depletion of metal phytotoxicity along with extracellular precipitation, intracellular aggregation, confinement or bioprocessing and also in the bio-conversion of toxic compounds to non-toxic compound (Khare et al. 2018). Hence, hosts continue to have stable synergy with endophytes to produce different compounds that enhance the growth of plants and support them to adapt according to the surrounding environment (Davies and Graves 1998).

7.3.3

Cyanobacterial Association with Host

Cyanobacteria, also known as photosynthetic bacteria, often exist as free-living (non-symbiont) organisms in pioneer habitats such as soils in deserts or associated with lichens in another pioneer habitat. Cyanobacteria can establish a symbiotic association with another organism such as Azolla and Cycades. The symbiotic association with Azolla involves cyanobacteria driven into the leaves and is important for nitrogen fixation in rice paddies during the fern-growing phase and then is cultivated into the soil to release nitrogen. Several kinds of free-living bacteria grow

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in close association with plants; for example, Azotobactor and Azospirillum (Bhat et al. 2015).

7.3.4

Interaction of Endophytes to Increase Resistance to Biotic and Abiotic Stress

When plants experience nutrition deficiency, their growth and development rates decline. Many factors are responsible for the inhibition of physiology in plants, for example, an increase or decrease in pH can give rise to acidity or salinity, which results in toxicity. Drought conditions may lead to a slower rate in germination and membrane integrity loss, which causes osmotic stress (Latch 1993). Nowadays, due to population increase, the problem of food security is increasing daily. The majority of crop yields are decreasing by many folds due to natural or anthropogenic biotic and abiotic factors. Endophytes play critical roles in protecting the host plant from various abiotic and biotic stresses and provide tolerance. Moreover, while associating with their host, they also promote plant growth by producing some natural growth promoting compounds. Bacterial endophytes impart tolerance by altering some physiological activities in a plant while facilitating modifications in root morphology, by increasing nitrogen accumulation, increasing metabolism, and maintaining homeostatic conditions with respect to the external environment. Because it is a mutualistic relationship, endophytes are facilitated by the plant’s nutrition and spread through the next generation.

7.4

Host Associated Biotic Factors and Endophyte Ecology

The rhizosphere is an active and competitive micro-ecosystem for microbes to inhabit and obtain nutrients for their survival from the plants (Raaijmakers et al. 2002). Consequently, these microbes act as either pathogens or symbionts and compete to colonize inside the plant and acquire food. These will multiply in the micro-ecosystem and influence the growth and development of plants (Haas and Keel 2003). In context with endophytes, plants will have a positive synergy in their growth and development. The establishment of the association between the endophytes and plants involves three factors: (1) affinity factors elicited by plants; (2) entry of endophytes into plants; (3) colonization of endophytes within the plants and establishment of a mutual relationship. Some significant factors such as compounds produced by plants, e.g., polysaccharides, and motility of the bacteria in the microenvironment of the rhizosphere are recognized as relevant characters that influence the establishment of Azospirillum brasilense and Alcaligenes faecalis growth inside the roots of the plant (Bashan and Holguin 1993; You et al. 1995). Various mechanisms were developed by endophytes for entry into plants, especially through roots (Fig. 7.1). Besides entering through the root system, endophytes acquired through seeds from the parent are also prominent in many cases (Bashan and Holguin 1994). General ways of entry of endophytes inside the tissues of the

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Fig. 7.1 Various modes of entry through wounds or cracks in the roots of a plant and producing enzymes by endophytes

plant are by wounds and cracks in the tissues of the root during the plant growth (Sprent and de Faria 1988; Ringelberg et al. 2012; Santoyo et al. 2016). Endophytes have a wide range of establishment on various tissues of plants. Mitter et al. (2013) reported that utilization of endophytes sprayed on flowers of the plant showed the growth and establishment inside fruits and seeds. Further, the endophytes also enter the plant via openings in leaves and stems, especially in young plants (Roos and Hattingh 1983); gas exchange holes such as lenticels in woody stems; radicles of germinated seeds (Scott et al. 1996; Gagne et al. 2011). Newly raised lateral roots and hair cells are the other ways of endophyte’s entry into the plants (Huang 1986). Hallmann et al. verified that the entry of Enterobacter asburiae JM22 inside the plant tissues occurred with the help of enzyme action that degraded the cellulose present in the cell wall (Hallmann et al. 1997). Although, Herbaspirillum seropedicae cannot produce the enzyme hydrolyzing the cell wall due to the absence of the respective genes (Pedrosa et al. 2011). Diverse endophytic bacteria establish their relationship with plants and colonize various tissues of a plant. Some of the endophytes first colonize on the root of a plant and continue spreading throughout the stems and leaves.

7.4.1

Endophytes Attracted by the Host Plants

Plants selectively secrete chemicals into the soil to facilitate a relationship with microbes. This mutualism could benefit the plant system as well as the endophytic microbe. The biochemical signals via released chemicals attract endophytic bacteria to move toward the plants by chemotactic movement. Biochemical signals support the plant to select the beneficial microbes to establish the colonization inside and symbiotic relationship with the plant’s roots (Bais et al. 2004). In addition to this, leaking of metabolites from the wounds in the roots of the plant also attracts the bacteria toward the site of the wound (Hallmann et al. 1997); however, in this case

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there is a chance that pathogenic microbes might enter into the plant system. Rice seedlings release phytohormones and flavonoids to enhance the Serratia spp. colonization (Balachandar et al. 2006). Rudrappa et al. (2008) reported that Bacillus subtilis FB17, which is selectively attracted by Arabidopsis thaliana and B. subtilis FB17, releases malic acid to control pathogenic infection and act as a biocontrol agent (Rudrappa et al. 2008). Once the efficient bacteria are attracted toward the roots of the host plant and attach to the surface, bacteria start multiplying and attaining a certain level of population leads to the formation of a biofilm. This biofilm permits non-sporeforming bacteria in the soil to make their colony surrounding the environment. The biofilms contain mostly bacterial cells and water. The extracellular polymeric substance (EPS) matrix acts as a physical barrier to prevent the defense compounds diffusion from the host plant and protect from various environmental stresses. In some cases the lysis of cells leads to the release of cellular components, such as DNA, proteins, and other compounds, and helps in special activities (Rinaudi and Giordano 2010). In Gluconacetobacter diazotrophicus PAL5, a nitrogen-fixing bacterium, gumD gene is responsible for the biosynthesis of exopolysaccharide and is required for the formation of biofilm and colonization on the root of the rice plant, and this was identified by the knockout of the gumD gene (Meneses et al. 2011). Moreover, the recognition of bacteria with plants is controlled by effectors of bacteria transported inside the plant tissues through the type III protein secretion system (TTSS) (Carvalho et al. 2012). Hurek and Reinhold-Hurek (2004) studied the Azocarus sp. association and N2 in infected plants via point of emergence of lateral roots and tips of a root by the enzyme endoglucanase (Hurek and Reinhold-Hurek 2004). Moreover, the endoglucanase activity blocked colonization in transposon mutants. They also continued research on glutathione reductase (gr) and superoxide dismutase (sod) genes important for the colonization inside rice plants by G. diazotrophicus PAL5 (Alquéres et al. 2013). The signals of soil bacteria can also be recognized by plants, and they are primarily facilitated by the receptors-like kinase (RLK) in plants such as lectin receptor-like kinases (Lec RLKs), leucine-rich repeat–receptor-like kinases (LRR–RLKs), Lys-motif receptors (LysM), and wallassociated kinases (WAK). The studies also suggest that small interfering RNA (siRNA) and small RNAs (sRNA) such as miRNA are also involved in the signal processing.

7.4.2

Entry of Endophytic Microbes Through Root Into the Plants

Entry sites of endophytes are through the root (Compant et al. 2010) and by open aerial parts of the plants (Chi et al. 2005). Subsequently, after the start of colonization upon entering via roots, endophytes access internal tissues and travel to the stem and leaf tissues. Those endophytic bacteria may move through a pathway of root tips or via mid lamella of epidermal cells (Compant et al. 2010). The root entry of nitrogen-fixing bacterial species happens in three ways: (1) through the site of wounds in lateral or adventitious roots; (2) via root hairs; (3) cracked cells in the

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epidermis (Cocking 2003). Furthermore, there are other possibilities to enter and infect by producing enzymes such as cellulase and pectinase, which degrade the cellulose and pectin present in the plant cells, respectively (Hallmann et al. 1997). This mode of entry is called “crack entry.” The entry is permitted to only a few endophytes that passively enter inner tissues by junctions of epidermal cells. In this system, the entry is possible through root hair and nearby epidermal cells or through damaged endodermal cells, by the result of emerging lateral root development. This mechanism of the entry (sometimes combined with active entry) has been observed in some species, for example, Bacillus (Ji et al. 2008), Burkholderia (Govindarajan et al. 2006), and Herbaspirillum (James et al. 2002). Interestingly, the way of entry is an ancient mode followed by Rhizobium that colonize legumes (Huang 1986; Fabra et al. 2010). Contrastingly, the endophytic Pseudomonas spp. starts colonization by root hairs of olive, and recognized changes of root hair morphology stimulated by Rhizobium in legumes were not seen (Prieto et al. 2011).

7.4.3

Colonization of Endophytes by Roots of the Plant

Endophytes are attracted by the plant root system through exudates and colonize the surface of the root. Hori and Matsumoto (2010) explained that polysaccharides, pili, and bacterial adhesins are involved in the binding on the surface of a plant root (Hori and Matsumoto 2010). Potentially attracted endophytes move toward and must attach to the root of a plant. Attachment of bacteria and the establishment of a colony with the surface of the host cell occurs by type IV pili of the bacteria. One of the studies on rice plants revealed that the removal of pilT and pilA genes led bacteria to lose their twitching and motile properties and prevented the colonization in plant roots (Dörr et al. 1998; Böhm et al. 2007). PilT and pilA genes are responsible for the retraction protein of pilus and structural protein of pilin, respectively. Bacterial surface components of gram-negative bacterial lipopolysaccharide (LPS) and exopolysaccharide (EPS) facilitates the attachment on the surface and make the colony on the root of the attracted plant. Entry and colonization of endophytic bacteria inside the tissues of a plant occurs by utilizing the organic metabolites for their food and using some mechanism to escape from the host defense system.

7.4.4

Entry and Colonization of Endophytes by Aerial Parts of the Plants

Besides the root entry method, some of the endophytes spread and establish their mutualism via aerial parts of the plants (Hallmann et al. 1997). Compant et al. (2010) studied the density of the endophytes’ population on stem and leaf and found the maximum population up to 104 CFU per gram of fresh weight of the leaf. Movement of bacteria inside plant tissues occurs by flagella movement and transpiration opening of plants. The movement of bacteria is also enhanced by the cell wall breaking enzyme’s production such as pectinase and cellulase (Compant et al.

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2010). Nevertheless, without the production of cell wall breaking enzymes, it can move by perforated plates of xylem. Finally, the endophytes reach the leaf tissues and start colonization. A few endophytic bacteria also enter through the phyllosphere by stomata and colonize in leaf tissues.

7.5

Physiology and System Diversity in Association

The interaction between plants and the bacteria residing in them has long been understudied; however, this interaction has now been proved as a beneficial one by many researchers, but the knowledge about the complete mechanism of interaction, advantages of these interactions, and the diversity of the system remain limited. In addition, the whole research field holds the capacity to discover novel properties of certain host plants and the microorganisms residing in them. Consequently, the work of many authors proves that these interactions are beneficial to plant growth, health, and that the host plants could choose their specific beneficial colonizers (Santoyo et al. 2016). These microorganisms can be categorized as endophytes and epiphytes; among these, endophytes are described as those microorganisms that reside inside the plant tissue and can be isolated from inner tissues or plant organs, whereas epiphytes can be described as those microorganisms that reside on the surface of roots or leaves. The molecular high throughput techniques used in recent studies have made it possible to study the diversity of these endophytes, and hence it has been proved by many authors that endophytes are capable of interacting with different plant parts or organs such as stems, fruits, and root nodules (Ibáñez et al. 2017). Bacterial endophytes (positively) affect plant growth mainly by two mechanisms: (1) Direct influence; (2) indirect influence. In the direct influence, the growth promotion mechanism involves the procurement of essential nutrients or modulation of hormonal level in plants, nutrient acquisition, and assist uptake of nitrogen, phosphorus, iron, etc., and hormonal modulation includes the synthesis of plant growth-promoting hormones such as auxins, cytokinin, and gibberellins or decreases the level of ethylene by producing the counter enzymes through the endophytic bacteria or fungi. On the other hand, the indirect mode of growth promotion remains unclear and understudied. Hypotheses on this mechanism still need to be established to completely understand the mechanisms of action of an indirect effect of bacterial endophytes. However, the attachment of bacteria to the plant surface is the primary step of colonization, and for its establishment, bacteria holds the structures in such a way that they support the attachment to the plant surface using flagella, fimbriae, or cell surface made up of polysaccharides. Bacterial endophytes generally occupy the intercellular spaces of a plant because it contains a good quantity of carbohydrates, amino acids, and inorganic nutrients (Kandel et al. 2017).

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Crucial Changes in Plant Physiology Due to Bacterial Endophytes

Referring to the above context, we can easily assume that the endophytic association leads to beneficial changes in most of the plants. Some examples stated below validate this point. Microbial endophytes are known to be present in all known plant species. The ability to thrive in the plant tissues makes endophytes unique, showing multi-dimensional interactions within the host plant. Several essential activities are affected by the endophytes; they can promote plant growth, elicit defense response against pathogen attack, and can remediate abiotic stress. Most research hints that plant and endophytic association is a plant growth-promoting (PGP) association. Some fascinating issues regarding endophytes that need to be studied in depth are the ability of host plants to produce metabolites, the impact of the association on the host’s gene expression, metabolism, and other physiological aspects essential in conferring resistance against biotic and abiotic stresses (Ogbe et al. 2020).

7.5.2

Diversity of Endophytic Bacteria and Their Association with Host

After several studies conducted on the association and interaction of endophytic microorganisms with the host, it is has been presumed that every plant species is associated with microorganisms. The association and variations in the associations were observed with the different species. The diversity of endophytic microorganisms and the plant species depends on numerous factors such as environmental factors, climatic conditions, plant growth stage, nutrient availability, and geographical location. Based on these factors, host-endophyte associations tend to include various microorganisms and affect the bacterial diversity in the host plant. The factors and their influence on the diversity in a host and the diversity of endophyte are briefly discussed below. Table 7.1 summarizes the endophytes and associated host plants.

7.5.3

Factors Affecting the Diversity in Host Plants

As mentioned above various factors influence the diversity of endophytes that could be colonized in the host. The endophyte species, species of plants, bacterial species, and environmental specific factors affect the diversity and are as follows.

7.5.3.1 Endophytic Species and Strains The compatibility between the endophytic microorganism and the host plant is the prime factor that influences the association; moreover, different plant organs can have colonies of different endophyte species. Hence, the distribution of endophytes varies accordingly. In 1998, Germida et al. showed that wheat plants and canola are

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Table 7.1 List of some selective fungal and bacterial endophytes diversity in different plant species (Arora and Ramawat 2017) S. no 1.

Endophytes Fungal endophyte

Endophyte species Penicillium sp. Aspergillus sp.

Acremonium sp. Colletorichum sp. C. gloeasporiodes

Cladosporium sp. C. herbarum

Curvalaria sp. Phyllosticta sp.

Phomopsist sp.

2.

Bacterial endophyte

Pantoea sp. Pantoea agglomerans Pseudomonas putida Pseudomonas fluorescens Pseudomonas citronellolis Salmonella enteric Klebsiella sp. Citrobacter sp. Burkholderia sp. Rhizobium leguminosarum Rhizobium (Agrobacterium) Azospirillum amazonense Bacillus spp. Bacillus megaterium Clostridium Paenibacillus odorifer Staphylococcus saprophyticus

Host plant species Lycopersicum esculentun Mill. Huperzia serrata Datura stramonium Moringa olifera Prosopis chilensis Taxus chinensis Huperzia serrata Triticum aestivum Citrus plants Chnnamomum camphora Pasania edulis Ginkgo biloba L. Tectona grandis and Samanea saman Huperizia serrata Cinnamonum camphora Lycoperisicum esculentum Mill. Opuntia ficus indica Cinnamomum camphora Lycopersicum esculentum Mill. Triticum aestivum Datura stramonium Moringa olifera Citrus sp. Pasania edulis Coffea Arabica Quercus variabilis Pasania edulis Ginkgo biloba L. Tectona grandis Rice, soybean Citrus plants, sweet potato Carrot Carrot Soybean, Banana, rice maize, sugarcane Carrot Soybean Rice Carrot, rice Citrus plants Maize, carrot, citrus plants Grass Miscanthus sinensis Carrot

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grown in a field where different diversities of bacterial endophytes are present. Later, in 2013, Ding et al. supported this finding and identified that the host species is the most important factor in the selection of its endophytic communities. Another study on bacterial species by Garner et al. (2003) reported that four different species of Brassica were grown in the same soil, but they found that all four species of the plant have different endophytic diversity. Furthermore, it was believed that the soil used for the growth of a plant can influence the endophytic diversity. Also, the endophytic diversity was observed in peanut cultivar grown in different fields (Gouda et al. 2016). The cultivation of a single cultivar of tomato in more than one agronomic soil resulted in the association of diverse endophytes in each case. Perhaps this provides the evidence that plants can establish the association in diverse microbial ecosystems. Moreover, this is ruled by the host plant factors rather than the microbial influence. However, the regional environmental conditions that influence the diversity of the bacteria in the field also play a critical role in this selection.

7.5.3.2 Host Plant Host plants strongly affect the diversity of endophytes flourishing in them. The host factors such as plant age, species, cultivar, a tissue of specific region where the endophyte is supposed to live, and the geographical location will allow that plant to choose the type of endophyte to be associated (Liotti et al. 2018). In 2001, Siciliano et al. showed that host plants grown in a high petroleum hydrocarbon contaminated soil allow only those endophytic bacteria that have the gene capable of degrading hydrocarbon. Moreover, it has been reported that host effector molecules are an important component for the reconstruction of endophytic association and diversity. The assortment of endophytic bacterial diversity is a vigorous process that is strongly controlled by the host plants (Berg and Hallmann 2006; Trivedi et al. 2010; Bogas et al. 2015). 7.5.3.3 Environmental Conditions Apart from the bacterial species and plant host, various environmental factors also greatly affect the diversity of endophyte communities. Those factors include climate, drought, salinity, and other soil conditions that lead to immense changes in the diversity of endophytes. Therefore, studies have been conducted to show that the same type of plant varieties grown in different soil conditions have different species of endophytes thriving in them. The recruitment of endophytes by the host plant depends on the type of stress it is under and what type it might face in its life span (Gehring and Whitham 1992). However, host plant growth stages also determine the endophytic diversity of a plant, where plant stages enriched in nutrient availability tend to have increased bacterial diversity. On the other hand, climatic conditions also affect the endophytic diversity of plant leaf tissue. It has also been observed that the range of bacteria isolated depends on the length, various concentration, and time of the disinfecting agents used, which usually minimized the endophyte communities. Furthermore, endophytic bacterial diversity can be studied to determine the overall diversity of microorganisms. Those methods are in vitro study-based methods and take place using cultural isolation and cultivation in the laboratory conditions. To

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determine the endophytic diversity several steps should be followed, including isolation of host-associated organism followed by inoculation, growth in different media, and different parameters. The end study includes identification via 16sRNA sequencing, which is the most reliable method of molecular identification and estimation of diversity among the endophytes. Furthermore, the diversity of endophytic bacteria has also been reported in a variety of plant species, and it is revealed that proteobacteria are the predominant phylum among the endophytes isolated from plants. It includes several classes of alpha, beta, and gamma proteobacteria. Among all these, the gamma proteobacteria are highly diverse and dominant (Miliute et al. 2015; Santoyo et al. 2016).

7.6

Molecular Events During the Endophytes Association

The study of the molecular events is a major aspect of interest in endophyte association with plants. Though many significant studies have been conducted in this context, conclusive information and a mechanism of association remains limited because each association has their selective characteristics. Thus, generalization is very difficult in this system. However, a few categories of most frequently associated molecular events are discussed here in this section. The concept of plant microbiome study has facilitated the “modern omics” concept. The factors involved in the life changes in the plant with various combinations of microbes and their interaction in the molecular level have greatly changed plant science. The colonization of endophytes involves competitive and strong molecular interaction with the host plants. The molecular interaction starts when an endophytic microorganism attacks the host plant, and this occurs because the affinity molecules exudate from the host plant is the first selective molecular interaction between the host plant and prospective endophyte candidate. For successful association and acceptance of prospective endophyte by host plant, truthful crosstalk and shared strong signal molecules must occur between each (Schauer and Kutschera 2011). Various studies have reported that they show the chemotactic responses between host plant and endophytes. The chemotactic responses are elicited by the host plant via roots and release various biomolecules that attract or are recognized by endophyte microbes. For example, flavonoids are the most popular biomolecules and are produced by a wide genera of the plant kingdom for the purpose of chemoattractant crosstalk. This initiates the first step of the molecular event of association and begins the interaction of endophyte with root hairs (Hardoim et al. 2015). Also, flavonoids are well known for the colonization of non-rhizobial endophytes in the roots of rice and wheat. In the case of Azorhizobium caulinodans and Serratia sp., Nod factors, also called lipochitooligosaccharides (LCO), act as signal molecules and activate the common symbiotic pathways in legume and arbuscular association (Li et al. 2017). This LCO is detectable through the lysin-motif (LysM) receptor and provides a signal for the activation of the signaling pathway also known as common symbiotic pathways that control the arbuscular mycorrhizal and rhizobia-legume association. Furthermore, the LysM receptor is required for both nodulation and mycorrhization

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and together the signal and the LysM receptor family show the main function for both nodulation and mycorrhization. Apart from this, the plant gene expression and various specific micro-RNAs triggered during the association suggested that, in a plant, the gene expression response and the molecular pathways depend on the endophytic microbe interaction. However, signaling pathways of ethylene/salicylic acid/jasmonic acid, etc., work irrespective of the type of endophytic microbes (Glandorf et al. 2001).

7.6.1

Molecular CounterAction Upon Endophytic Association in Plants

Upon association of the endophytes with the host plant, many molecular events initiate in the plant system as counteractions. More often than not, these counteractions are beneficial to the host plant. These activities vary with the host and endophytes associated with it. A few of the most common events are discussed in this section.

7.6.1.1 Defensive Response of Host Plant The defensive mechanism in the host plant is mediated by bacterial endophytes and fungal endophytes, which are then attached with cell surface receptors and trigger the signal to activate the cytoplasmic kinase and cell surface receptor kinases. Once these kinases are activated, they phosphorylate and send the molecular signal to the ethylene/jasmonic acid or salicylic acid against microbes. In this host defensive mechanism, the early detection of phytopathogen will be triggered by the cell surface receptor kinase (RK). This further leads to the cytoplasmic kinase (CK) mediated intracellular response and triggers the ethylene/jasmonic acid transduction pathways. Also, ET/JA triggers the signal to release the chemokines such as phytoalexin and PR proteins and important plant defensive molecules. These provide the active signal for plant defense. A good example in this category is Aspergillum sp., which can induce the systematic disease resistance in wheat and rice. Furthermore, the gene expression analysis showed that ET molecular signaling triggered, which is needed for acquiring systemic resistance in wheat and rice. Down regulation of genes has been observed throughout the plant defending pathway during the colonization of plants via mutualistic interaction such as arbuscular mycorrhizal fungi or rhizobium (White et al. 2018). Although, it is also observed that during the mutual interaction microbes are protected from transgressing and overpowering the plant. Furthermore, it is reported that the hormone-response pathways were also activated due to the mRNA induction in the host which led to the activation of hormoneresponding pathways. It is also shown that the disruption of gibberellic acid (GA) signaling pathways occurs during the infection of arbuscular mycorrhizal fungal and the gene miRNA upregulates and leads the disruption of gibberellic acid (GA) signaling pathways (Plett and Martin 2018).

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7.6.1.2 Activation of Plants Immune System The process of endophytes invading into the host plant remains unclear; however, the mechanism of how the endophyte gets inside the plant host can be explained as they have to pass through the first line of the defense system of the host plant immune system. In this process, a conserved molecule is being recognized by the microbes and plant, and this will assist the positive interaction. The molecules that play a significant role in the endophyte-associated molecular pattern are usually lipopolysaccharides, beta-glycan, a few classes of elongation factor, flagellin, bacterial cold shock protein, beta-glucans, and chitin. These molecules trigger the molecular events required for the microbes associated molecular patterns (MAMPs) (Newman et al. 2013). In this kind of molecular event, the MAMPs are recognized by the surface of plant cells via pattern recognition receptors (PRRs). Furthermore, the fungal endophytes, with chitin-specific receptors (PR-3) on the plants, identify the chitin oligomers present on the cell wall of fungi, and later this triggers the defensive reaction against the pathogens. However, the endophytes upon association begin protecting themselves using the plant defensive mechanism, as illustrated in Fig. 7.2. 7.6.1.3 Biotic and Abiotic Stress Remediator In this process, the environmental stress signal plays an important role and sends the signal for the induction and positive expression of the gene responsible for stress tolerance, and those genes are also known as stress-responsive genes. Due to the positive expression of stress-responsive genes, a high amount of callose deposition occurs on the surface of the plant cell wall. However, phytohormones play a vital role in the tolerance of abiotic stress in plants. ABA (abscisic acid) is one such kind of phytohormone, which initiates the negative signals that trigger the systemic acquired resistance (Rudrappa et al. 2008; Wang and Dai 2010; Vinagre et al. 2019). In this case, the endophyte modulates the stress via the downregulation of ABA. On the other hand, gibberellin (GA), which is a phytohormone synthesized by the plant, also triggers and assists in the stress tolerance induction. Endophytes inhibit the effects of DELLA proteins; however, they promote the stimulation of growth factors. The seed germination is inhibited by DELLA protein in the plant, and this is characterized by the presence of DELLA motif as (aspartate-glutamateleucine-alanine) or D-E-L-L-A which is a single letter of amino acid code. Upon association of endophytes, the seed germination is positively triggered. 7.6.1.4 Protection from Reactive Oxygen Species The reactive oxygen species (ROS) are generated by the plant and are to be deactivated by the various plant’s enzymes. These enzymes are produced by various cell signaling pathways involved in the osmotic stress. Effective production of these proteins leads to genetically superior stress tolerant ones. Endophytes assist the scavenging ROS by triggering the molecular pathways which in turn helps in upregulation of the ROS scavenging enzymes and biomolecules. The most significant enzymes involve in scavenging of ROS are catalases, peroxidase, superoxide dismutase, and glutathione-s-transferase. In this process, the various combinations of

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Fig. 7.2 Molecular events elicited by the plant upon association with endophytes and their synergetic benefits to the host

enzymes, including the enzymes produced by endophytic cells, symmetrically detoxify the ROS and protect the plant host from oxidative rupture/burst (Santoyo et al. 2016).

7.6.1.5 Modulation of Protein Secretion Systems The protein secretion system found in the bacteria alters the plant immune system by reducing the protein secretion into the plant. There are four different classes of secretion systems found in a bacterial cell, but only type III secretion and type IV secretion systems are required for the delivery of a special type of protein known as effector proteins (EF) through which the pathogenic bacteria enter into the plants. However, it is found that this protein is present in a low amount in mutualistic endophytic bacteria (Green and Mecsas 2016). In this process, the endophytic bacterial secrets effector protein, and this effector protein mediates the colonization and assists in the root nodulation process in host plants. Also, the endophytic

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bacteria have a gene to code for aspartate, maltose, and dipeptide metabolism. Altogether, they assist in protecting the host plant system.

7.6.1.6 Phytoremediation Mediated by Endophytic Microbes in Association with Host Plant Phytoremediation is a process of precipitating the toxic metals and chemicals in the soil environment and detoxifying to a neutral level. It can be mediated by endophytic bacteria and play an important role in the remediation of heavy toxic materials into non-toxic materials (Deng and Cao 2016). In this process, the bacterial cell wall captures the heavy metals and makes a complex structure known as a siderophoremetal complex (SMC). The extracellular precipitation of heavy metals accumulates on the surface of the bacterial cell wall. Furthermore, these accumulated heavy metals are being attached through the bilayer membrane and make the biosurfactant complex of heavy metals (Mukherjee et al. 2018). Also, those attached heavy metals are being transported inside the endophytic cell through metal transporter channels and then bind with metallothionein-like protein and make a large complex and assist in intracellular sequestration of metal, and via biotransformation, it leads to the conversion into non-toxic metals (Khare et al. 2018).

7.7

Application of Endophytes

Due to the mutualist relationship with the host plant, endophytes play an important role in the fields of agriculture, industry, pharmaceuticals, and environmental aspects such as bioremediation. In this section, a brief discussion is given on each of these aspects.

7.7.1

Endophytes in Agriculture

It has been found that the plant usually maintains a symbiotic relationship with the variety of endophytic microbes that promote plant growth and protect the host against various abiotic and biotic stresses (Rosenblueth and Martínez-Romero 2006; Brader et al. 2014). However, those symbiotic endophytic microbes can be lost, especially during housetraining and long-term cultivation of the same crop. It was observed that the continuation of the crop cultivation and seeding cause the a great loss of symbiotic microbes and increased the level of diseases due to fungal pathogen residing in the soil such as Alternaria and Fusarium (Kaul et al. 2016). By many ways, it has been observed that endophytes improve the plant health by suppressing the growth of pathogens and maintain the fitness of the host plant. The suppression of growth involves many mechanisms, including direct antagonism via competing with the pathogen for shelter, uptake of the nutrition by a synthesis of a variety of metabolites such as antimicrobial molecules, and via induction of the systemic resistance/enhancing the resistance in the host plant against pathogens by upregulation of the host immune system genes or defense genes. Apart from this, it

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has been reported that endophytic microbes produce secondary metabolite compounds such as alkaloids, which are able to reduce the herbivory by several insects and herbivores (Hardoim et al. 2015). It has been reported that endophytes can reduce weed growth. Endophytes undergo adaptation with the host and endobiome interference occurs with the non-adapted host. Aureobasidium pullulans is a fungal endophyte, which was isolated from the root of a weed—a native species Froelichia gracilis—and was introduced through seeding inoculation into the cells and into the tissues of an exotic plant and growth suppression was observed (White et al. 2018). Additionally, to extend the weed suppression from micrococcus luteus, which was isolated from the tomato seeds and seedlings, it was transferred to seedlings from where they enter into the root cell of a number of cells, and decreased the growth of the seedling itself; this could be applied in weed reduction in agriculture. Endophytes are found most suitable as bio-pests, especially fungal endophytes such as Unidifilum, which produces a highly toxic alkaloid known as swainsonine that is used as a powerful anti-herbivore. Moreover, in the advanced era of science, it is possible to take advantage of transgenically modified endophytes by utilizing the genome of an endophytic microorganism. Hence, it can be a powerful strategy and can be used as an alternative tool to manipulate the genome of the host plants (Li et al. 2017). By utilizing transgenic modification, the gene of interest can be successfully introduced into endophyte microbes that can exhibit the new characteristics and can be useful for biocontrol of host plant pathogen. The endophytic bacteria Clavibacter xyli sub sp. Cynodontids that colonizes the xylem of several host plants was successfully modified and introduced into the Bacillus thurengenesis gene coding endotoxin, which was used to control insects (Glandorf et al. 2001). Considering the above statements, the future efforts for crop management can encompass the exploitation of genetically modified endophytes.

7.7.2

Endophytes in Industry

Endophytes not only have applications in the fields of agriculture, pharmaceuticals, and nanotechnology but also in the biotechnology industry. The extracellular enzyme produced by endophytes or plants in association have several industrial applications. The endophytic bacteria and fungi produce a variety of extracellular enzymes such as hydrolase, oxidoreductase, lyases, and transferase that have industrial applications (Traving et al. 2015). Extracellular enzymes break down the biomolecules such as lignin, protein, organic phosphate, and carbohydrate to micromolecules. These micromolecules are transported to a cell to continuously regulate their metabolism and assist the host for the symbiotic process (Strong and Claus 2011). It has been shown that the hydrolase enzyme enhances the plant’s response toward pathogen infection (Leo et al. 2016). Moreover, there are several enzymes associated with an endophytic organism that lyse the cell wall of the pathogenic fungi and bacteria; thus, it can be used as an effective biocontrol agent (Wang et al. 2014). In addition, it has been observed that the production of various enzymes such as cellulase, protease, amylase, xylanase, lipase, phosphatase, and

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glucosidase caused a reduction of pathogens and promoted cell growth (Fouda et al. 2015; Ayob and Simarani 2016; Pereira et al. 2016). Furthermore, the endophytic fungi from medicinal plants can assist plant growth to overcome the adverse conditions via secreting extracellular enzymes. Bacterial endophytes also have been studied to produce several enzymes, including cellulase, amylase, esterase, protease, deaminase, pectinase, lipase, phytase, xylanase, and asparaginase which have various applications in biotechnology industries (Carrim et al. 2006; Gupta et al. 2013).

7.7.3

Endophytes in Nanobiotechnology

Nanobiotechnology is gaining considerable attention owing to its application in numerous fields, including medical and diagnostics, applications in the environment, in agriculture and waste management for heavy metal sequestration, pesticide detoxification, and toxicity management. Nanobiotechnology draws much attention due to its high efficacy and the higher surface area with minimum size of 0.01 nm to 100 nm. Nanobiotechnology is now focusing on integrating the nanoparticle with endophytic organisms, assuming that it can be explored for many applications such as drug delivery, pathogen detection, and protein and enzyme engineering (Edelstein et al. 2000; Nam et al. 2003). To expand this knowledge, nanoparticles are being immobilized with endophytic organisms, especially fungal and bacterial endophytes. It has been reported that Bacillus species can be used to produce nanoparticles such as AgNPs by reduction of a AgNO3 solution. Moreover, the synthesized nanoparticles showed antibacterial activity against E. coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, and Klebsiella pneumonia (Shankar et al. 2003; Sunkar and Nachiyar 2012). Furthermore, the endophytic fungi have been explored to synthesize gold nanoparticles by using geranium leaves and observed that the terpenoid present in the leaves acted as a reducing and capping compound. Nanoparticles synthesized from endophytic fungus Colletotrichum sp. utilizing enzymes and polypeptide acquired spherical shape and indicated that the shape of nanoparticles can be controlled (Sangaru et al. 2003).

7.7.4

Endophytes in Pharmaceutical

The world’s human population is increasing at an alarming rate, and a variety of new types of health issues are surfacing. For instance, an increase in several drugresistant bacteria is a cause for concern. The use of therapeutic plant species in traditional medicine is as old as mankind; and currently, it is strongly believed that all types of plant species across the plant kingdom do harbor endophytic bacteria (EB). Some of the natural products obtained from endophytic microbes are found to be antimicrobial, antiviral, anticancer, antioxidants, anti-diabetic, and immunosuppressant. The fungal endophytes are known to produce these types of natural products. Endophytic bacteria appear to be a potential source of novel antibiotics.

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It is a well-known fact that to date soil bacteria have been the source for most antibiotics. They are known to boost the growth and development of most plants in varied environmental ecological conditions. There is an urgent need to understand the applications of bioactive compounds present in plant species in association with endophytic bacteria. There are various organic compounds produced by endophytic bacteria that show a varying capacity of antibacterial and antiviral activities. The demand for pharmaceutical compounds is considered as one of the important applications of microbial-based organic products. The microbes located in the intracellular plant tissues are an essential source of secondary metabolites with major applications in the healthcare industry. Endophytic bacteria are considered as an important source for bioactive pharmaceuticals. Some of the important medicinal and pharmaceutical compounds such as antimicrobial, bioactive, and anticancerous compounds are briefly discussed in this section.

7.7.4.1 Antimicrobial Compounds Antimicrobial compounds produced by the endophytes have a significant role in acting as a bioactive drug. Several antimicrobial metabolites belonging to different classes such as terpenoids, flavonoids, steroids, and peptides were reported (Yu et al. 2010). The new compounds formed by the endophytes can play a role in counteracting the rising level of multiple drug resistance (Singh et al. 2017). Zhang et al. (2011) have isolated a huge number of endophyte actinomycetes from 26 different medicinal plants stating various antimicrobial properties (Zhao et al. 2010). The nanoparticles used as antimicrobial carriers synthesized from endophytic bacteria have efficiently been used in the research field of pharmaceutical engineering. Nanoparticles synthesized from endophytic strains of bacteria have the potential of antimicrobial properties and to act against various viral diseases (Singh et al. 2017).

7.7.4.2 Anti-Cancerous Compounds Several plant-derived compounds from endophytic bacterial strains have been shown to exhibit inhibitory properties in cancerous cells. One such example is the endophytic strain isolated from red peppers that shows a tremendous effect in inhibiting tumor cells (Hercegová-Firáková et al. 2007).

7.7.4.3 Antibiotics Various compounds are produced by microorganisms as secondary metabolites to inhibit the disease-causing microorganisms. The pharmaceutical industry has significantly targeted microorganisms such as bacteria and fungi to produce antibiotics. Endophytic bacteria can efficiently produce antibiotics such as pseudomycin, kakadumycin, etc. (Christina et al. 2013).

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Conclusion

Endophytic microorganisms have the specific function to assist not only plant growth but also to protect the host plant from diverse biotic and abiotic circumstances. The endophytes can be explored in a form of inoculants to ease the abiotic stresses resulted from the unpredictable environmental changes. Endophytes can also be considered as a good source for a variety of pharmaceutical compounds, which can successfully replace the chemical drugs that elicit undesired side effects and exert environmental harm during their synthesis. Endophytes are a boon for the agriculture fields where chemical pesticides and insecticides are being replaced with biocontrol agents because endophytes are believed to be safe to use as biocontrol agents. In addition, endophytic microorganisms can act as phytoremediators by converting heavy toxic compounds to non-toxic compounds. The secretion of several industrially important enzymes is one great application among many, and those enzymes are found to be highly active. A few classes of an enzyme have application in biological detoxification of various toxic chemicals. Endophytes can be investigated as a complementary tool for host plants that helps them adapt to many stresses such as salinity, drought, nutrient, heavy metals, and different temperature conditions. Moreover, the detailed study and investigation of endophytes will provide a clear understanding of their relationship with the host and will open the door for maximum utilization as biocontrol agents, the ability to protect the environment, and they can be utilized as the future bicellular factories for the production of various natural pharmaceuticals without harming the environment. Acknowledgments The authors would like to thank the DBT-India for providing research funding under the Ramaling-reentry fellowship. The authors also would like to thank Prof. Rajendra Kumar Pandey (Vice-Chancellor), Amity University Chhattisgarh, Dr. Ravi Kant Singh (Director, Amity Institute of Biotechnology) for their kind support and encouragement.

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Genetic Basis of Fungal Endophytic Bioactive Compounds Synthesis, Modulation, and Their Biotechnological Application Anuj Ranjan, Abhishek Chauhan, Vishnu D. Rajput, Rupesh Kumar Basniwal, Tatiana Minkina, Svetlana Sushkova, and Tanu Jindal Abstract

Endophytes produce varieties of secondary metabolites with diverse pharmacological, medicinal, and biotechnological applications. Many of them have been extensively explored for the novel bioactive compounds, yet the majority are unexplored. The secondary metabolite compounds are well known for their use as antimicrobial, antitumor, antidiabetic, plant growth promoters and enzymes, and other applications. Many of them have got the commercial use and they are a big success. They are also an important source of new drug discovery. The biosynthesis of such bioactive compounds or secondary metabolites involves specialized genes or biosynthetic gene clusters (BGCs), for example, nonribosomal peptide synthase, Indole diterpene gene clusters, and polyketide gene clusters. Such gene clusters are complex and help to encode the required proteins or enzymes to carry out the synthetic pathways. The omics tools such as comparative genomics, transcriptomics, and metabolomics altogether have added another dimension in exploring such potential bioactive compounds. The current chapter intends to compile and highlight the gene clusters-based biosynthesis processes and biotechnological applications of important bioactive molecules.

A. Ranjan · V. D. Rajput (*) · T. Minkina · S. Sushkova Academy of Biology and Biotechnology, Southern Federal University, Rostov Oblast, Russia A. Chauhan · T. Jindal Amity Institute of Environmental Toxicology, Safety and Management, Amity University, Noida, Uttar Pradesh, India R. K. Basniwal Amity Institute of Advanced Research and Studies (M & D), Amity University, Noida, Uttar Pradesh, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. K. Singh et al. (eds.), Bacterial Endophytes for Sustainable Agriculture and Environmental Management, https://doi.org/10.1007/978-981-16-4497-9_8

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Keywords

Bioactive compounds · Biosynthetic gene clusters · Gas chromatography · Enzymes · Mass spectroscopy

Abbreviations BGCs GC ITS MS NMP NRP NRPS PKS

Biosynthetic gene clusters Gas chromatrography Indole terpene synthase Mass spectroscopy Nuclear magnetic resonance Nonribosomal peptide Nonribosomal peptide synthase polyketide synthase

8.1

Introduction

Microorganisms which colonize a plant’s internal tissues and spend part of its life or whole life without harming the plant are usually termed as the “endophytes.” The term is derived from the Greek word “endon which meant within” and “phyte which meant plants.” This term was introduced by de Bary in the year 1866. Such endosymbiosis can be exhibited by the fungus as well as bacteria (Hardoim et al. 2008; Wilson 1995). Endophytes are ubiquitous and they are found in all plant species, though most of them have not been studied so far. They are also known to enhance the growth of host plant (Hardoim et al. 2008, 2015; Varma et al. 1999), provide the nutrients (Behie and Bidochka 2014; Waqas et al. 2012), help in combating abiotic stresses (Lata et al. 2018), and curbing the biotic stress by providing resistance from pathogens and insects (Chadha et al. 2015). Endophytes have been reported in plants from hot deserts, arctic tundra, mangroves, forests (both temperate and tropic) grasslands, savannas, and also on cropland plants. They have also been reported on mosses, ferns, conifers, and other nonvascular seedless plants. The diversity of endophytic fungi is huge, particularly in temperate and tropical rainforests. These fungi are known to be found in almost 0.3 million plant species and each plant has more than one endophyte (Rodriguez et al. 2009). The association of fungal endophytes with the plant is a unique adaptation that allows them to synchronize their growth with the host plants (Verma et al. 2012). Endophytes are usually from the taxonomically and ecologically heterogeneous group from Ascomycota, coelomycetes, and hyphomycetes (George et al. 2005; Verma et al. 2007). They have been classified into two groups based on variation in taxonomy, host plant, colonization and transmission behavior, tissue specificity, and ecological importance (Schaechter

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2009). The two groups are namely Clavicipitaceous and Non-clavicipitaceous endophytes. They are also referred to as C-endophytes and NC-endophytes, respectively. The C-endophytes (Class I) or the Clavicipitaceae is a family of fungi (Hypocreales; Ascomycota) including free-living and symbiotic species associated with insects and fungi or grasses, rushes, and sedges (Stone et al. 2000). Members of these families produce alkaloids with toxic effects on animals and humans. It was first hypothesized by Guerin and Vogl in the year 1898 in the seeds of Lolium temulentum, L. arvense, L. linicolum, and L. remotum (Guerin 1898; Vogl 1898). This was later confirmed by the Bacon et al. (1977) in Neotyphodium coenophialum on tall fescue (Festuca arundinacea) plants. The grazing of cattle to this plant had caused the summer syndrome. The mycelium of C-endophytes has been reported in rhizomes, culms, within the leaf sheath (intercellular spaces), and also on leaf blades. The C-endophytes have been reported to have several effects such as insect deterrence (Clay 1996; Kuldau and Bacon 2008; Spiridon 2020), mammalian herbivores deterrence (Bamisile et al. 2018; Gundel et al. 2020), nematodes deterrence (Kuldau and Bacon 2008; Rodriguez et al. 2009), imparting resistance to the pathogen (Lee 2010) and also improves the ecophysiology (resistance to abiotic factors) of the plants (Naik 2019). Similarly, the NC-endophytes (Class II) are much more diverse endophytic fungi found on tropical trees as well as nonvascular and seedless vascular plants, conifers, and woody and herbaceous angiosperms (Arnold et al. 2000; Fröhlich and Hyde 1999; Gamboa and Bayman 2001). Most of the endophytic fungi belong to Ascomycetes and a relatively much lower number of Basidiomycetes. The NC-endophytes can help the host plant by fighting with abiotic stress (Lledó et al. 2015; Mei and Flinn 2010; Rodriguez et al. 2009), increase in biomass of root and shoot by modulating the plant hormone synthesis (Tudzynski and Sharon 2002), and providing defense against several fungal pathogens (Campanile et al. 2007; Danielsen and Jensen 1999; El Mansy et al. 2020; Pavithra et al. n.d.).

8.2

The Genetic Basis of Secondary Metabolites Production

Endophytes do promote the plant’s growth by providing essential nutrients and enzymes and it also accumulates the secondary metabolites. Most of these compound or secondary metabolites produced by entophytic fungi the product of complex pathways which is driven by a set of enzymes and proteins (Kusari et al. 2012). These enzymes and proteins are often encoded by a set of genes or clusters of genes usually referred to as biosynthetic gene clusters (Remali et al. 2017; Spiering et al. 2005). The production of secondary metabolites is the result of the activation of such silent BGCs (Khare et al. 2018). These gene clusters are helpful in the modulation of chemical scaffolding, regulations, resistance, and transporting the gene products (Dinesh et al. 2017; Kaneko et al. 2010) and they are arranged contiguously for which the concept of gene clusters has come up. Advanced bioinformatics approaches are very helpful in identifying those clusters and even though only a fraction of bioactive compounds are known properly out of a wide range of

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bioactive compounds produced by fungi (Ali et al. 2014; Bhargavi et al. 2014). The first such gene cluster was reported in Aspergillus nidulans and Penicillium chrysogenum. An endophyte Talaromyces radicus (CrP20) found on the leaves of Catharanthus roseus has a gene that codes for an enzyme tryptophan decarboxylase (TDC). This enzyme is involved in the biosynthesis of terpenoid indole alkaloids and able to produce vincristine and vinblastine (Palem et al. 2015). Endophytic fungi have co-confined and contiguous BSGs in its genome that act as signatures for its genetic identification. The most frequent properties attributed to endophytes due to BSGs are specialized metabolism for the production of bioactive compounds, using uncommon nutrients and unique ecological adaptation (Malinowski and Belesky 2000; Sziderics et al. 2007). The endophytes are involved in varieties of pathways where BSGs are playing a crucial role. These pathways have specialized enzymes for biosynthesis of bioactive or secondary metabolites such as polyketide synthases, terpene synthases, terpene cyclases, and prenyltransferases (Dinesh et al. 2017; Sudhakar et al. 2013).

8.2.1

Important Biosynthetic Gene Clusters in Endophytes

Endophytic fungi produce secondary metabolites which can be categorized as (a) Nonribosomal peptides and amino acid-derived bioactive compounds, (b) polyketides and fatty acid-derived bioactive compounds, and (c) terpenes. The gene clusters responsible for the biosynthesis of these three classes of bioactive metabolites are commonly referred to as (a) NRPS gene clusters, (b) PKS gene clusters, and (c) ITS/TS gene clusters respectively (Hoffmeister and Keller 2007).

8.2.1.1 NRPS Gene Clusters Nonribosomal peptides are small peptides synthesized by the enzyme nonribosomal peptide synthetases. Such peptides are not dependent on translation from messenger RNA. The NRPS is exclusive to one type of peptide compound (Süssmuth and Mainz 2017). The peptides synthesized may have variation in structures viz. cyclic, branched, non-proteinogenic amino acids, methylated, formylated, acylated, hydroxylated, and halogenated (Schwarzer et al. 2003; Walsh et al. 2013). Oxidation, reduction, and dehydration have also been observed in nonribosomal peptides. Such small nonribosomal peptide synthesis is a product of NRPS gene clusters that possess a set of domains that are useful and commonly referred as modules (Marahiel et al. 1997; Schwarzer et al. 2003). In NRPS gene clusters, modules and their domain are in the following order (Fig. 8.1): 1. Initiation Module: *Formylation (F)/*N-methylation (NMT)-Adenylation (A)Thiolation and peptide carrier protein with attached 40 -phospho-pantetheine (PCP)-. 2. Elongation modules: -Condensation forming the amide bond (C)/*Cyclization into thiazoline or oxazolines (Cy)- *N-methylation (NMT)- Adenylation

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Fig. 8.1 Illustrates the initiation, elongation, and termination module and their domains of NRPS gene cluster. Each domain contributes to specific amino acids and modifies the growing peptide

(A)-Thiolation and peptide carrier protein with attached 40 -phospho-pantetheine (PCP)- *Epimerization into D-amino acids (E)-. 3. Termination module: Termination by a thio-esterase (TE)/*Reduction to terminal aldehyde or alcohol (R). *Represents the optional domain and it may vary from organism to organism. Endophytes have been reported for producing a range of nonribosomal peptides and they have been associated with an enormously broad range of biological activities. They also have been extensively explored for potential pharmacological properties. NRPs based antibiotics, cytostatics, and immunosuppressants have been approved for their commercial use (Finking and Marahiel 2004; Kessler et al. 2020; Romero-Diaz et al. 2020; Walsh 2004).

8.2.1.2 Polyketide Synthases Gene Clusters for Maklamicin Biosynthesis Polyketides are a huge group of secondary metabolites produced by endophytic fungi. These metabolites are known to be the product of Polyketide synthases (PKSs) which possess multidomain enzyme complexes (Khosla et al. 1999). The PKSs have also been reported in plants, bacteria, and in a few animals as well. The polyketide biosynthesis has very similar characteristics of biosynthesis of fatty acid (Jenke-Kodama et al. 2005). The PKSs have distinct modules and their domains with precise functions that have been separated by short spacers. The PKSs modules are in the following order (in the order N-terminus to C-terminus): 1. Starting or loading module: Acyltransferase (AT)—Acyl carrier protein (ACP)-. 2. Elongation or extending modules: Keto-synthase (KS)-Acyltransferase (AT)[Dehydratase (DH)-Enoylreductase (ER)-Ketoreductase (KR)]-Acyl carrier protein (ACP)-. 3. Termination or releasing domain: Thioesterase (TE).

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Fig. 8.2 PKS BGCs representing the putative PKS genes. This schematic representation also shows the comparative similarity of genes with BGCs. (a) Citrinine BGC, (b) Sorbicillin BGC, and (c) Depudicin BGC. Arrows represent the orientation of the gene and same color has been given to common types of genes among BGCs. Distance and size are not scaled

Gene cluster for synthesis of polyketides in Clonostachys rosea is an example of PKS gene cluster. Clonostachys rosea is a fungus from the division Ascomycota, which is known to act as a biocontrol agent (BCA) against many pathogenic fungi of the plants (Jensen et al. 2004, 2016; Knudsen et al. 1995), Compounds like epipolysulfanyldioxopiperazines, polyterpenoid glisoprenins, and TMC-151-type polyketide have been reported from this fungus, which has nematicidal activity, appressorium formation inhibition activity, and antibacterial activity respectively (Rodríguez et al. 2011; Thines et al. 1998). Genetic analysis using bioinformatics approaches have revealed that the PKS cluster has 31 PKS genes. The cluster also has one PKS-NRPS hybrid gene and 20 putative PKS BGCs. The PKS BGCs span from 36.1 to 59.6 kb in size (Fig. 8.2) (Fatema et al. 2018).

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8.2.1.3 Indole-Diterpenes Gene Cluster Indole-diterpenes are a bioactive compound produced by a few ascomycetes fungi especially Aspergillus, Penicillium, Epichloe, and Claviceps (Parker and Scott 2004; Prakash and Srinivasan 2020). These compounds have diverse chemical structures and some of them are potential mammalian toxins (Kozák et al. 2020; Steyn and Vleggaar 1985). Such compounds have a unique cyclic core of diterpene as a skeleton which is a derivative of geranylgeranyl diphosphate. The indole moiety in the structure is derived from indole-3-glycerol phosphate which is a common precursor of tryptophan amino acid (Byrne et al. 2002; Tagami et al. 2013). Neotyphodium lolii is a fungal endophyte of perennial ryegrass that has been explored by many authors for the Indole-diterpenesgene clusters (Fig. 8.3). The N. lolii has three distinct gene clusters of ltm gene which has been separated by a large stretch of AT-rich sequences. Cluster 1 of ltm has ltmG, ltmM, and ltmK genes and they are flanked by relics of a Type-I retrotransposon. To the right side to this cluster, a Rua retro-element insertion of 17.2 kb is located (Young et al. 2006). A polyketide synthase pseudogene is also present next to the retro-element with further AT-rich sequence. It delineates the right-side boundary of the gene cluster ltmK gene (Young et al. 2005, 2006). To the left of cluster 1, there is another AT-rich sequence of Rua retro-element which has a length of nearly 35 kb. The cluster of the ltm gene has five genes namely ltmP, ltmQ, ltmF, ltmC and ltmB. Four of these genes have been reported to be orthologues of paxilline biosynthetic genes of P. paxilli (Young et al. 2001). Cluster 2 and 3 have been separated by two imperfect repeats of 16 kb. Cluster 3 has two distinct genes ltmE and ltmJ and they are unique to the genus Epichloe. The left boundary of cluster 3 has an AT-rich sequence. Details of the ltm gene clusters have been included in Table 8.1. There are many other gene clusters responsible for the biosynthesis of paspalinederived indole diterpene compounds which have been identified. Some of them have been nicely discussed by Kozák et al. (2018). These bioactive compounds are

Fig. 8.3 LTM locus for Indole-diterpenes Gene Cluster of the N. lolii represented by a physical map. The boxes represent the boundary of each gene ltm gene cluster. Above the map, the details of lambda clones is represented which have been used for sequencing and mapping. The arrows’ direction shows the direction of transcription and black bands represent the exons. The retrotransposons have been represented using colored blocks right above the sequence and percent AT content has been shown right below the sequence. Between cluster 2 and 3, two imperfect repeats can be seen. (The image has been adapted from Young et al. (2005))

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Table 8.1 LTM gene clusters of Neotyphodium lolii Gene name ltmB ltmC ltmE ltmF ltmG ltmJ ltmK ltmM ltmP ltmQ

Cluster name 2 2 3 2 1 3 1 1 2 2

Function Terpene cyclase Prenyl transferase Multi-functional prenyl transferase Prenyl transferase GGPP synthase Cytochrome P450 monooxygenase Cytochrome P450 monooxygenase FAD-dependent monooxygenase Cytochrome P450 monooxygenase Cytochrome P450 monooxygenase

Homologous genes paxB paxC paxC/paxD paxD paxG NA NA paxM paxP paxQ

penitrems, paxilline, shearinines, aflatrems, paspalitrems, terpendoles, and lolitrems. Their clusters have the orthologues genes of paxG, paxM, paxC, and paxB genes which are involved in the biosynthesis of paspaline. Figure 8.4 illustrates the BGCs of these paspaline derivatives in respective organisms.

8.2.1.4 Loline Biosynthetic Gene Cluster Loline is an alkaloid produced by many Neotyphodium/Epichloë endophytes of grasses. It is a very potent broad-spectrum insecticide (Malinowski and Belesky 2019; Song et al. 2020) with a unique structure having a saturated 1-aminopyrrolizidine-ring system (Blankenship 2004; Petroski et al. 1989) with a bridge between C2 and C8 carbons. This compound is found in Lolium and other related grasses (Bush et al. 1997; Christensen et al. 1993). The gene clusters of LOL gene (Fig. 8.5), that is LOL-1 and LOL-2 are involved in the biosynthesis of loline alkaloid. It has a span of 25 kb where nine genes namely lolF-1, lolC-1, lolD-1, lolO1, lolA-1, lolU-1, lolP-1, lolT-1, and lolE-1. LOL-2 (in order) have been identified in cluster LOL-1, whereas the LOL-2 has lolC-2 to lolE-2 genes in similar order and orientation. Table 8.2 includes the gene of lol clusters along with its closest similar genes and their identifiers. Genes in LOL-2 cluster are homologs to LOL-1 genes (Kutil 2010).

8.3

Potential of Endophytic Bioactive Compounds

Endophytes are an under-investigated group of microbes and synthesizers of the natural compound which are beneficial to plants (De Carvalho et al. 2019). They are being exploited for the novel bioactive compounds with potential use in medical, agriculture, industries, and research (Nandhini et al. 2020; Strobel et al. 2004). After the discovery of a compound Taxol, it was developed as an anticancer drug from endophytic fungi, such as Taxomyces andreanae, Pestalotia spp., and Pestalotiopsis spp. (Stierle et al. 1993; Strobel et al. 1996). The endophytes have attracted considerable attention from researchers, mycologists, and chemists (Aly et al. 2010, 2011).

Genetic Basis of Fungal Endophytic Bioactive Compounds Synthesis,. . .

Fig. 8.4 Physical map of ITD clusters: Red: paspaline biosynthesis (genes B, C, G, and M); Green: oxidation of the paspaline rings E and F, Blue: prenylation of C20, C21, or C22 of paspaline (genes D, O, idtF, and ltmE), Yellow: Poorly understood membrane-associated processes (genes A and S) and Black: Indole diterpene group tailoring (Kozák et al. 2020). (The figure is adopted from Kozák et al. (2018))

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Fig. 8.5 Gene of lol 1 clusters (lolF-1, lolC-1, lolD-1, lolO-1, lolA-1, lolU-1, lolP-1, lolT-1, and lolE-1), LOL-2 clusters genes are homologs to LOL-1 genes. (Image adopted from Kutil (2010))

In the past 30 years, hundreds of bioactive compounds have been isolated and identified from the fungi which have anti-cancer properties. There are varieties of applications where endophytic bioactive compounds obtained from fungi have found their use, for example, antibacterial and antiviral, antidiabetic and anti-obesity, antioxidants, immunomodulatory, immunosuppressant, and insecticide (Chadha et al. 2015; Kharwar et al. 2011; Strobel et al. 1996; Varma et al. 1999). Javanicin as an antibacterial isolated from Chloridium of A. indica (Kharwar et al. 2009). Subglutionol and collutelin-A as immunosuppressant Fusarium subglutinans of Triptergium wilfordii and Colletotrichum dematium of Pteromischum sp., respectively (Lee et al. 1995; Shukla et al. 2014) and Cytonic acid as an antiviral obtained from Cytonaema sp. (Guo et al. 2008) are a very good example of endophytic bioactive compounds with potential use. These bioactive compounds produced by endophytic fungi have varieties of uses. Recently, there are numerous reports which have reported the potential of endophytic compounds with diverse applications viz. anti-microbial, anti-diabetic, anti-cancer, and insecticide (Fig. 8.6). Some of the notable uses of such bioactive compounds have been discussed below:

8.3.1

Antioxidant Activity

Endophytic fungi are an excellent source of antioxidant compounds. Different extracts obtained from endophytic fungi have been evaluated for ntioxidant potential. Researchers have screened various strains of endophytic fungi isolated from various parts of plants for the characterization and identification of antioxidants compounds. In a recent study (da Silva et al. 2020), 315 endophytic fungi obtained from the leaves of Passiflora incarnata were screened for the presence of chemical constituents exhibiting antioxidant properties. Ethyl acetate and n-Butanol extracts have significantly produced flavonoids, phenolic compounds, and tannins. DPPH free radical scavenging assay was performed with maximum activity (95.8%). Orcinol (3.19%) sorbicillin (33.59%) and Four-methoxymethylphenol (4.79%) were produced from ethyl acetate fraction from the Aspergillusstrain. In another study, Botryosphaeria dothidea isolated from Brazilian forest was evaluated for the production of antioxidant compounds using submerged fermentation. The broth showed varied scavenging activity (94.47%, 94.87%, and 89.78%) and also reported

Predicted function FAD-containing monooxygenase FAD-containing monooxygenase γ-Type PLP enzyme γ-Type PLP enzyme PLP enzyme/decarboxylase PLP enzyme/decarboxylase Oxidoreductase/dioxygenase Oxidoreductase/dioxygenase Amino acid binding Amino acid binding Possible DNA-binding protein Possible DNA-binding protein P450 monooxygenase P450 monooxygenase Class V-aminotransferase PLP-enzyme Class V-aminotransferase PLP-enzyme Epoxidase/hydroxylase Epoxidase/hydroxylase

Size 540 540 473 473 420 415 362 362 209 210 495 506 496 184 454 464 256 256

Most similar match 1,2-Cyclopentanone monooxygenase 1,2-Cyclopentanone monooxygenase O-Acetylhomoserinethiol-lyase O-Acetylhomoserinethiol-lyase Ornithine decarboxylase Ornithine decarboxylase Probable oxidoreductasef Probable oxidoreductasef Aspartate kinase KIBYD; C-terminal domain Aspartate kinase KIBYD; C-terminal domain A. nidulans predicted protein A. nidulans predicted protein Pisatin demethylase Pisatin demethylase Isopenicillin N epimerase Isopenicillin N epimerase Epoxidase subunit A Epoxidase subunit A

Accession ID, identity, and E-value have also been provided for the closest match of LOL genes

Gene lolF-1 lolF-2 lolC-1 lolC-2 lolD-1 lolD-2 lolO-1 lolO-2 lolA-1 lolA-2 lolU-1 lolU-2 lolP-1 lolP-2 g lolT-1 lolT-2 lolE-1 lolE-2 EAA61586 EAA61586 Q12645 Q12645 P18549 P18549 BAA75924 BAA75924

GenBank accession no CAD10798 CAD10798 P50125 XP381593 CAC80209 P27121 NP248837 NP248837

Table 8.2 Genes of LOL clusters and their predicted function with the closest matching enzyme is provided in the table below Identity 35 482 35 482 54 431 53 426 35 401 36 428 25 359 25 359 28 160 28 160 21 484 20 489 28 477 28 152 24 388 25 400 39 242 39 242

Evalue 7e-89 2e-89 1e-126 1e-124 3e-69 6e-69 2e-21 2e-23 7e-08 5e-08 7e-04 5e-04 1e-47 1e-12 1e-13 2e-13 2e-42 2e-42

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Fig. 8.6 Schematic illustration showing the interaction of host and endophytes in a certain environmental condition, which leads to the synthesis of bioactive compounds with diverse applications

for the production of different volatile compounds such as 1.2-benzenedicarboxylic acid bis(2-methylpropyl) ester, 3.6-bis(2-methylpropyl)-2.5-piperazinedione, and hexahydropyrrolizin-3-one (Druzian et al. 2020). The extracellular crude extract of Fusariumoxysporum isolated from a Colombian medicinal plant (Otoba gracilipes) was evaluated for potential antioxidant activity (Caicedo et al. 2019). In this study, crude extracts were prepared using two different media i.e. potato dextrose broth (PDB) and potato dextrose–yeast extract broth (PDBY). The scavenging effect was studied using DPPH (free radical 2,2-diphenyl-1-picrylhydrazyl) assay with 51.5% scavenging activity in the extract of PDB culture and 26.4% in the extract of PDBY culture. Similarly, Penicillium oxalicum an endophytic fungus isolated from Citrus limon exhibited antioxidant properties with maximum scavenging activity (7970%) (Kaur et al. 2020). A table (Table 8.3) has been attached below that exhibits a list of endophytic fungi along with its host plant and also compound(s) or extracts identified for its antioxidant activities.

8.3.2

Antimicrobial Activity

Nowadays, the most important challenge for healthcare research organizations is to combat the infections caused by multiple antibiotic-resistant microorganisms. The World Health Organization (WHO) in February 2017 published a list of resistant bacteria for which new antimicrobial compounds are urgently needed (World Health Organization (WHO) 2017). Endophytic fungi benefit their host and provide favorable environmental conditions in the production of novel bioactive compounds. Secondary metabolites produced by endophytic fungi showed chemically diverse

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Table 8.3 Summary of the studies conducted for the evaluation of endophytic fungi for antioxidant properties and compounds Compounds secreted/ extracts Phenolic compounds

Endophytic fungi Ochrocladosporium elatum Talaromyces Colletotrichum, Cochliobolus, Diaporthe, Bjerkandera, Phaeophlebiopsis, Curvularia, and Xylaraceae Aspergillus minisclerotigens AKF1 and Aspergillus oryzae DK7

Host plant Schinus terebinthifolius Raddi Campomanesia xanthocarpa, Lafoensia pacari, Siparuna guianensis and Guazuma ulmifolia

Chaetomium globosum

Adiantum capillus

Penicillium citrinum CGJ-C1, P. citrinum CGJ-C2, Cladosporium sp. CGJ-D1, and Cryptendoxyla hypophloia CGJ-D2 Alternaria sp. (ML4)

Tragia involucrata Linn

Dihydropyran and 4H-Pyran-4-one, 5-hydroxy-2(hydroxymethyl(CAS) Kojic acid EtOAc extract (phenolic compound) L-ascorbic acid

Mussaenda luteola L. (Rubiaceae) Sargassum wightii

Phenolic and flavonoid compounds Ethyl acetate extract

Calotropis procera, Catharanthus roseus, Euphorbia prostrate, Vernonia amygdalina, and Trigonella foenumgraecum Alpine plants

Gallic acid

Cladosporium cladosporioides Aspergillus sp.

Rhodiola spp.

Mangifera casturi Kosterm

Aspergillus niger, A. flavus, Alternaria alternata A. niger, Penicillium sp. and Trichoderma sp.

Lannea coromendalica

Fusarium solani (ERP-07), Fusarium oxysporum (ERP-10),

Pigeon pea [Cajanus cajan (L.) Millsp.

Tabebuia argentea (Bignoniacae)

Favonoids

Salidrosides, ptyrosol, and rosavins (phenolic and flavonoid) EtOAc extract (phenolic compound) Tannins, flavonoids, steroids, alkaloids, phenols Cajaninstilbene acid

References Rocha et al. (2020) Santos et al. (2020)

Nuraini et al. (2019)

Selim et al. (2018) Danagoudar et al. (2018)

Gunasekaran et al. (2017) Hulikere et al. (2016) Khiralla et al. (2015)

Cui et al. (2015)

Premjanu and Jaynthy (2014) Govindappa et al. (2013) Zhao et al. (2012) (continued)

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Table 8.3 (continued) Endophytic fungi and Fusarium proliferatum (ERP-13) Hypocrea, Penicillium, Tolypocladium, Chaetomium, Xylaria, Nemania, and Creosphaeria Phyllosticta sp.

Host plant

Compounds secreted/ extracts

References

Liverwort Scapania verrucosa

Ethyl acetate extracts

Zeng et al. (2011)

Guazuma tomentosa

EtOH extract (phenol)

Srinivasan et al. (2010)

and structurally different properties against a variety of pathogenic microorganisms (Toghueo 2020). Various studies have been conducted for the development of new antimicrobial compounds globally; researchers studied the antimicrobial activity of extracts obtained from endophytic fungal species belonging to phyla Ascomycota, Basidiomycota, and Zygomycota. Most of the studies aimed to identify extracts that could be further explored in isolating potential antimicrobial compounds with their safety characteristics (Table 8.4).

8.3.3

Antiviral Activity

Viral diseases have been the main cause of high mortality rate in the human race worldwide. The current COVID19 pandemic has caused a significant loss of human lives and the situation is still challenging. Few drugs have been approved in past decades but prolonged use has caused the rapid emergence of drug resistance. The antiviral potential of endophytic fungi isolated from a variety of plants has been studied extensively. Extracts and compounds obtained from the culture of fungal endophytes showed diverse antiviral potential (Table 8.5). Metabolites identified using various spectral techniques are being utilized for therapeutical purposes. The current coronavirus pandemic situation enforces us to think about novel antiviral compounds from a novel source.

8.3.4

Anticancer Activity

When a healthy cell loses its control over growth then its divisions and ability to spread into different parts of the body result in the formation of a primary tumor, which invades and destroys adjacent tissues. The collective term for such a process is known as cancer. DNA damage, mutation, and environmental agents are responsible for this cancer process (Wiseman et al. 1995). As per the record of the International Agency of Research on Cancer, globally, 1.1 million men were diagnosed with prostate cancer in 2012. Cancer Research UK recorded 356,860 cancer cases and an

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Table 8.4 Antimicrobial compounds isolated from endophytic fungi have been listed below along with the host plants and authors Antimicrobial compounds 3-phenylpropionic acid

Plant host Zygophyllum mandavillei Leaves of wheat

Shen et al. (2020)

Pelargonium sidoides Juniperus communis C. alata

Aboobaker et al. (2019) Morehouse et al. (2019) Khan et al. (2018)

Fusarium sp. PN8 F. equiseti

Mentha longifolia L. P. notoginseng Padina pavonica

F. chlamydosporium

Anvillea garcinii

Equisetin

Fusarium sp.

O. dillenii

Isoflavonoids

Phomopsis sp.

Terpenoid

Phomopsis sp.

Erythrina cristagalli Plumeria acutifolia

Ibrahim et al. (2018) Jin et al. (2017) Hawas et al. (2016) Ibrahim et al. (2016) Ratnaweera et al. (2015) Radji et al. (2011)

7-Amino-4methylcoumarin Cryptocandin

Xylaria sp. YX28

Ginkgo biloba.

Cryptosporiopsis sp.

1-Butano,3methylacetate

Muscodor albus

Cryptosporiopsis quercina Cinnamomum zeglanicum

Ophiobolin Dibutyl phthalate Poly(3R,5Rdihydroxyhexanoic acid) Fusarubin, bostrycoidin, anhydrofusarubin Fusaripeptide A (antifungal) Ginsenoside w-hydroxyemodin and cordycepin Fusarithioamide A

Producing fungi Cladosporium cladosporioides Bipolaris species TJ403-B1 Penicillium skrjabinii Annulohypoxylon multiforme (TC2-046) F. solani Fusarium sp.

References Yehia et al. (2020)

Nithya and Muthumary (2010) Xu et al. (2008) Guo et al. (2008) Strobel and Daisy (2003)

estimated 45% mortalities due to cancer in the year 2014. Leukemia cancer cases were dominant in the USA and an estimated 24,500 people to die from leukemia in the year 2017 (Howlader et al. 2021). According to Berit & Rolf, proto-oncogenes, tumor suppressor genes and genes involved in DNA repair mechanisms play a key role in cancer tumor initiation (Tysnes and Bjerkvig 2007). Change in apoptosis regulating genes or DNA repair systems leads to abnormal expression and transformation of target cells (Carlo and Croce 2008; Knudson 2001). To treat cancer, conventional cancer chemotherapy is very risky, expensive, and non-specific for target cells (Nygren and Larsson 2003). Nowadays, pharmaceutical companies are harvesting medicinal plants at a high pace rate for the extraction of cancer drugs like combretastatin, camptothecin, and taxol (Mbaveng et al. 2011). Over-harvesting, illegal exploitation, and destruction of ecological habitat are the responsible factors

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Table 8.5 Antiviral bioactive compounds isolated from endophytic microorganisms Endophytic microorganism Talaromyces purpureogenus

3.

Bioactive compound (s) Polycyclic meroterpenoids talaromyolides E-K 14-Nordrimane sesquiterpenoid Cytosporone U

4.

Podophyllotoxin

5.

Podophyllotoxin

6.

Mullein

Chaetomium globosum Fusarium oxysporum Penicillium janczewskii

7.

Sequoiatones C–F

8.

Brefeldin A and B

9.

Cytonic acid A and B

10.

Lactone S

S. No. 1.

2.

Phoma sp. Phomopsis sp.

Aspergillus parasiticus Aspergillus clavatus Cytonaema sp. Microsphaeropsis sp.

Host plant Grateloupia filicina C. Ag (Wulf.), (seaweed) Aconitum vilmorinianum. Brucea javanica Sinopodophyll um hexandrum Juniperus recurva Prumnopitys andina

Sequoia sempervirens Taxus mairei Quercus sp. Buxus sempervirens

References Cao et al. (2020) Liu et al. (2019) Tan et al. (2017) Wang et al. (2017) Kour et al. (2008) SchmedaHirschmann et al. (2005) Zhou et al. (2004) Wang et al. (2002) Guo et al. (2000) Tscherter et al. (1988)

for the reduction of conventional natural medicinal resources (Kala 2000). Thus, this alarming situation enforces us to conserve endangered medicinal plant species and look for new alternative paths for the development of cancer drugs. Scientists are now focussing the research on endophytic fungi for the solution of the cancer drug problems. Endophytes (saprophytes, latent pathogens, and mycorrhizal fungi) are those organisms that reside in plant internal tissue without harming them (Newman et al. 2003; Petrini and Fisher 1990). Endophytes are great sources of bioactive secondary metabolites like Xanthones, benzopyranones, quinones, tetralones, alkaloids, and flavonoids (Tan and Zou 2001). A recent literature survey specified that 51% of the bioactive metabolites isolated from endophytic fungi were earlier unidentified compared to 38% from soil fungi. This has changed the direction of current research towards finding a reliable, economical, environmentally safe, and alternative method for the isolation of endophytic fungal bioactive compounds from these microorganisms. Secondary metabolites like camptothecin (Kusari et al. 2009; Puri et al. 2005; Rehman et al. 2008), vincristine, and podophyllotoxin (Lingqi et al. 2000; Puri et al. 2006) isolated from different endophytic fungi had been already recognized as anticancer agents. In Table 8.6, anticancer bioactive secondary metabolites in different chemical forms

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Table 8.6 Anticancer bioactive natural products from endophytic microorganisms

Swainsonine (SW) Duclauxamide A1 Acremoxanthone E

Endophytic microorganism A. alternata MGTMMP031 Epicoccum nigrum TXB502 Alternaria sect. Undifilum oxytropis Penicillium manginii YIM PH30375 Acremonium camptosporum

Panax notoginseng Bursera simaruba

6.

3-epi-Waol A

Libertella blepharis

Olyralati folia

7.

Dothiorelone F

Dothiorella sp.

8.

Cercosporene F

Cercospora sp.

9.

Multirostratin A

10.

Vincristine

Phoma multirostrata EA-12 Fusarium oxysporum

11.

Vinblastine

Fusarium oxysporum

12.

Penicillenols

Penicillium spp.

13.

Phomopsin A

Phomopsis spp.

14.

Xylariaquinone A

Xylaria spp.

15.

Nigerasperone

Aspergillus niger

16.

Aspergillus clavatus

17.

Brefeldin A and B Paclitaxel (Taxol)

Aegiceras corniculatum Fallopia japonica Eupatorium adenophorum Catharanthus roseus Catharanthus roseus Aegiceras corniculatum Excoecaria agallocha Sandoricum koetjape Colpomenia sinuosa Taxus mairei

18.

Torreyanic acid

S. No. 1. 2. 3. 4. 5.

Bioactive compound(s) Alternariol methyl ether Taxol

Taxomyces andreanae P. microspore

Host plant Vitex negundo Taxus baccata Locoweeds

Taxus brevifolia Torreya taxifolia

References Palanichamy et al. (2019) El-Sayed et al. (2020b) Ren et al. (2017) Cao et al. (2015) MeléndezGonzález et al. (2015) Adames et al. (2015) Du and Su (2014) Feng et al. (2014) Chen et al. (2015) Kumar et al. (2013) Kumar et al. (2013) Lin et al. (2008) Huang et al. (2008) Tansuwan et al. (2007) Zhang et al. (2007) Wang et al. (2002) Strobel et al. (1996) Strobel et al. (1993)

viz. Polyketides, Alkaloids and Nitrogen-Containing Compounds, Lactones, Xanthones, Peroxides, Quinones, Terpenoids, Others, Pyrans, Pyrones, Coumarins, and Phenolic Compounds have been listed.

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Antidiabetic Activity

Endophytic fungi are also known for the production of secondary metabolites with antidiabetic properties. A recent study revealed Xanthone, an antidiabetic compound produced from Penicillium canescens isolated from Juniperus polycarpos (Malik et al. 2020). Similarly, compounds such as biosynthetic compounds, (S)-(+)-2-cis-4trans-abscisic acid (1),, 70 -hydroxy-abscisic acid (2), and 4-des-hydroxyl altersolanol A (3) obtained from Nigrospora oryzae of Combretum dolichopetalum leaf exhibited potential antidiabetic activity (Uzor et al. 2017). Ethyl acetate extracts of various endophytic fungi (Aspergillus terreus XJA8, Schizophyllum commune XJA1, Aspergillus sp. XJA6, Talaromyces sp. XJA4) isolated from a Chinese medicinal plant named Vernonia anthelmintica have also been evaluated with strong antidiabetic activity (Rustamova et al. 2020). The antidiabetic potential of crude extract of Acremonium coenophialum obtained from Acremonium coenophialum has also been assessed by α-amylase activity (Hateet 2020). Ranganathan and Mahalingam (2020) in their recent study reported the production antidiabetic compound i.e. 2,4,6-triphenylaniline (TPA) from Alternaria longipes strain VITN14G isolated from the plant Avicennia officinalis. In another study, 36 fungi isolated from a medicinal plant (Acacia nilotica) were evaluated for the creation of alpha amylase and glucosidase inhibitors (Singh and Kaur 2016). These studies have shown that extracts and secondary metabolites obtained from different strains of endophytic fungi exhibited strong antidiabetic activity. These recent studies also reported that endophytic fungi could also be an alternative source for the control and management of diabetes.

8.3.6

Insecticidal Activity

Endophytic fungi are known for their insecticidal potential. Studies have been conducted using potential endophytic fungal isolates from different medicinal plants. In a recent study El-Sayed et al. (2020a) 45 strains of endophytic fungi isolated from 15 medicinal plants were evaluated for potential insecticidal activity. Among all isolated strains, EtOAc extracts of Sarocladium strictum showed strong insecticidal activity, reported with four major compounds namely Cis-13-octadecenoic acid, sebacic acid, pentamethoxy flavone, and n-hexadecanoic acid. A study conducted by Guo et al. (2017), ethyl acetate extract of mangrove plant endophyte Aspergillus fumigatus JRJ111048 was assessed for insecticidal activity against Spodoptera litura with five known compounds; α-ethyl glucoside, spiculisporic acid, spiculisporic acid B, C, spiculisporic acid, and secospiculisporic acid B and one new lipid amide 11-methyl-11-hydroxyldodecanoic acid amide. Jadhav and Pardeshi (2017) in his study, used ethyl acetate and methanolic extracts of endophytic fungi were studied against Callosobruchuschinensis. It was observed that the mortality increases with an increase in concentration of endophytic fungi. Eighty-six endophytic fungi isolated from T. wilfordii were studied for their insecticidal activity (Han et al. 2013). In another study, Thakur et al. (2013) evaluated the insecticidal

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Table 8.7 List of insecticidal compounds isolated from endophytic fungi Insecticidal compounds Cis-13-octadecenoic acid, sebacic acid, pentamethoxy flavone and nhexadecanoic acid

Producing Fungi Sarocladium strictum, Aspergillus nidulans

Nthraquinone derivatives (compounds 1–4), including a previously undescribed dimethylated derivative of bipolarin, 6,8-di-O-methylbipolarin Alkaloids (PIAs) with pyrano[2,3-g] indole moieties, amoenamide C and sclerotiamide B, and four known biosynthetic congeners Xanthene derivatives, penicixanthenes A–D (1–4) α-Ethyl glucoside, spiculisporic acid, spiculisporic acid B, C, spiculisporic acid, and secospiculisporic acid B and one new lipid amide 11-methyl-11hydroxyldodecanoic acid amide Nodulisporic acids (NAs)

Acremonium vitellinum

Fungus fusarium sambucinum TE-6L and griseofulvin and beauvericin Penicillium sp. JY246

Plant Host Cynancum acutum, Lantana camara –

References El-Sayed et al. (2020a) Yuan et al. (2020)

Nicotiana tabacum L.

Zhang et al. (2019)

Mangrove

Bai et al. (2019) Guo et al. (2017)

Aspergillus fumigatus JRJ111048

Mangrove

Hypoxylon pulicicidum sp. nov

–

Bills et al. (2012)

potential of the endophytic fungus Cladosporium uredinicola isolated from Tinospora cordifolia. Ethyl acetate extract of this fungus was found to be effective against S. litura at a concentration of 1.25–2.00μl g1. A list of some selected insecticidal compounds isolated from plant endophytic fungi has been summarized in Table 8.7.

8.4

Strategy for Production of Endophytic Secondary Metabolites

Although it is always difficult to find out desirable compounds from endophytic organism due to their vast diversity, during the last 7 years, scientists have explored more than 300 endophytes with their potential therapeutic valuable active compounds (Gunatilaka 2006) like Paclitaxel (Taxol) (Stierle et al. 1993), Camptothecin (Cui et al. 2012; Kusari et al. 2009; Puri et al. 2005; Ren and Dai 2012), and Azadirachtin (Kusari et al. 2012). Plants grow in high biodiversity areas of the world are likely to have a chance of novel endophytic secondary metabolites (Strobel et al. 2004; Verma et al. 2009) that can be used for various biotechnological and pharmacological applications. Thus Western Ghats of India, the Amazon basin, tropical rain forests, coastal line with mangroves are the possible places for searching a novel endophytic plant. Endemic

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plants with unusual longevity and growing in extreme environments such as saline, acidic (unusual lower pH) or alkaline soils (unusual higher pH) can be also a novel source of novel drug compounds (Kumaresan and Suryanarayanan 2001). Following are the schematic ways for bioactive metabolite production using endophytic microorganisms • Selection of Endophytic Plant: Selection of endophytic plant is based on habitat, ethnomedicinal importance, and geographical importance. Then the selection is also made based on their part like root, stem, or leaf for endophytic microorganism. • Identification of endophytic microorganisms: Identification of endophytic microorganisms is carried out with the help of microscopic and molecular techniques. • Culturing on laboratory media: Culturing of isolated endophytic microorganism is done on nutrient-rich media. Further, they grow on either solid-state or submerged condition’s media for large-scale production of bioactive secondary metabolites. • Extraction of desire secondary metabolites: After culturing, the culture broth is filtered through filter paper and checked for biological activities. • Screening of biological activity: Screening of biological activities like antiviral, anticancer, antioxidants, and other pharmacological properties through existing standard laboratory methods. • Characterization of Bioactive Compound: The presence of bioactive compounds like Polyketides, Alkaloids, Xanthones, Peroxides, and Lactones can be confirmed through various characterization techniques like spectroscopy, NMR, GC-MS, and other existing techniques.

8.5

Major Obstacles to the Production of Bioactive Compounds from Endophytes

As per Kusari et al., despite the enormous biodiversity of endophytes and the comprehensive research we failed to convert these discoveries into industrial bioprocess for sustainable production of pharmaceutically vital metabolites (Kusari et al. 2014). Natural endophytes grow in adverse and unreached fields. While culturing endophytes in a laboratory, the fulfillment of specific chemical components in the media that mimic their natural environment is a challenging task. Successful in vitro cultivation of endophytic microorganisms will provide a new source of microbial metabolites with diverse biological activities. The following major obstacles come into the path during the commercial production of bioactive compounds. 1. Conventional isolation and cultivation procedures are backbreaking, timeconsuming, and only support the growth of specific bacteria or fungi.

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2. Filamentous ascomycetes are the major source of bioactive compounds. It is mandatory to understand the complete mechanism and physiology of filamentous fungi for the bioprocess design because uncontrolled mycelial growth of fungi can decrease the metabolite production rate. 3. Sustainable metabolite production depends on the constant expression of biosynthetic genes. 4. Generally, we use the axenic monoculture system for industrial production of bioactive compounds, but endophytes never present in an axenic form in their natural ecological habitats. 5. The selection and isolation of competent endophytic microorganisms is always a challenging task for microbiologists.

8.6

Conclusion and Future Prospects

It has been projected that less than 1% of endophytes are currently known, suggesting that millions of endophytic microorganisms are still untapped. Therefore, time requires to identify and isolate them for useful biological and pharmacological applications. Microorganisms living in interstitial spaces of healthy plants have a complex interaction with their host plant and can be a useful source of bioactive metabolite compounds. Existing antimicrobials and antiviral drugs are being ineffective against continuous morphology changing viruses like HIV and COVID-19 or in the case of bacteria that are gaining antibiotic resistance or multiple drug resistance. Currently, the nCoV-2019/COVID19 pandemic situation enforces agencies, researchers, and scientists to think about new approaches with novel endophytic fungal strain for the isolation of natural active antiviral compounds. Nowadays, Omics-based molecular techniques are highly useful and reliable for finding the drug-like compounds, drug targets, and also support high-throughput screening. In the future, genetic engineering, upgraded cultivation, and fermentation techniques would allow scientists to isolate unexplored endophytic fungal strains for novel bioactive compounds. Genetic engineering coupled with omics approaches would be highly likely to obtain optimal production of secondary metabolites. Further, manipulating their metabolic pathway for mass production of bioactive compounds could be a game-changer. Acknowledgment The research was financially supported by the Ministry of Science and Higher Education of the Russian Federation within the framework of the state task in the field of scientific activity (no. 0852-2020-0029).

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Endophytic Bacteria for Plant Growth Promotion Nitin Bohra, Rupesh Kumar Singh, Raksha Jain, Lav Sharma, Eetela Sathyanarayana, Francisco Roberto Quiroz-Figueroa, and Vishnu D. Rajput

Abstract

Endophytic bacteria survive in close association with their host plants. These bacteria become an integral part of the host tissue system. Almost every plant species is found to be in association with these bacteria. These are the promising agents that promote their host growth even in stressed environments, like in phytoremediation. They flourish their host’s growth either directly or indirectly. Directly by accumulating nutrients and modulating the level of phytohormones. Indirectly, by producing antibiotics, cell wall degrading enzymes, nutrient

N. Bohra Department of Agriculture Engineering, Indian Institute of Technology, Kharagpur, India R. K. Singh (*) Centro de Química de Vila Real (CQ-VR), Universidade de Trás-os-Montes e Alto Douro, Vila Real, Portugal e-mail: [email protected] R. Jain Anand Agriculture University, Anand, Gujarat, India e-mail: [email protected] L. Sharma Syngenta Ghent Innovation Center, Devgen NV, Ghent, Belgium E. Sathyanarayana Yerevan State University, Yerevan, Armenia F. R. Quiroz-Figueroa Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional Unidad Sinaloa (CIIDIR-IPN Unidad Sinaloa), Laboratorio de Fitomejoramiento Molecular, Instituto Politécnico Nacional, Guasave, Sinaloa, Mexico V. D. Rajput Academy of Biology and Biotechnology, Southern Federal University, Rostov-on-Don, Russia # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. K. Singh et al. (eds.), Bacterial Endophytes for Sustainable Agriculture and Environmental Management, https://doi.org/10.1007/978-981-16-4497-9_9

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limitations, and activating resistance system of the host plant against the broad spectrum phytopathogens. Today these endophytic bacteria grab the attention of scientists, as these are the potential source of sustainable agriculture. Instead of using agrochemicals which are the main cause of soil health and environmental depletion, farmers should start using these endophytes as biocontrol agents, biofertilizers, biofungicides, in seed treatment, and in the reclamation of soil contaminated with heavy metals (phytoremediation). This application promotes sustainable agriculture and prevents the growing population of the world from future hazards in agriculture. Agriculture is the basic need for human survival so it requires the attention to be secured. In this chapter, we will go to the depth of the above-mentioned topics and more about the endophytes. Keywords

ACC deaminase · Endophytes · Phytoremediation · Sustainable agriculture

9.1

Introduction

The hypothesis about bacteria living inside the plant tissue goes back to Perotti (1926), but studies have been done on non-symptomatic bacteria residing inside plant tissues by Pasteur and others in the late nineteenth century (Hollis 1949). Over the course of evolution, plants have developed associations with different species of microorganisms for their benefits, which makes them compatible in a hostile environment (Santoyo et al. 2016). These beneficial microorganisms can be found both outside and inside the plant tissue. Bacteria living outside the host plant are generally known as epiphytes while those living inside the hostplant are called endophytes (Compant et al. 2010; Hardoim et al. 2008). Bacteria isolated from healthy internal plants tissue comprise 129 different bacterial species from 54 genera, with Bacillus, Pseudomonas, Enterobacter, and Agrobacterium as the most common isolates (Gardner et al. 1982; Hallmann et al. 1997). Endophytic bacteria are considered to be the subset of rhizospheric bacteria, and they are commonly known as plant growth-promoting bacteria (PGPB) (ReinholdHurek and Hurek 1998). The most acceptable functional definition of endophytic bacteria is the bacteria which is isolated from the surface-sterilized plant internal tissue (Hallmann et al. 1997). Rhizopheric bacteria share common traits with endophytic bacteria and both can have plant growth-promoting beneficial effects on their host plant. However, it is understood that endophytic bacteria can have usually greater beneficial effects compared to their rhizospheric counterparts (Chanway et al. 2000). Endophytic bacteria can be found in various tissues of the plant, both above and below the ground (Chebotar et al. 2015). It has been reported that almost 300,000 species of plants have one or more endophytes associated with them (Ryan et al. 2008). Endophytes help plants to promote growth, cope with stress conditions, and also show allelopathic effects against other competing plant species (Cipollini et al. 2012; Rosenblueth and Martínez-Romero 2006). Thus, endophytic

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bacteria are very useful for host plants to make them survive in a hostile environment (Mei and Flinn 2010). In this chapter, we will discuss the different mechanisms employed by endophytic bacteria to promote plant growth and health such as nutrient acquisition, phytohormone production, ethylene level maintenance, and other indirect mechanisms. We will also explain how 1-aminocyclopropane-1-carboxylase (ACC) deaminase plays an important role to promote plant growth. After this, we will briefly look into the aspect of which genes are responsible to give bacteria their endophytic behavior. With all this background information, authors will then look into the potential application of endophytes in agriculture.

9.2

Ecology and Diversity

Bacterial endophytes are present in almost all plants studied so far, plants without endophytes are very rare in nature. Endophytes-free plants are incompetent to survive in nature because they are susceptible to phytopathogens and various stress conditions (Hallmann et al. 1997). Bacterial endophytes are considered to be the subset of rhizospheric bacteria which has an added advantage on their rhizospheric counterparts (Germida et al. 1998; Marquez-Santacruz et al. 2010). Bacterial endophytes colonize the living tissue of their host plants thus always in close contact with the plant’s cell. Therefore, endophytes are able to provide direct beneficial effects to their host plants. Bacterial endophytes need to constantly compete with other microorganisms in the rhizosphere. It has been reported that endophytes are able to interact with their host plants in a very efficient way thus promoting their colonization in root tissue (Compant et al. 2010; Rosenblueth and Martínez-Romero 2006). Endophytes colonize different parts of the host plants such as roots, stems, leaves, and fruits (Zinniel et al. 2002). To gain entry inside the host plant, endophytes employ several mechanisms such as through stomata, lateral roots, germinating seedling, and young stems but the most common method for entry is through wounds and root cracks (Agarwhal and Shende 1987; Sprent and de Faria 1998). Wounds and root cracks of host plants exudate plant secondary metabolites which attract the bacterial endophytes to colonize the host plant tissues (Hallmann et al. 1997) (Fig. 9.1).

9.3

Mechanism of Plant Growth Promotion

Endophytic bacteria employ various mechanisms to promote plant growth. Broadly, all the mechanisms can be classified into direct and indirect mechanisms (Gamalero and Glick 2011). The direct mechanism of plant growth promotion by endophytic bacteria helps plants to acquire nutrients from the surrounding or modulating plant growth by regulation of plant growth hormone. Whereas, the indirect mechanism consists of producing antibiotics, protecting plants from pathogenic agents, and

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Fig. 9.1 Possible entry sites of endophytes in to the host plant tissues

production of volatile compounds (Glick 1995, 2012, 2015). We will look into different mechanisms in more detail in the subsections that follow.

9.3.1

Direct Mechanism

9.3.1.1 Nutrient Acquisition Endophytic bacteria have been reported to acquire nutrients such as nitrogen, phosphorus, and iron for their host plants in nutrient-deficient soil to promote plant growth (Glick 2012). For nitrogen acquisition, nitrogenase activity plays important role in N2 fixing bacteria (Montanez et al. 2012). Though endophytes are not as efficient as Rhizobium, they perform better than rhizosphere microorganisms in nitrogen-limiting environments. Azopirillum brasilense, Gluconacetobacter diazotrophicus, and Burkholderia spp., have been reported to increase nitrogen biomass in controlled experiments (Bhattacharjee et al. 2008). Endophytes also help plants in phosphorus uptake from the insoluble pool of phosphorus from the soil (Ahemad 2015). They employ several different mechanisms such as acidification, ion-exchange, chelation, and secretion of acid phosphatase to mineralize organic phosphorus for the uptake of phosphorus from the soil (Nautiyal et al. 2000; Van Der Heijden et al. 2008). As phosphorus is an essential micronutrient for plants and endophytes increase the availability of phosphorus to the host plant, it ultimately promotes the growth of the hostplants and maintains their health. Iron is present in several enzymes which control various physicochemical processes and hence its availability directly impacts the plant’s growth (Ma et al. 2016). Endophytic bacteria produce siderophore, which is an iron-chelating agent. Plants

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can then uptake these insoluble ferric ions with the help root-based ligand exchangers (Rajkumar et al. 2009). It has been reported in maize that siderophore from the endophytes plays a major role in fulfilling the iron requirement and has a strong correlation with plant biomass (Marques et al. 2010; Radzki et al. 2013). Siderophore production is also associated with inhibiting the growth of phytopathogenic molds, may be due to iron depletion.

9.3.1.2 Phytohormone Production Recent studies have shown the possible role of plant hormone production by plant endophytic bacteria in increased plant nutrient uptake and biomass (Gravel et al. 2007; Shi et al. 2014). Among the five plant hormones, namely cytokinins, ethylene, auxin (IAA), abscisic acid, and gibberellins, IAA and ethylene play a major role in plant–endophyte interaction. Indole-3-acetic acid (IAA) is the major plant hormone that is involved in various physicochemical processes such as metabolite production, defense mechanism, cell signaling, and development (Navarro et al. 2006; Spaepen et al. 2007). Regulation of IAA can directly affect the production of other plant hormones such as ethylene, which has a profound impact on plant health and development (Woodward and Bartel 2005). It has been reported that endophytic bacteria isolated from orchids produced IAA which improved plant root biomass and surface area in control experiments. Patten and Glick (2002) showed a mutant strain of Pseudomonas putida GR12-2 in IAA synthesis unable to increase root growth. It is worth mentioning that while the lower amount of IAA production is good for plants, a higher amount of IAA resulted in stunted growth. Higher IAA concentration has also triggered the production of pant stress hormone ethylene (Malik and Sindhu 2011). Several studies also have reported that plant endophytic bacteria can produce gibberellins and cytokinins (Cohen et al. 2009).

9.3.1.3 Ethylene Level Maintenance and Role of 1-Aminocyclopropane-1-Carboxylase (ACC) Deaminase In abiotic and biotic stresses ethylene plays a major role in controlling plant development. Some of the processes in which ethylene is required are cell elongation, fruit ripening, leaf senescence, and auxin transport (Glick et al. 1998). When a plant is subjected to abiotic and biotic stress it increases the ethylene production which ultimately inhibits root elongation and negatively impacts the plant growth. 1-aminocyclopropane-1-carboxylate (ACC) is the precursor molecule of ethylene (Honma and Shimomura 1978). Endophytes produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase which is an enzyme that can hydrolyze ACC. Endophytes present in the root can degrade this ACC molecule into α-ketobutyrate and ammonia, thus preventing excess production of ethylene and hence help in alleviating plant stress. Hydrolyzed molecules are then used by bacteria as their nitrogen source. Thus, the production of ACC by endophytes is related to the increased growth of the plant (Glick 2012, 2015).

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Indirect Mechanism

Bacterial endophytes prevent the host plant from several pathogenic agents like fungi, bacteria, nematodes, and pests, which indirectly results in promoting plant growth. Endophytic bacteria provide resistance to their host plant by producing antibiotics, cell wall degrading enzymes, lowering plant growth inhibiting hormones like ethylene and siderophores, and synthesizing pathogen-inhibiting volatile compounds (Glick 2015). Bacteria belonging to genera Actinobacteria, Bacillus, Enterobacter, Paenibacillus, Pseudomonas, and Serratia are commonly known to have antimicrobial activity towards the phytopathogens. Prevention from fungal attack is done by producing cell wall-degrading enzymes like chitinase and glucanases (Zarei et al. 2011). Some endophytic bacteria and fungi are known as biocontrol agents, like Pantoea vagans C9-1 for the fire blight (Smits et al. 2011) and Trichoderma harzianum, T. viride against soil-borne pathogens, foliar pathogens respectively. Control of burrowing nematode by endophytic bacteria Bacillus megaterium BP17 and Curtobacterium luteum TC10 was reported by Aravind et al. (2009). Another mechanism referred to as Induced Systemic Resistance (ISR) is used by plant beneficial bacteria to protect the host plants against bacteria, fungi, and virus (Alvin et al. 2014). ISR induces the plant defense mechanism at its highest level, thus helping in protecting unexposed plant parts from future attacks. Endophytic bacteria induce this defense mechanism by using salicylic acid (SA), jasmonic acid (JA), and ethylene (ET)-mediated pathways (Pieterse et al. 2012).

9.4

Gene Responsible for Plant Growth

There are several genes involved in making a bacterium exhibit endophytic behavior. Several studies have been done to understand the role of different genes using techniques like the bioinformatics approach (Ali et al. 2014), gene overexpression (Fan et al. 2013), real-time PCR (Yousaf et al. 2011; Zhao et al. 2016), and gusA fusion reporter system (Roncato-Maccari et al. 2003). A bioinformatics approach is used to compare endophytic and rhizospheric plant growth promoting bacteria. DNA sequences of different Burkholderia spp. were compared and genes from rhizhopheric strain subtracted from the endophytic strain to find out which genes are responsible for plant growth behavior. Nearly 40 different genes encoding various transporter channel proteins, ATPase, transcriptional regulation genes, secretion and delivery system were identified (Ali et al. 2014) (Table 9.1). Studies on Burkholderia phytofirmans strain PsJN show that they require ACC deaminase, quorum sensing, and IAA degradation to colonize the host plant (Sun et al. 2009). During gene expression profiling studies, it has been revealed that the gene responsible for cellular homeostasis, energy production, transcriptional regulation, and oxidative stress are upregulated (Sheibani-Tezerji et al. 2015). Other studies have shown that gene upregulation is also induced by root exudates from

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Table 9.1 Gene responsible for endophytic behavior and plant growth promotion Bacteria Azoarcus sp. strain BH72

Gene eglA, eglS (endoglucanse)

Function Systemic colonization

pilT (type IV pili)

Endophytic colonization by twitching motility Systemic colonization

Bacillus

Pectinase

B. phytofirmans PsJN

acds (ACC deaminase) iacC N-acyl-homoserine lactone synthase Ferritin

Pseudomonas fiuorescens strain WCS417r Enterobacter ludwigii Burkholderia sp. KJ006 Pseudomonas sp., Rhodococcus sp. Pseudomonas sp. strain G

References ReinholdHurek et al. (2006) Böhm et al. (2007) Fan et al. (2013)

Stress reduction IAA degradation Quorum sensing Iron storage

Zhao et al. (2016) Zhao et al. (2016) Zhao et al. (2016) Duijff et al. (1997)

TonB-dependent siderophore receptor L-ornithine5monooxygenase O-antigenic side chain

Siderophore-mediated iron uptake Siderophore synthesis

CYP153 genes (alkane degradation) aiiA (N-acylhomoserine lactonase) alkB (alkane monooxygenase) carAB

Petroleum hydrocarbon degradation Pathogen quorum sensing disruption

Afzal et al. (2011) Cho et al. (2007)

Diesel degradation

Andria et al. (2009) Newman et al. (2008)

Attachment

Pathogen cell–cell signaling disruption

the host plant which then helps bacterium to colonize plant tissue and confer growth benefits (Shidore et al. 2012).

9.5

Agricultural Application of Bacterial Endophytes

9.5.1

Growth Promotion

Conventional farming depends upon pesticides, fertilizers, and other agrochemicals. Long-term application of these chemicals deteriorates both soil and plant health. These problems threaten human life. To overcome this upcoming threat, biofertilizers, bioherbicides, and biocontrol agents attract the attention of researchers. These natural-based products are being widely used to enrich soil health. These natural products are derived from different groups of microbes like

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bacteria, fungi, and protozoans. In this context, we discuss bacteria in detail. Various genera of PGPB are used as biofertilizers for a few decades to increase crop yield and productivity. Genera like Azotobacter, Bacillus, Klebsiella, Anthrobacter, Enterobacter, and Pseudomonas have been used as biofertilizers (Singh et al. 2019). PGPB-derived biofertilizers provide essential nutrients like nitrogen, phosphorous, and potassium and promote plant growth. Production of HCN and NH3 by PGPB are considered as an important activity to promote plant growth. Produced HCN is used in the formation of chelating agents of metal ions as well as directly involved in phosphate availability (Rijavec and Lapanje 2016). NH3 producing PGPB are responsible for the accumulation and provision of nitrogen to their host plant which later promotes their shoot–root elongation, thus increasing the biomass (Marques et al. 2010). Interestingly, few strains have the ability to synthesize both HCN as well as NH3 and promote the growth of their host plant in a synergetic manner (Kumar et al. 2016). PGPB enriches the host plant with nitrogen by directly fixing atmospheric N2 to their rooting system and delay the play senescence (Kuan et al. 2016). Endophyte Alcaligenes faecalis sub sp. faecalis str.S8 is used as a biofertilizer in the host plant Withania somnifera which promotes plant growth as it has PGP (plant growth-promoting) activities of phosphorus solubilization and IAA production (Abdallah et al. 2016). Rhizosphere strain Serratia Ureilytica Bac5 work as a biofertilizer in Ocimum sanctum with PGP activities of siderophores formation, ACC deaminase, and phosphorus solubilization (Barnawal et al. 2012). Rhizosphere strain Pseudomonas BA-8, Bacillus OSU-142, Bacillus M-3 work as biofertilizer in strawberry with PGP activity of auxin and cytokinins production. The abovementioned examples show the variation among different bacteria to promote plant growth and yield. These PGPB-derived biofertilizers are also applied in seed treatment, which helps in promoting seed germination and yield. Seeds of various crops like carrot, cucumber, pea, beet, and tomato inoculated with biofertilizers show a significant increase in seed germination and vigor. An experiment on maize shows similar results, seeds were inoculated with biofertilizer containing active compounds of two strain: B. megaterium var. phosphaticum and Azotobacter chroococcum with concentrations of 1 ml L 1 and 3.5 ml L 1 respectively. Seeds are treated with 3.5 ml L 1 biofertilizer by following two methods—direct and through filter paper seed treatment. Inoculated seeds start germinating from the second day; significant difference is observed on the second day of the experiment—the result shows 37% and 34% more germinated seeds in direct and filter paper treatment respectively than control seeds (distill water treated). Similarly, cucumber seeds grown in filter paper soaked with biofertilizers show 20–25% high germination rate as compared to control seeds (distill water treated). Seed inoculation in wheat with Azotobacter and Bacillus resulted an increased yield by 30% and 43% respectively (Bákonyi et al. 2013).

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Phytoremediation

The utilization of plants and their associated microbes or PGPB for the remediation of metal contaminated soil is termed as phytoremediation. After the year of experiments, some plant species have been identified to absorb and accumulate higher concentration of heavy metals like Pb and Cd, known as hyperaccumulators. The following are some examples of hyperaccumulators: Pteris vittata, Sedum plumbizincicola, Thlaspi caerulescens, and Solanum nigrum (Kong and Glick 2017). Unlike organic contaminants, heavy metals cannot be destroyed or decomposed by microbial activity. Heavy metals remain as such in the environment for years and cause toxicity, which implies deleterious effects on human welfare and natural resources. Researchers have found a way of phytoremediation to reclaim contaminated soil. But this method also has some limitations like it only removes metal from the top layers, deep layers below the root zone remain contaminated.

9.6

Conclusion

Bacterial endophytes have shown great potential in agriculture application to promote plant growth, as a biofertilizer, and for disease prevention. But these endophytes fail to give consistent results when applied in the field. One of the major reasons is that most of the scientists studied rhizospheric bacteria instead of endophytes due to their similarity in plant promotion mechanism. However, endophytes have different environments from their rhizospheric counterparts such as temperature, nutrient availability, pH, light emission, oxygen concentration, and interaction with other microorganisms. Therefore, the mechanism of plant promotion focused on endophytes need to be understood more clearly. Hence, it will be easy to design a new methodology for using bacterial endophytes in agriculture practices and decrease our dependence on chemical fertilizers and pesticides. Acknowledgement The reported study was funded by RFBR (no. 20-55-05014) and SC RA (no. 20RF-036).

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Ali S, Charles TC, Glick BR (2014) Amelioration of high salinity stress damage by plant growthpromoting bacterial endophytes that contain ACC deaminase. Plant Physiol Biochem 80:160–167 Alvin A, Miller KI, Neilan BA (2014) Exploring the potential of endophytes from medicinal plants as sources of antimycobacterial compounds. Microbiol Res 169:483–495 Andria V, Reichenauer TG, Sessitsch A (2009) Expression of alkane monooxygenase (alkB) genes by plant-associated bacteria in the rhizosphere and endosphere of Italian ryegrass (Lolium multiflorum L.) grown in diesel contaminated soil. Environ Pollut 157(12):3347–3350 Aravind R, Kumar A, Eapen S, Ramana K (2009) Endophytic bacterial flora in root and stem tissues of black pepper (Piper nigrum L.) genotype: isolation, identification and evaluation against Phytophthora capsici. Lett Appl Microbiol 48:58–64 Bákonyi N, Bott S, Szabó A, Jakab A, Tóth B, Makleit P (2013) Using biofertilizer to improve seed germination and early development of maize. Pol J Environ Stud 22:1595 Barnawal D, Bharti N, Maji D, Chanotiya CS, Kalra A (2012) 1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing rhizobacteria protect Ocimum sanctum plants during waterlogging stress via reduced ethylene generation. Plant Physiol Biochem 58:227–235 Bhattacharjee RB, Singh A, Mukhopadhyay S (2008) Use of nitrogen-fixing bacteria as biofertiliser for non-legumes: prospects and challenges. Appl Microbiol Biotechnol 80:199–209 Böhm M, Hurek T, Reinhold-Hurek B (2007) Twitching motility is essential for endophytic rice colonization by the N2-fixing endophyte Azoarcus sp. strain BH72. Mol. Plant Microbe Interact. 20:526–533 Chanway C, Shishido M, Nairn J, Jungwirth S, Markham J, Xiao G, Holl F (2000) Endophytic colonization and field responses of hybrid spruce seedlings after inoculation with plant growthpromoting rhizobacteria. For Ecol Manag 133:81–88 Chebotar V, Malfanova N, Shcherbakov A, Ahtemova G, Borisov AY, Lugtenberg B, Tikhonovich I (2015) Endophytic bacteria in microbial preparations that improve plant development. Appl Biochem Microbiol 51:271–277 Cho HS, Park SY, Ryu CM, Kim JF, Kim JG, Park SH (2007) Interference of quorum sensing and virulence of the rice pathogen Burkholderia glumae by an engineered endophytic bacterium. FEMS Microbiol Ecol 60:14–23 Cipollini D, Rigsby CM, Barto EK (2012) Microbes as targets and mediators of allelopathy in plants. J Chem Ecol 38:714–727 Cohen AC, Travaglia CN, Bottini R, Piccoli PN (2009) Participation of abscisic acid and gibberellins produced by endophytic Azospirillum in the alleviation of drought effects in maize. Botany 87:455–462 Compant S, Clément C, Sessitsch A (2010) Plant growth-promoting bacteria in the rhizo-and endosphere of plants: their role, colonization, mechanisms involved and prospects for utilization. Soil Biol Biochem 42:669–678 Duijff BJ, Gianinazzi-Pearson V, Lemanceau P (1997) Involvement of the outer membrane lipopolysaccharides in the endophytic colonization of tomato roots by biocontrol Pseudomonas fluorescens strain WCS417r. New Phytol 135:325–334 Fan X, Yang R, Qiu S, Hu F (2013) Over-expression of pectinase gene in endophytic Bacillus strains and its effect on colonization. Chin J Appl Environ Biol 19:805–810 Gamalero E, Glick BR (2011) Mechanisms used by plant growth-promoting bacteria. In: Maheshwari MK (ed) Bacteria in agrobiology: plant nutrient management. Springer, Berlin, pp 17–46 Gardner JM, Feldman AW, Zablotowicz RM (1982) Identity and behavior of xylem-residing bacteria in rough lemon roots of Florida citrus trees. Appl Environ Microbiol 43:1335–1342 Germida JJ, Siciliano SD, Freitas JR, Seib AM (1998) Diversity of root-associated bacteria associated with field grown canola (Brassica napus L.) and wheat (Triticum aestivum L.). FEMS Microbiol Ecol 26:43–50 Glick BR (1995) The enhancement of plant growth by free-living bacteria. Can J Microbiol 41:109–117

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Bacterial Endophytes and Bio-nanotechnology

10

Shruti Rathore, Mansi Ujjainwal, Ajeet Kaushik, and Jyoti Bala

Abstract

Bio-nanotechnology is of great interest and a promising area for biomedical research and applications. Bacterial entophytes have tremendous future prospects in the biomedical sector. Recent advances in nanotechnology have shown several important applications in different areas such as biomedical sector, agriculture, cosmetic, industry, and environment. In recent times, novel biological systems have been developed for the production of NPs. Recently, endophytic microbes have been utilized as bio-factories of nanoparticles (NPs) production. Endophytes are microbial symbionts that reside in plants and play a vital role in plant growth, nitrogen fixation, phytohormone production, nutrient regulation, and stress control. Endophytic bacteria have a promising role in bio-nanotechnology. Current development shows its prospect in biomedical area. This chapter offers a comprehensive view on bacterial endophytes, their recent biomedical scope, synthesis, associated challenge, and their implication in bio-nanotechnology. Bacterial endophytes have incredible future prospects in the biomedical sector and bio-nanotechnology. Keywords

Bacterial endophytes · Bio-nanotechnology · Nanomaterials · Plant–microbe Interaction · Bio-remediation S. Rathore · M. Ujjainwal Amity Institute of Biotechnology (AIB), Noida, Uttar Pradesh, India A. Kaushik Division of Sciences, Arts & Mathematics (SAM), Department of Natural Sciences, Florida Polytechnic University, Lakeland, FL, USA J. Bala (*) Rapture Biotech International Pvt Ltd., Noida, Uttar Pradesh, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. K. Singh et al. (eds.), Bacterial Endophytes for Sustainable Agriculture and Environmental Management, https://doi.org/10.1007/978-981-16-4497-9_10

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Introduction

Nanobiotechnology holds significance in medical, pharmaceutical, and food industries (Wenlong et al. 2008; Singh et al. 2017a). Hence, development of reliable as well as safe processes of nanoparticle (NPs) synthesis becomes an essential aspect of the field of nanotechnology. Using biologically synthesized NPs such as bacterial endophytes enables protection for nanotechnology users. The NPs derived are of variable sizes, composition, and shapes, thus providing innumerable applications such as receptors, catalyzers, and bio-labellers to researchers. The influence of the microbial world has provided a possible alternative to the primitive chemical methods used. Endophytes are microbes living in symbiotic relationship with the plant tissues in their roots, leaves, stem, seed, fruits, etc., even though the population density of endophytes is the highest in roots. This excludes the pathogenic and nodule-producing microorganisms that cause harm to their host plants. Bacterial endophytes are known to promote plant growth by phytohormone production, nutrient regulation, and by providing resistance to biotic and abiotic stress (Santoyo et al. 2016). Additionally, these endophytic bacteria are also associated with the production of antimicrobial metabolites, nanoparticle synthesis (NPs), and other pharmaceutical products, which make them a promising candidate in biomedical sector (Mohamad et al. 2018). Recently, Nano-biotechnology has gained widespread recognition in the scientific field due to its extensive significance in various sectors, including medicine, diagnosis, therapeutics, pharmaceuticals, drug-delivery, bio-imaging nanocomposites, bio-sensing, electronics, cosmetics, and space industries (Clarance et al. 2020; Rahman et al. 2019; Singh et al. 2014). This technology is based on the synthesis of very small particles known as ‘nanoparticles’ (NP) which are in-demand for their perceptible properties such as their altering mechanical and thermal properties, high surface-to-volume ratio, catalytic metal characteristics, optical, physical, and other chemical features, which enhances valuable diverse applications (Khan et al. 2019; Kumbhakar et al. 2014; Toranzos et al. 2017). Moreover, they are well-known for their variations in shape and size, and surface parameters of their internal structure. The structural configuration of NPs comprises of three layers. The surface layer is made up of polymers, surfactants, small molecules, or metal ions. The shell layer consists of chemically synthesized materials different from other materials used in the core layer. Finally, the core, which makes up the chief composition of the NP (Shin et al. 2016). These NPs are usually made up of metals, alloys, ceramics, nanoparticle dispersions, nanopowders, carbon-based semiconductors, nitrides, oxides, dendrimers, etc. (Mohajerani et al. 2019; Wahyudi et al. 2018). Some of the most widely used metal NPs are made up of silver, gold, platinum, zinc, titanium, thallium, cerium, iron, and their oxides, nitrides, phosphates, fluorides, hydroxides, sulfides, and chlorides (Zhang et al. 2016; Gawande et al. 2016; Slavin et al. 2017). They are significant for their opto-electrical properties and a broad absorption band in the electromagnetic spectrum. Gold nanoparticles can enhance the electronic scheme and are thus used in sampling of the Scanning Electron Microscope (SEM) to generate a high resolution SEM image (Dreaden et al. 2012).

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NPs are synthesized by top-down and bottom-up approaches. Briefly, in the top-down synthesis, larger molecules are destructed into smaller units which are then converted into NPs. It involves methods such as electro-explosion, sputtering, chemical etching, mechanical milling, and laser ablation (Priyadarshana et al. 2015). On the other hand, bottom-up synthesis is opposite to top-down, where smaller and simpler substances are combined together to form NPs. This includes Laser pyrolysis, spinning, template support synthesis, plasma/flame spraying synthesis, atomic/ molecular condensation, and Chemical Vapor Deposition (CVD) (Wang et al. 2016). Moreover, bottom-up synthesis approach also involves ‘biological synthesis’ using plants, fungi, bacteria, algae, yeasts, etc. (Wang and Xia 2004). The traditional physical and chemical methods of NP synthesis make use of hazardous chemicals that are highly toxic and not environment friendly. Hence, there is a need to develop eco-friendly, cost-efficient, safe, and effective NPs. This is done by using the biological methods of NPs synthesis. This is also referred to as ‘green synthesis’ of nano-materials (Devatha and Thalla 2018). Using plants, fungi, bacteria, yeast, and other biological source for NPs production. Environmental performance of existing industries, toxicity concerns could be improved, and thus energy consumption would be minimized. For green synthesis of NPs, bacterial endophytes have proven beneficial in the past with promising experimental results, and current developments also establish their promising role in bio-nanotechnology sector (Rahman et al. 2019; Dong et al. 2017).

10.2

Applications of Bacterial Endophytes

Bacterial endophytes are known to be a great source of antibiotics and other pharmaceutical products. Because of their high tolerance towards biotic and abiotic stresses, they enhance thermal and other physical properties of NPs. They play important roles in heavy metal phytoremediation and bioaccumulation (Ma et al. 2016a). These endophytes are involved in extracellular production of enzymes at a large scale (Khan et al. 2017). In addition to this, they also help in the formation of water insoluble complexes and bio-reduction of ions to enhance their defence mechanism against toxicity (Ma et al. 2016b). Endophytic bacteria provide wide varieties of bioactive secondary metabolites, which enhance their application in the production of agrochemicals, antibiotics, antiparasites, antioxidants, and immune suppressants (Fig. 10.1). The emergence of antibiotic resistance is a major concern and global issue. Development of novel effective antibacterial agents could be a great resolution. Resistant microbes are more complex to treat; therefore, alternative agents are highly essential. Several studies have shown NPs synthesized from bacterial endophytes as potential anti-bacterial materials. Several antimicrobial metabolites are also discovered using endophytic bacteria (Ek-Ramos et al. 2019). This has helped to tackle with the increased levels of drug resistance in various plant and human pathogens (Christina et al. 2013). Bacterial endophytes-based antibacterial NPs production has prospect to tackle the (antimicrobial resistance) AMR challenge and its implication in biomedical area. Hence, several of such

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Plant growth stimulation Extracellular production of enzymes

Disease supression

Alkaloids, terpenoids, quinones, etc.

Anticancerous compunds

Applications of bacterial endophytes

Antimicrobial drugs, antibiotics, etc. Bioactive secondary metabolites

Nanoparticles

Fig. 10.1 The diagram represents various applications related to bacterial endophytes

effective antimicrobial drugs have been studied using endophytic bacteria, while some new candidates are still in their developmental phase (Beiranvand et al. 2017). They are also used for the production of alkaloids, terpenoids, phenolic acids, quinones, flavonoids, benzopyrones, steroids, tetralones, and xanthones. Some of these bacterial endophytes are also being studied for their role in cancer therapeutics research (Singh et al. 2017b). They have been associated with phyto-remediation or rhizoremediation, which is the degradation of accumulated environmental pollutants from plants (Mukherjee et al. 2018). These endophytes can degrade hydrocarbons, herbicides, and explosives. Moreover, atmospheric fixation, nitrogen fertilizers are expensive to be implemented, though bacterial endophytes have been studied and observed to have nitrogen fixation characteristics that are cost-effective, safe, effective, and eco-friendly. These are known to enhance the cereal biomass of the soil without the use of nitrogen fertilizers (Padda et al. 2019). NPs synthesized by traditional chemical and physical methods have various drawbacks. Especially metal NPs are heavily toxic materials that can have hazardous consequences in human body, as well as plants. NPs are used as excellent drug-delivery systems in various diseases. Metal NPs such as Silver (AgNPs) and Gold (AuNPs) are toxic in nature, and since they have the capability to cross the blood brain barrier, there is a

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risk of these toxic materials entering the brain of the patient and cause neurotoxicity (Karmakar et al. 2014). Hence, green synthesis of NPs from bacterial endophytes is a highly researched field to overcome these drawbacks of metal and carbon-based NPs-associated concerns, which can be overcome by using bacterial endophytesbased NPs.

10.3

Biosynthesis of Nanoparticles Using Endophytic Bacteria

Several reports that shows the NPs synthesis by using endophytic bacterial. Various methods been standardized for optimal bulk production of endophytic bacteria derived NPs. Endophytic bacteria are found in various regions of the plant such as roots, stem, fruits, and seeds. For instance, Curtobacterium and Pseudomonas are found abundantly in the aerial parts of the Poplar plant (Kandel et al. 2017). Similarly, dominant endophytes such as Flavobacterium, Serratia, Collimonas, and Burkholderia are found in abundance in Sphagnum mosses (Shcherbakov et al. 2013). These endophytic bacteria can be isolated either from surface plant tissues or internal plant tissues (Fig. 10.2).

Isolation of the bacterial endophytes from plant

Cultured bacterial plates are incubated and stored

Culture filtrate is mixed with aquoeous metallic salt solution

The mixed solution is incubated and centrifuged (depending upon extracellular or intracellular process) Color change in the solution indicates formation of nanoparticles

Nanoparticles are capped and stabilized and extracted by ultra-sonication and centrifugation Fig. 10.2 The process of biosynthesis of metal nanoparticles by using bacterial endophytes

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10.3.1 Isolation of Endophytic Bacteria from Plant Leaves Taking the example of extracting bacterial endophytes from a tea leaf sample, the isolation of these bacteria have been shown by Yan et al. (2018). Briefly, the leaf sample is first cleaned under the running flow of water to ensure the removal of debris from the leaf surface. This is followed by sterilization of the leaf using isolation media. Finally, incubation is carried out, after which the bacterial colonies are selected, and re-streaking is done on Agar media plates. As a result, endophytic bacteria were successfully isolated from tea leaves. Another similar experiment was carried out by de Oliveira Costa et al. (2012) where endophytic bacteria had been extracted from leaves of Phaseolus vulgaris. Tissue extract culture was prepared from the leaf sample which was regularly monitored to determine the presence of microbial colonies. Once the endophytic bacteria are isolated successfully, they are mixed with metal salt solution for the formation of nanoparticles.

10.3.2 Biosynthesis of Nanoparticles Using Endophytic Bacterial Culture The biosynthesis of NPs using bacterial endophytes can be done either by extracellular or intracellular method (Rajeshkumar et al. 2013). In the extracellular method, the culture filtrate collected by centrifugation is mixed with an aqueous metallic salt solution. The mixed solution is kept and observed for color change that is an indicator of NP formation. For example, if the solution color changes from light yellow to dark brown, it signifies the formation of AgNPs (Vanaja et al. 2013). Various factors affect this formation of NPs such as pH, temperature, incubation period, and salt concentration of the metal ion. Once the NPs are formed, they undergo capping and stabilization to generate the final product of bacterial endophytic-based NPs. For intracellular biosynthesis, the biomass is washed thoroughly with distilled water after the microbe has been cultured under optimal growth conditions. This microbe culture is then incubated with the metal ion solution which facilitates the formation of NPs. Like extracellular synthesis, color change acts as an indicator for NPs formation in intracellular production too. These NPs can finally be collected by ultra-sonication and centrifugation methods, washed, and selected (Hassan et al. 2019). Green synthesis of NPs using plant tissue extracts results in an elevation of metal ion concentration due to the presence of biomolecules such as benzopyrones, xanthones, steroids, terpenoids, quinones, and alkaloids (Ahmed et al. 2017).

10.4

Advantages Over Conventional Nanoparticles

Metal NPs have been used in diverse fields for their renowned characteristic features including their opto-electrical and magnetic properties. They are widely used in the medical field, especially for their excellent properties that make them a suitable

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candidate for drug delivery. An interesting feature of NPs is that they can cross the blood-brain barrier in human system which allows target-mediated delivery easy and effective in neurological disorder therapy. However, due to heavy toxicity involved with metal nanoparticles, it becomes harmful for the patient. Hence, these nanomedicines are associated with neural toxicity in many drug delivery systems used in neurological therapeutics (Sawicki et al. 2019). Metal NPs are also known to kill some useful bacteria in the environment which is responsible for environmental imbalance. They are not eco-friendly (Bahadar et al. 2016). One of the most hazardous effects of metal NPs is their cellular toxicity which is responsible for causing cell-membrane damage, inhibition of Electron Transport Chain (ETC), formation of Reactive Oxygen Species (ROS), causing oxidative stress, destabilization of ribosomes, mitochondrial dysfunction, and DNA damage that subsequently contributes to cell death (Huang et al. 2017). This cellular toxicity is known to have carcinogenic properties. Apart from metal and carbon-based NPs, polymeric NPs cause occasional cytotoxicity, while dendrimers have a complex synthetic route. Liposomes can induce an immunogenic response in the host organism which would make the liposome-mediated drug delivery ineffective. In addition to this, the chemical- and physical-based syntheses of NPs are costly and extensive processes. These drawbacks are overcome by the NPs formed by green synthesis using bacterial endophytes. These NPs are eco-friendly and do not cause any toxicity. The biologically synthesized nanomaterials are involved in biomedical area, waste-water treatment, nano-bioremediation, pollutant scavenging, polymer degradation, and act as environment catalysts (Karpagavinayagam et al. 2020). Such NPs can induce oxidation-reduction mechanisms in their environment which promotes biochemical conversions (Shah et al. 2015). They are widely used in medical field, in the production of antimicrobials and antibacterial drugs. Some of these nanomaterials are also being used extensively in cancer therapy as they do not have any toxic effects in the body. Additionally, they are also used in wound healing and dentistry (Ankit et al. 2014). Bacterial endophytic-based NPs allow easy scale-up process for large synthesis in a cost-effective manner. They lack any limitations related to energy consumption, pressure alterations, or chemical toxicant usage. For their industrial applications they are used as nanocomposites, cosmetics, and nanopigments. They are also utilized in the production of superplastic ceramics, antifouling coating, and paper coating (Li et al. 2011). Table 10.1 shows a list of some of endophytic bacteria derived from plant species, which have been used for NPs synthesis.

10.5

Discussion

Contemporary studies focus on the biosynthesis of silver nanoparticles from a group of microbes (Rana et al. 2020; Iravani 2014). The synthesis of NPs by chemical and physical methods is especially costly; this becomes another contributing factor apart from dangerous threats these chemicals hold to the ecosystem. The inorganic NPS include silver (AuNPs) commonly used as bactericide and gold (AgNPs) commonly

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Table 10.1 List of some bacterial endophytes used for nanoparticle synthesis Name of the endophytic bacteria Pantoea ananatis Bacillus cereus Pseudomonas poae Bacillus megatherium

Plants derived from Eucalyptus, maize, melon, rice, onion, pineapple Garcinia xanthochymus, tomato plant roots Allium sativum—garlic plant Barley, rice, bean plants

Applications Synthesis of AgNPs, antimicrobial agents against multidrug-resistant bacteria (Monowar et al. 2018) Synthesis of AgNPs, antibacterial properties (Sunkar and Nachiyar 2012) Synthesis of AgNPs, antifungal properties against wheat head blight pathogen (Ibrahim et al. 2020) Synthesis of AuNPs (Sanpo et al. 2013)

used for its catalytic activity. The inorganic NPs are also gaining significance because of their role in medical imaging and disease therapeutics. Shifting focus from traditional inorganic to organic NPs (microorganisms) is required to establish more cost-efficient and eco-friendly input to nanobiotechnology. The research studies around the world are focused on bacterial endophytes; these are groups of microorganisms capable of growing intra- as well as inter-cellular in the tissues of higher plants. Endophytes do not cause any symptom or disease to their host plants (Iravani 2014; Baker 2012). In their mutual relationship with higher plants, endophytes provide surfeit of substances to their host plants. The substances produced by endophytes have shown potential for uses in fields of agriculture, medicine, and commercial industries. Bacteria are potential synthesizing sources of NPs like gold, silver, palladium, titanium dioxide, magnetite, and cadmium sulphide. They also possess the ability to reduce heavy metal ions. These properties make bacteria potential and successful candidates for NPs synthesis. This approach of deriving NPs from microorganisms particularly bacteria and fungi can be concluded to be a safe, environmentally benign approach with applications in agricultural, textile, medicinal, therapeutic, biochemical, and other allied industries. The current examples of green biosynthesis of nanoparticles using bacteria include silver NPs produced using Aeromonas sp. SH10, gold NPs produced using Bacillus subtilis, and cadmium sulphide NPs produced using Escherichia coli. Certain strains of Lactobacillus are capable of producing gold-silver alloy NPs (Iravani 2014; Baker 2012). The sizes and shapes of the NPs produced depend upon the microorganism used and experimental conditions of biosynthesis process. NPs have established their usefulness in optical product formations, disinfectants, food packaging materials, biosensors, cosmetics, electronics (Khandel and Shahi 2018; Sedaghat et al. 2016). The NPs of noble metals like gold and silver are fulfilling use in daily human life. Consumer goods like creams, soaps, toothpastes contain NPs of palladium, platinum (Sedaghat et al. 2016). Biofertilizers and biopesticides are currently derived from NPs that help in controlling crop diseases. One major advantage is that all of these are eco-friendly and confer safety to their consumers. These products have provided

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relevant information on anti-inflammatory effects of the biosensors, thus optimal for medicinal, drug delivery, genetic engineering, and related purposes (Ghiassi et al. 2018). Despite their diverse use, bacteria in biosynthesis of NPs possess certain drawbacks. The future prospects of bacterial endophytes in NPs require overcoming these challenges. A prominent application of NPs is in bioremediation and treatment of toxic chemicals, heavy metals, and pollutants. Future prospects include aspects such as selection of the best bacterial candidates. Large-scale production of NPs will require greater amounts of enzymes that can be produced in optimal conditions for cell growth and enzyme activity. Further studies can be made on the inheritable and genetic properties of the bacterial endophytes to understand the size and morphology of these biocatalysts and NPs (Mittal et al. 2013; Gaur et al. 2014). The world-wide research over sustainable, non-hazardous, non-toxic, inexpensive NPs promises a significant role for bacterial endophytes as well as their large-scale production.

10.6

Future Prospects

Green synthesis of NPs has various advantages over the traditional synthesis methods. With wide applications in the field of agriculture, medicine, industry, and environment these bacterial endophytic NPs are being researched and studied extensively. Selection of the best candidate in terms of their biocatalyst state for endophytic bacteria can be done by extensive research focused on the growth rate of the bacteria, its enzymatic properties, and biological pathways associated with it. Rate and quality of such NPs can be determined based on such study. Few studies are done on the extraction and purification methods involved in endophytic bacteria used for NPs synthesis could be explored in details and determination of soil microbiota and metagenomic data could also help to explore this area further which will impact the wider usage of bacterial endophytes derived NPs. These nanoparticles produced by green synthesis using endophytes have great stabilization characteristics, which might be due to the enzymes and proteins secreted by the microbes, which requires extensive research support. The safety and efficiency of these NPs is yet to be assessed on a larger scale to elevate its industrial applications in the near future. Nonetheless, this is a new and rather unexplored area of biotechnology which has exceptional potential for the growth of nano-biotechnology in various sectors. Considering the high toxicity levels of metal NPs, bacterial endophytic NPs are clearly a better choice for sustainable progression of nanotechnology. Conflict of Interest The authors declare no conflict of interest.

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Role of Endophyte Metabolites in Plant Protection and Other Metabolic Activities

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Neha Singh, Santosh Kumar Mishra, Priya Ranjan Kumar, Narendra Kumar, and Dhirendra Kumar

Abstract

Endophytes are generally symptomless microorganisms and may be of bacterial as well as fungal origin and have been found in almost all living plant species. Symbiotic association of these microorganisms with host plant by colonizing the internal tissues play valuable role in agricultural practices resulting in the improvement of crop production. Fungal endophytes produce specific secondary metabolites that promote the growth and development of host plant. Secondary metabolites of both plant and its endophytic microorganisms are produced due to symbiotic association including phenolic, flavonoids, tannins and saponins which inhibit the growth of other plant pathogen and microorganism. This chapter highlights the role of endophytes and their symbiotic association that plays a significant role in the plant defence system and other important metabolic activities. Keywords

Bioremediation · Metabolites · Phytohormones · Rhizospheric · Antagonoistic

11.1

Introduction

The growth and survival of plants in any environmental condition largely depends on their association with other components of the respective ecosystem. The plants uptake all the essential nutrients through their roots present in the soil. The soil has

N. Singh · S. K. Mishra (*) · P. R. Kumar · N. Kumar Department of Biotechnology, IMS Engineering College, Ghaziabad, UP, India D. Kumar Department of Biotechnology, R.R. Institute of Modern Technology, Lucknow, UP, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. K. Singh et al. (eds.), Bacterial Endophytes for Sustainable Agriculture and Environmental Management, https://doi.org/10.1007/978-981-16-4497-9_11

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an intricate ecosystem with diverse statistics of various bacteria, fungi, protists, and animals. These soil microorganisms play a significant role in the communications between plant roots and soil (Chandra and Enespa 2019). The interactions of soil microbial communities with plants are of various types, including pathogenic, symbiotic, and associative, which helps in plant productivity, stress tolerance, and disease resistance. Bacteria play a vital role in soil ecosystem and provide enormous benefits to the host plants by helping them to deal with the biotic and abiotic stresses that can challenge their growth. These beneficial bacteria can either be epiphytic, living on the plant leaf surfaces or roots (Compant et al. 2010), or endophytic, inhabiting inside the host plant especially inside roots. The endophytic bacteria secrete phytohormones and help in the nourishment of host plants using bidirectional nutrient transfer resulting in the protection of plants against various phytopathogens (Andreozzi et al. 2019; Shen et al. 2019). The bacterial endophyte and plant interection also trigger the host-plant adaptation for the certain stresses such as presence of heavy metal and drought (Khan et al. 2019; Kushwaha et al. 2019). Recent findings on plant gene expression and micro-RNAs (miRNAs) suggest that in plants the response of genes and pathways depend upon the endophytic microbe. Experiments have shown that inoculating endophytes in the host plants results in biomass production and thus boosting commercial agriculture (Santoyo et al. 2016; Shen et al. 2019). These endophytes help the host plants by generating a variety of bioactive metabolites, which can be used commercially as biocontrol agents, antimicrobial agents, antitumour agents, immune suppressants, and antiviral agents. Because of the production of secondary metabolites, the abundant endophytic microorganisms, present in nature, are continuously attracting the attention of biotechnology industries. These bioactive metabolites can be used in enzymesbased industries, agriculture, and medicine (Khan et al. 2014; Rajamanikyam et al. 2017).

11.2

Endophytic Bacteria

Endophytic bacteria have been recognized as a specialized group of rhizobacteria that have the ability to invade its host plants and promote its growth (ReinholdHurek and Hurek 1998), thus known as growth promoting rhizobacteria. They usually promote plant growth more than the rhizospheric bacteria in stressed conditions (Chanway et al. 2000). Perotti has described the endophytic bacterial growth as a specific stage when bacteria infect and develop a close mutualistic relation with plants (Perotti 1926). Now, the endophytic bacteria are described as the plant-host dependent symbiotic bacteria isolated from surface-sterilized plant tissues (Santoyo et al. 2016). These bacteria can exist within any part of the host plant and have a significant impact in promoting the growth and development of the plant (Chebotar et al. 2015). The bacteria use plant endosphere as a protective environment to provide consistent environmental conditions that affect rhizospheric and epiphytic bacteria (Senthilkumar et al. 2011). It has been reported that most endophytic bacteria have a biphasic life cycle. About 300,000 plant species on the

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earth are considered to be host to one or more endophytes, which include both fungi and bacteria (Ryan et al. 2008; Reinhold-Hurek and Hurek 2011; Singh et al. 2011). The endophytic bacteria involve in the growth promotion of the host plant, they are also helpful in tolerating stress conditions for host, and they produce allelopathic effects against other competing plant species (Cipollini et al. 2012; Mei and Flinn 2010; Rosenblueth and Martínez-Romero 2006).

11.3

Plant Colonization by Endophytic Bacteria

The endophytic bacteria first establish a rhizospheric population and then invade the plant roots, where they grow and form colonies. The process of colonization is complex and mediated by the specific metabolites produced by plant root exudates. The plant produces root exudates to communicate with the beneficial bacteria present in the soil for its own ecological advantage (Compant et al. 2005, 2010; de Weert et al. 2002; Rosenblueth and Martínez-Romero 2006). The colonization process of endophytic bacteria is similar to rhizospheric, but also involve several environmental and genetic factors (Hallmann et al. 1997; Compant et al. 2010). Besides the roots, endophytes can also enter into various aerial parts such as stems, leaves, flowers, and cotyledons (Zinniel et al. 2002). Once inside the roots, endophytic bacteria can now systemically infect the adjacent plant tissues. The bacterial traits like motility and polysaccharide production play a key role in the rhizosphere colonization. It helps the bacteria to attach to the root surfaces in a string form covering the whole root surface (Hansen et al. 1997). The endophytes produce a variety of metabolites to adapt to the range of nutrients available and to compete with the rhizosphere for their growth in root exudates. Once these bacteria form biofilms around the root exudates, they start penetrating inside the root interiors using different mechanisms. They can either directly enter via cracks, root tips, or deleterious areas or can enter by secreting cell wall-degrading enzymes and attaching using flagella, pili, or quorum sensing. To eliminate being recognized and attacked by host defence, the endophytic bacteria produce low level of cell wall-degrading enzymes and maintain low cell density in comparison to phytopathogens (Elbeltagy et al. 2000; Zinniel et al. 2002). Active colonization process of endophytic bacteria depends on the genetic characteristics of the bacterial species leading to plant and bacterial relationships. During this association plants play a critical role in the selection of endophytic partner where formation of specific compounds by roots and selective defence response of plant are considered as an important and critical factors in the selection of specific endophytes (Rosenblueth and Martínez-Romero 2006). After the entry of endophytic bacteria into the plant system through roots, it spread in a systematic way. During this process only a few bacteria colonize in the aerial part of plant tissues (Hallmann 2001). Plant interacts different types of microorganism during it's growth and promots different responses within microbial community member via a complicated network. Most recent researches reveal that endophytic fungi have the

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ability to support the defence mechanism of their host plants. This interaction also helps plants to adapt against both biotic and abiotic stresses (Yan et al. 2019).

11.4

Endophytic Bacteria Diversity

An estimate of plant diversity suggests that about 300,000 plant species are present and majority of these plants contain endophytes (Smith et al. 2008). It is also suggested that the bacterial endophytes have different genetic makeup compared to rhizospheres, which helps them to penetrate and colonize inside the plant roots. No defined genes or gene groups have been confirmed for this purpose and neither a list of genes with possible roles in endophytic behaviour is available (Ali et al. 2014a, b). Comparative genomic analysis provides insights into endophytic behaviour. A study reported that endophytes involve diverse and abundant genes for anabolic pathways, while the catabolism-related genes are prominently involved in the invasion of the host (Hardoim et al. 2015). This has been observed that co-existence of genes for nitrogenase and ribulose bisphosphate carboxylase or oxygenase worked as a specific marker for endophytes and are having symbiotic nitrogen fixation capabilities (Karpinets et al. 2014). Lateral gene transfer plays a key role in promoting genetic diversity and results in the acquisition of characteristics that are important for colonizing of the endophytes inside the plants. This process successively produces some important secondary metabolites that may play a significant role in the symbiotic relationship (Tisserant et al. 2013; Arora et al. 2018). The effect of lateral gene transfer was reported in several bacterial endophytes for mannitol dehydrogenase gene against phytopathogenic fungi (Wu et al. 2011). The endophytic Enterobacter sp. is reported to have genes for amino acid/iron transport, hemolysin, and hemagglutinin, which are important for host–bacterium interactions, on a large conjugative plasmid (Taghavi et al. 2009). The location of potent genes can thus give clues about endophytic lifestyle. Metagenome study reveals the colonization of bacteria in the plant endosphere. The reduction in genome size up to half of the normal soil bacteria has been observered in some of the endophytic bacteria (Sessitsch et al. 2012; Brewer et al. 2016). It has also been observed that not a single factor or mechanism is responsible for this endophytic behaviour. Genotype of host plant and species diversity influence the micobiome endosphere (Rodriguez-Blanco et al. 2015). Research reveals that the precise external or endogenous factors that are responsible for fungal transition from endophyte to pathogen are not well understood. The bacterial competence, host plant, soil and climatic conditions are the factors determining the growth of bacterial endophytes (Hallmann and Berg 2006). Various studies have reported that the plant species growing in the same field have different spectrum of endophytic bacterial species (Germida et al. 1998; Ding et al. 2013; Granér et al. 2003) and the same plant cultivar growing in the different soil shows diverse endophytic bacteria (Song et al.

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1999; Rashid et al. 2012). Activities of endophytic community can also be imposed by the plant host in response to soil and stress conditions.

11.5

Bioactive Compounds synthesized by Bacterial Endophytes

The mutual relationship of host plants and endophytes over a long duration results in the production of bioactive metabolites (Jia et al. 2016). The communication of endophytic communities with the host plant significantly influences the physiological activities of the plant i.e. activation of silent genes leading to the synthesis of certain secondary metabolites. Various studies have proved that plant-endophytes are able to produce an array of common secondary metabolites from the same precursors. These secondary metabolites belong to various classes such as antibiotics, antitumour and anticancer agents, podophyllotoxin, insecticides, and azadirachtin (Kusari et al. 2012; Puri et al. 2006). There are several reports on the precise effect of endophytes on host plant in secondary metabolites production. However these mechanisms are quite unknown. One possible hypothesis is that the homologous gene clusters present in plants and microorganisms may get cross-activated by stress-induced molecules from plant hosts or endophytes under certain conditions resulting in abundant secondary metabolites production (Howitz and Sinclair 2008). An endophytic actinobacterium Pseudonocardia induced artemisinin production in Artemisia plant by inducing the expression of cytochrome P450 monooxygenase and cytochrome P450 oxidoreductase genes (Liu et al. 2012). A recent research finding suggests that endophytes such as Acinetobacter sp. and Marmoricola sp. of Papaver somniferum L. upregulate the expression of key genes for the biosynthesis of benzyl isoquinoline alkaloid (Pandey et al. 2016a, b). Some studies reported that endophytes are involved in the gene expression of homospermidine synthase, terpenoid indole, and pyrrolizidine alkaloids responsible for the plant’s chemical defence (Pandey et al. 2016b; Sreekanth et al. 2017; Irmer et al. 2015). There may be possibilities that many other secondary metabolites could be synthesized by the plants in the presence of endophytes with novel properties.

11.6

Modulation of Plant’s Defence by Endophytes

The researchers use different methods to study various endophytic mechanisms which help it to get differentiated from pathogens and it’s recognition from the first line defense of the plant immune system. This involves pattern recognition of conserved molecules, characteristic of many microbes also known as microbe or pathogen-associated molecular patterns, including Flagellin, elongation factor, peptidoglycan, lipopolysaccharides, bacterial cold shock proteins, bacterial superoxide dismutase, beta glycan, β-glucans from oomycetes, and chitin (Newman et al. 2013). The endophytic bacteria either modify their recognition molecules or produce their

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own MAMPs, which are not recognized by plant’s receptors resulting in a comparatively weak and transient defence reaction compared to pathogenic interactions (Vandenkoornhuyse et al. 2015). In case of oxidative burst or generation of reactive oxygen species as plant defence system, the production enzymes such as superoxide dismutases, catalases, peroxidases, and glutathione-S-transferases (GSTs) of endophytes play a key role in the plant defence mechanism (Zeidler et al. 2004; Trda et al. 2015). Modulation of the immune system of plants is also governed by the protein expression system in bacteria. Among all known expression systems, type III secretion system of proteins and type IV secretion system are essential for delivering effector proteins by the pathogenic bacteria into the plant; however, these may be absent or present in low abundance in mutualistic endophytic bacteria (Green and Mecsas 2016; Liu et al. 2017). Several studies have proved that there is downregulation of plant defence pathways during the colonization of plants by mutualistic partners such as rhizobia and some mycorrhizal fungi (Fouad et al. 2014; Benhiba et al. 2015; Sarkar et al. 2016). Overstepping and overpowering of the plant during this mutualistic approach from microbe has been observed due to late induction of signalling pathways (Plett and Martin 2018).

11.7

Endophytic Metabolites in Plant Protection

Bacterial endophytes live in a symbiotic relationship with the plants. They take essential nutrients from plants for their survival and generate a variety of metabolites that help plants in their growth and survival. Endophytes also helps plants by suppressing the phytopathogens via its antagonistic activities (Miller et al. 2002; Gunatilaka 2006). Endophytes are also known to play roles in induced systemic resistance (ISR) against phytopathogens (Kloepper and Ryu 2006). Foliar endophytes are also reported to regulate the genetic expression of host by affecting physiological responses of plants and defensive pathways (Van Bael et al. 2012; Estrada et al. 2013; Salam et al. 2017). These endophyte metabolites help in plant protection by two mechanisms, direct or indirect, as shown in Fig. 11.1.

11.7.1 Direct Mechanism The endophytes can directly eliminate the phytopathogens through secretion of different bioactive metabolites such as lytic enzymes or allelochemicals, which can degrade the cell wall of pathogens or inactivate any essential pathway of pathogenic organisms. These endophytic bioactive compounds are used in pharmaceutical industries as an antimicrobial drug, an anti-cancer drug, an anti-viral drug, anti-diabetic agents as well as they help the host plant (Guo et al. 2008). The direct mechanisms implied by endophytes are characterized by the type of host, endophytes, and incoming pathogens. The direct defence system has been discussed here.

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Bacterial Endophytic Metabolites for Plant Protection

Direct Mechanism

Indirect Mechanism

(Elimination of Plant Pathogens)

(Increase in Plant’s Strength to Resist)

Antibiosis Lytic Enzymes Essential Nutrients: Availability and Competition

Phytohormone Modulation Bioremediation Induced Systemic Resistance

Fig. 11.1 Role of bacterial endophyte metabolites in the protection of plant

11.7.1.1 Antibiosis The endophytic bacteria can protect the plants from phytopathogens and pests by producing substances, with antagonistic properties, such as antibiotics, toxins, siderophores, and antimicrobial volatile organic substances (Gunatilaka 2006; Sheoran et al. 2015). The diversity of microbial species in any host plant stimulates the secretion of metabolites by the endophytes or the host to inhibit the growth of microbes that are harmful (Kusari et al. 2012). Plants are also well known to synthesize low-molecular-weight antimicrobial molecules known as phytoalexins, which are included in various groups of metabolites (Gao et al. 2010). Various studies demonstrated that the endophytic bacteria belonging to the genera Actinobacteria, Bacillus, Enterobacter, Pseudomonas, Paenibacillus, and Serratia successfully produce antimicrobial compounds against bacterial and fungal pathogens (Liu et al. 2010; Aktuganov et al. 2008; Lodewyckx et al. 2002). Different endophytic metabolites such as peptides, flavonoids, alkaloids, terpenoids, quinones, steroids, phenols, and polyketides have been studied to screen their antimicrobial properties (Mousa and Raizada 2013; Suryanarayanan 2013; Daguerre et al. 2016; Lugtenberg et al. 2016). The siderophores produced by endophytes, iron-chelating compounds for plants, also showed biocontrol activities (Rajkumar et al. 2010). Siderophore producing Rhizobium helps in the biological control of phytopathogen Macrophomina phaseolina which cause charcoal rot in several crops. In some instances, the host plant and endophytes are shown to use distinct pathways for enhancing the production of the metabolites, while some use induced metabolic process that helps in the metabolization of the product (Kusari et al. 2012; Arora et al. 2001; Ludwig-Müller 2015). Later it was concluded that many of the endophytes cannot produce these metabolites independently (Heinig et al. 2013). The endophytic bacteria living in plants like potato, wheat, and black pepper have been reported to eliminate their fungal disease (Coombs et al. 2004; Aravind et al.

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2009; Sessitsch et al. 2004). It was thought that these bacteria produce fungal cell wall targeting enzymes, namely chitinase, proteases, and glucanases (Zhang et al. 2012; Zarei et al. 2011). The elimination of bacterial pathogens was reported by Bacillus subtilis BSn5 that targeted the plant pathogen Erwinia carotovora subsp. carotovora. However, the mechanism of action against the pathogen was unknown (Deng et al. 2011; Dong et al. 1994). Much current biotechnology research is booming to identify endophyte metabolites for possible commercial use. Pantoea vagans C9-1, an endophytic bacterium, has been commercially produced as a bacterial biocontrol agent against fire blight (Smits et al. 2011). The inhibition of phytopathogenic burrowing nematode (Radopholussimilis thorne) by Bacillus megaterium and Curtobacterium luteum is an example of endophytic bacteria which was reported by Aravind et al. (2009). The elimination of plant pests by genetically modified endophytic Pseudomonas fluorescens that expresses the Serratia marcescens chitinase and Bacillus thuringiensis toxin effectively target the Eldana saccharina (a Sugarcane Borer) larvae (Downing et al. 2000). Endophytic bacteria produce several volatile organic compounds demonstrating the broad-spectrum antimicrobial activity against the phytopathogenic bacteria, fungi, as well as against nematodes. The black pepper plant-associated endophytic bacteria Pseudomonas putida BP25 has been reported for the secretion of volatile substances and thereby inhibiting the phytopathogens, Phytophthora capsici, Gibberella moniliformis, Pythium myriotylum, Rhizoctonia solani, Atheliarolfsii, Colletotrichum gloeosporioides, and the plant-parasitic nematode, Radopholussimilis (Sheoran et al. 2015). The antagonistic activity of several other bacterial endophytes was reported earlier. These strains are Pantoea, Bacillus, Pseudomonas, Serratia, and Stenotrophomonas from wild pistachio showing control of Pseudomonas syringae and Pseudomonas tolaasii (Etminani and Harighi 2018). Mercado-Blanco et al. (2004) isolated endophytic P. fluorescence from roots of olive trees antagonistic against Vertcillium.

11.7.1.2 Synthesis of Lytic Enzymes About 1350 lytic enzymes including hemicellulose, proteins, DNA, and chitin are secreted by microorganisms for the hydrolysis of polymers to serve different purposes (Gao et al. 2010; Tripathi et al. 2008). To colonize the roots, the endophytes secrete lytic enzymes to sequentially hydrolyze the plant cell wall. These enzymes help in reducing phytopathogens indirectly and also aid the fungi cell wall degradation. The types of enzymes used for this purpose are chitinases, cellulases, hemicellulases, and 1,3-glucanases. The lytic enzymes that were produced by Streptomyces have shown a strong antagonizing effect on cacao witches broom disease (Macagnan et al. 2008). The mutation in the lytic enzymes producing gene 1,3-glucanase of Lysobacterenzymogenes reduced the biocontrol activity against the Pythium and tall fescue leafspot disease of sugar beet (Gao et al. 2010). It may be possible that these enzymes do not only act as an effective antagonizing agent but also enhancing the antagonistic activities when combined with other mechanisms. The pectinase enzyme was reported to reduce the pathogenesis in the plant (Babalola 2007).

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Some other studies also reported the insecticidal properties of endophytes because of a reduction in pest penetration of the stele by thickening the endodermal cell wall or by producing secondary metabolites (Azevedo et al. 2000; Gao et al. 2010). The toxic metabolites produced by endophytes are pyrrolizidine, alkaloids, pyrrolopyrazine alkaloid, peramine ergot alkaloid, and ergovaline (Wilkinson et al. 2000).

11.7.1.3 Essential Nutrients: Availability and Competition The endophytes compete strongly and systemically with plant pathogens to colonize the host tissues (Martinuz et al. 2012; Lata et al. 2018). For example, they act via colony formation and the lurking of available nutrients and by occupying space that can be used by pathogens for their activities (Rodriguez et al. 2009). The mechanism used for competition by most endophytes usually takes place in combination with other mechanisms, instead of acting independently (Arnold et al. 2003). One of the important nutrients for plant growth is phosphate, present in the soil but in an insoluble form. The uptake of phosphate by plants is driven by the solubilizing activity of endophytes. Most soil-related microorganisms are capable of solubilizing insoluble phosphate and converting them into various mineral compounds such as organic acids, protons, siderophores, carbon dioxide, and hydroxyl ions, thus enhance the production of phosphate and make it available for plant use (Alori et al. 2017; Olanrewaju et al. 2017). Numerous bacterial species have been reported earlier for releasing the accessible form of potassium from potassium-bearing minerals in soils—Bacillus mucilaginosus, B. circulans, Pseudomonas sp., Burkholderia, Paenibacillus sp., Acidothiobacillus ferrooxidans, Pantoea spp. and Bacillusedaphicus. The studies also reported that the endophytic microbes can be used as a biofertilizer and biocontrol agent (Yadav 2018). Similarly, it was reported that the endophytic actinomycetes carry out the phosphate solubilization and its availability to plants via acidification, chelation, mineralization, and by the redox changes of organic phosphorus (Jog et al. 2014; Singh and Dubey 2018). The iron plays many important roles in plants such as chlorophyll development, nitrogen fixation, energy transfer, plant respiration, and metabolism. The deficiency of iron leads to chlorosis and a decrease in the crop production cycle. Irons are present in complex forms in the soil and made available to the plants by chelating agents. The siderophores secreted by endophytes is a chelating agent and help in the fixing of nitrogen (Kraepiel et al. 2009). Effect of specific pseudomonas strain a siderophore producing endophyte was observed on Vigna radiate for the nutrition of iron and the plants showed a reduction in iron and chlorotic symptoms, where as an increase in chlorophyll a as well as chlorophyll b content in the plant which was inoculated with specific strain as compared to the control (Sharma et al. 2003). Some other endophytic actinomyces such as Streptomyces sp. Streptomyces sp. Nocardia sp. and Streptomyces sp. has been reported to produce siderophores (Singh and Dubey 2018). S. acidiscabies was found as an excellent producer for siderophore which enhances the growth of Vigna unguiculata under nickel stress (Sessitsch et al. 2013).

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11.7.2 Indirect Mechanism The indirect mechanism involves the endophytic role in the enhancement of the plant’s defence line to eliminate the phytopathogens. The endophytic organisms actively increase the regulation of phytohormones to tackle the entering pathogens. By increasing the stress tolerance of plants, the endophytic organisms enhance the health of the host plant. Some endophytes use essential nutrients preventing the growth of pathogens by non-availability of nutrients, which is discussed in this chapter.

11.7.2.1 Phytohormone Modulation Many times, plants are unable to uptake the essential nutrients from the soil which are needed for their growth. Many studies have reported that the endophytes play a key role in the regulation of phytohormones production and regulation (Gravel et al. 2007; Phetcharat and Duangpaeng 2012; Shi et al. 2014). Endophytes produce phytohormones to enhance plant growth, thus play a key role in the area of agricultural sustainability (Sturz and Nowak 2000). The mechanism of phytohormone production adopted by endophytes with the machinery of rhizobacteria promote the plant growth. Secretion of Gibberillic acid and other phytohormones including Indole Acetic Acid (IAA) and ethylene promotes the growth and protection of non-leguminous plants (Khan et al. 2014; Patel and Patel 2014; Babalola 2010; Kang et al. 2012). The functions of indole acetic acid in plants are the initiation of plant cell division, differentiation, and extension, stimulation of seed and tuber germination, increase in the root and xylem development rate, enhancement of lateral initiation, controlling the rate of vegetative growth, adventitious root formation, and formation of pigments and metabolites This also supports controlling the responses of plant towards gravity, light, and fluorescence, affects photosynthesis and resistance to extreme conditions (Gao et al. 2010). In the case when the plants are unable to conceal required auxin for the growth, endophytic organisms start secreting IAA, which can increase the length of root and its surface area, leading to better absorption of nutrients from the soil. In addition to that IAA production expands the cell walls of bacteria and enhances the exudate secretions that provide more nutrients to other beneficial bacteria present in the rhizosphere. Therefore, the IAA forming endophytic bacteria are recognized as effective phyto-stimulators for pathogenesis and plant–microbe interaction (Gao and Tao 2012). The studies also demonstrated that the endophytic actinomycetes are also responsible for the production of plant growth-promoting compounds like IAA, which enhance the formation and elongation of adventitious roots in plants (de Oliveira et al. 2010; Shimizu 2011). 11.7.2.2 Stress Tolerance The plant responds and adopts to different stress conditions through various mechanisms. The two resistance patterns of plants viz Induced systemic resistance (ISR) and the Systemic acquired resistance (SAR) have gained more attention from researchers. The ISR pattern is driven by ethylene or jasmonic acid and induced by some non-pathogenic rhizobacteria, while the SAR is caused by infections from

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pathogens mediated by salicylic acid and linked with the building up of pathogenesis-related proteins (Tripathi et al. 2008). These pathogenesis-related proteins consist of lysis enzymes such as 1,3-glucanases and chitinases, which not only degrade the cell wall of invading pathogens but also strengthen the plant’s cell wall, thus building the plant’s resistance power against pathogens and cell death (Gao et al. 2010). The endophytes ISR can be associated with the alteration of genes present in pathogenesis. Fusarium solani endophytes harbouring tomato roots are believed to prompt ISR against Septoria lycopersici, the causative agent of the tomato foliar pathogens, and it activates the PR genes PR7 and PR5 in roots (Kavroulakis et al. 2007). Kusajima et al. (2018) observed that the bacterial endophyte Azospirillum sp. B510 induces systemic disease resistance in rice and they indicated that ethylene (ET) signaling is required for endophyte-mediated ISR in rice by gene expression analysis. Thus, salicylic acid and jasmonic acid, in particular, are known to play vital roles during plant stress responses against phytopathogens (Khare et al. 2016). Gibberellin-producing endophytes were also found to confer resistance against the attack of phytopathogens and insects through salicylic acid and jasmonic acid pathways (Waqas et al. 2015). Kavroulakis et al. (2007) reported that Fusarium solani elicits induced systemic resistance (ISR) against Septoria lycopersici (tomatofoliar pathogen) via induction of pathogenesis-related genes expression in root tissues. Theobroma cacao inoculated with foliar endophytic fungi, Colletotrichum tropicale, showed a reduction in Phytophthora infection (Mejía et al. 2008). Inoculation of C. tropicale resulted in the elicitation of many components of the ethylene defence pathway and several other signaling genes responsible for disease resistance in T. cacao, A. thaliana, and other hostplants (Mejía et al. 2014). The endophytes also found to increase the stress tolerance against abiotic stress such as drought, salinity, extreme temperatures, heavy metal toxicity, and oxidative stress that are severe threats to the agroecosystems (Wang et al. 2003; Khare and Arora 2015). The endophytes use different molecular mechanisms to increase the stress tolerance in host plants, which include induction and expression of stressresponsive genes, synthesis of anti-stress metabolites, and generation of scavenger molecules like ROS (Lata et al. 2018). Burkholderia phytofirmans (PsJN) potato endophyte exhibited modulated gene expression associated with cell surface signaling element in response to changing environmental conditions, consequently modifying metabolic activities accordingly (Sheibani-Tezerji et al. 2015). This endophyte also upregulates transcription, cellular homeostasis, and detoxify reactive oxygen species to resist drought-induced osmotic stress. Stress-related metabolite levels and gene expression increased faster, earlier, and at higher levels in PsJN bacterized grapevine over non-bacterized control at low temperatures by harmonizing carbohydrate metabolism (Fernandez et al. 2012). In a recent study, de Zélicourt et al. (2018) found that a desert plant endophyte Enterobacter sp. SA187 colonizes both the inner and surface tissues of Arabidopsis shoots and roots and induces tolerance to salt stress by production of bacterial 2-keto-4-methylthiobutyric acid (KMBA), which modulates the plant ET signaling pathway. This unique mechanism is utilized by Enterobacter sp. SA187 and was

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found to be very effective in enhancing the yield of alfalfa crops under stress conditions of salt. Endophytes were reported to protect host plants from herbicide stress by manipulating metabolic activities. Different endophytic Proteobacteria have shown metabolism manipulation to tackle the stress generated by herbicides (Ngigi et al. 2012). Role of endophyte species Pseudomonas in response to 2,4-dichlorophenoxyacetic acid accumulation was studied (Germaine et al. 2006). The genome of Pseudomonas punonensis D1-6 reveals many metabolizing genes and herbicide-resistance that indicates an important role in herbicide resistance in plant hosts (Lafi et al. 2017). The ACC deaminase-producing endophytic bacterial species have been discovered in different genera that include Acinetobacter, Bacillus, Agrobacterium, Pseudomonas, Serratia, Ralstonia, Enterobacter, Rhizobium, Burkholderia, and Alcaligenes (Kang et al. 2012). The ACC deaminase-producing endophytic bacteria were checked to verify the ability to promote the growth of tomato plants in extremely high salt stress. The severe inhibitory stress of high salt is readily overcome by tested strains (Ali et al. 2014a). Other studies also reported the effectiveness of ACC deaminase-producing endophytic bacteria against the salt stress in Catharanthus roseus (Karthikeyan et al. 2012), copper stress in canola (Zhang et al. 2011), and osmotic stress in pepper plants (Sziderics et al. 2007). The comparative studies carried out by several workers showed that endophytescontaining plants have a relatively higher cellulose content and lamina density and relatively high leaf toughness, which result in reduced herbivory rates, specifically by the leaf-cutting ants (Van Bael et al. 2012; Estrada et al. 2013). The presence of these endophytes within host tissues improves resistance against pathogens by generating host response or by the synthesis of antagonistic metabolites themselves. The gene pools of endophytes and plant host work in tandem to develop a defence in the plant against parasites.

11.7.2.3 Bioremediation Plants employ several genetic as well as physiological strategies to grow in the contaminated soils, among which one is the recruitment of endophytic bacteria. These endophytes help plants to deal with the stress developed due to accumulation of contaminants. Studies suggest the role of endophytic bacteria in the reduction of metal phytotoxicity with cellular responses to endophyte in association with plant cell via several metabolic activities i.e. biotransformation, extracellular precipitation, or sometimes intracellular accumulation (Ma et al. 2016; Mishra et al. 2017). Several bacterial endophytes from the Zn/Cd hyper accumulator plant Sedum plumbizincicola and some selected isolates exhibit plant growth activities viz ACC deaminase activity, production of indole-3-acetic acid, synthesis of siderophores, and solubilization of phosphorus (Ma et al. 2015; Ullah et al. 2015). In addition, the strains showed high resistance to growth inhibition by heavy metals including Cd, Zn, and Pb. Further the study revealed that a specific strain of Bacillus pumilus significantly increased the ability of plants to uptake Cd also increasing the growth of root and shoot length and biomass compared to plants that are not inoculated with

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this specific Bacillus strain. This showed that endophytic bacteria help in the improvement of phytoextraction capacity and also promote the growth of the plant. It also has been observed by researchers that heavy metal-induced oxidative damage can also be prevented by endophytes with the help of modulating the formation of plant antioxidant enzymes as well as lipid peroxidation (Wan et al. 2012). It has been observed that rhizobial symbiosis promotes Cu accumulation in the shoots and roots of the plant (Kong et al. 2015). There are several plant genes involved in antioxidant responses in plants when treated with the bacterium in the presence of excessive amounts of Cu. Thus, the symbiosis with several microbes is not only responsible for improved plant growth and uptake of metal but also supports the plant’s antioxidative defence mechanism in the presence of excess amounts of Cu, which is a stress condition for plants. The decrease in heavy metal contaminants is followed by ingestion of iron from bacterial siderophores. The siderophores are known to chelate irons present in soil, thus making it absorbable by plant cells. It was found that some growth promoting bacterial species oxidize Fe2+ to Fe3+ siderophore complex in the membrane helping plants to directly absorb the required iron (Schmidt 1999; Gao et al. 2010). Research data reveals that inoculation of endophytic bacteria such as Methylobacterium oryzae and Burkholderia sp. reduced the toxicity and accumulation of Ni and Cd and play an important role in the translocation from roots to shoots in tomato plants (Madhaiyan et al. 2007).

11.8

Conclusion

It is proven that endophytes promote the growth of plants and improve the yield of crops. However these organisms play an important role in the suppression of pathogenic organisms as well as removal of contaminants. The beneficial role of endophytes in the assimilation of nitrogen in the plant and solubilising phosphate has been observed in several research work carried out in last few decades. The natural environmental condition of plants offers favourable conditions for interaction with indophytes and supports the colonization process. A recent molecular study reveals that plant defence responses limit bacterial populations in the plants. Expression of plant genes due to the presence of endophytes provides important information regarding the beneficial effects of endophytes and symbiotic association.

References Aktuganov G, Melentjev A, Galimzianova N, Khalikova E, Korpela T, Susi P (2008) Wide-range antifungal antagonism of Paenibacillus ehimensis IB-Xb and its dependence on chitinase and β-1, 3-glucanase production. Can J Microbiol 54:577–587 Ali S, Charles TC, Glick BR (2014a) Amelioration of high salinity stress damage by plant growthpromoting bacterial endophytes that contain ACC deaminase. Plant Physiol Biochem 80:160–167

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de Zélicourt A, Synek L, Saad MM, Alzubaidy H, Jalal R, Xie Y et al (2018) Ethylene induced plant stress tolerance by Enterobacter sp. SA187 is mediated by 2-keto-4-methylthiobutyric acid production. PLoS Genet 14:e1007273 Zhang YF, He LY, Chen ZJ, Wang QY, Qian M, Sheng XF (2011) Characterization of ACC deaminase-producing endophytic bacteria isolated from copper-tolerant plants and their potential in promoting the growth and copper accumulation of Brassica napus. Chemosphere 83:57–62 Zhang D, Spadaro D, Valente S, Garibaldi A, Gullino ML (2012) Cloning, characterization, expression and antifungal activity of an alkaline serine protease of Aureobasidium pullulans PL5 involved in the biological control of postharvest pathogens. Int J Food Microbiol 153:453–464 Zinniel DK, Lambrecht P, Harris NB, Feng Z, Kuczmarski D, Higley P, Ishimaru CA, Arunakumari A, Barletta RG, Vidaver AK (2002) Isolation and characterization of endophytic colonizing bacteria from agronomic crops and prairie plants. Appl Environ Microbiol 68:2198–2208

Role of Bacterial Endophytes in the Promotion of Plant Growth

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Isha Kohli, Swati Mohapatra, Prashant Kumar, Arti Goel, Ajit Varma, and Naveen Chandra Joshi

Abstract

The symbiotic interaction of plant microbial system is a complex relationship which has been under study for decades and has come up with many ways of improving the growth of plants system directly or indirectly. By now, it is clearly evident that these plant-associated microorganisms called as endophytes, colonize in the robust tissues of plants and have a positive impact on their life cycles (exempting a few). It is also believed that the mechanism selected for the plant growth promotion by endophytic bacteria is similar to that followed by rhizospheric bacteria. Diverse ranges of culturable endophytes are reported that help plants in various biotic and abiotic stress conditions. Several bioactive molecules and secondary metabolites produced by these organisms in the symbiotic system are not only important for plants but also have economic values for humans too. Some of these compounds are antibiotics, vaccines and drugs of high value. Endophytes similar to rhizospheric bacteria have found potential applications in phytoremediation, horticulture and agriculture also. Globally, work is done to study the molecular and genetic basis of the endophytic lifestyle to unveil their importance and applications more diversely in the plant system. Keywords

Plant growth promotion · Endophytes · Colonization · Molecular traits · Applications

I. Kohli · S. Mohapatra · P. Kumar · A. Goel · A. Varma · N. C. Joshi (*) Amity Institute of Microbial Technology, Amity University, Noida, India e-mail: [email protected]; [email protected]; [email protected]; [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. K. Singh et al. (eds.), Bacterial Endophytes for Sustainable Agriculture and Environmental Management, https://doi.org/10.1007/978-981-16-4497-9_12

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Introduction

The microorganisms, commonly referred as endophytes, use the symbiotic and mutual associations to colonize within the vigorous internal tissues of plants and make a positive impact by promoting plant growth (Hallmann et al. 1997; Ali et al. 2012). These microorganisms are being studied for more than a century now, and it has been seen that they can colonize in different tissues that include leaf, root, stem and even rhizospheres (Beckers et al. 2017).The endophytes can directly or indirectly benefit plant growth and developments.The most common endophytes belong to Enterobacter sp., Actinobacteria sp., Proteobacteria sp.,Colletotrichum sp., Bacteroidetes sp., Cladosporium sp., and Firmicutes sp., Salmonella spp. known to cause diseases in human, may act as endophyte for plants. Endophytes may help in removing contaminants, promoting nitrogen assimilation, phosphate solubilization in the plant system and also in stress conditions; these organisms can interact with plants more effectively than rhizospheric organisms (Coutinho et al. 2015). The recent molecular studies like studying the gene sequence of bacteria that are isolated from the DNA of plant tissue have shown that besides these laboratory culturable endophytes, there is richness in their diversity (Reiter et al. 2003; Chaturvedi and Singh 2016). In a molecular study done on the endophytes of wheat, it was seen that the Actinobacteria sp. was much more diverse than found by cultivating in the lab (Conn and Franco 2004). So, the information that we have today on endophytes is based on both modern molecular methods as well as traditional lab culture methods. This is yet not clear that if this class of bacteria is more benefitted living inside the plant system or on the surroundings of the plant root system in a free environment (Rosenblueth and Martínez-Romero 2006). Besides colonizing in plants, some endophytes are seed-borne too. Based on the host’s system, endophytes can be categorized as obligate and facultative for self-survival and growth. Sometimes these organisms are also termed as passenger endophytes (accidental entrance in the plant system) or opportunistic endophytes (occasional entry in the host for the benefits of nutrition or protection from competition) (Reinhold-Hurek and Hurek 1998). The role of genes involved in the endophytes is yet to be adequately established, but it is evident that for the internal colonization in plant different sets of genomic strategies are planned by endophytes than the rhizobium counterparts. In this chapter, we have attempted to introduce the insights of plant growth-promoting bacterial endophytes along with their applications in diverse fields.

12.2

Endophytes Biodiversity

More than 300,000 plant species have been identified and studied, and in all plant species, it is found that endophytes are present (Table 12.1) (Smith et al. 2008). There are some rarest of the rare plants that do not have a mutual relationship with the endophytes (Partida-Martínez and Heil 2011) and these plants are more permissible for the pathogenic attack as compared to plants having endophytes (Timmusk

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Table 12.1 Bacterial endophytes reported in different plant species Plant Oryza sativa L. cv. KDML105 Triticum aestivum L. Sugarcane Wheat

Bacterial Species 43 Bacillus sp.

Citations (Rangjaroen et al. 2019)

Bacillus, Rathayibacter, Flavobacterium, Micrococcus Herbaspirillum rubrisulbalbicans Streptomyces sp.

(Germida et al. 1998)

Sweet potato Kallar grass, rice

Pseudomonas Azoarcus sp.

Marigold

Microbacterium esteraromaticum

Lettuce Citrus plants Soybean

Escherichia coli Nocardia sp. Erwinia sp.

Banana

Pseudomonas fluorescens Pf1, P. fluorescens CHA0 Bacillus subtilis EPB22 Paenibacillus dauci sp. Pseudomonas synxantha Clostridium

(Wu et al. 2015) (Pirttila et al. 2005) (Miyamoto et al. 2004)

Bacillus cereus and Bacillus subtilis

(Hassan 2017)

Bacillus sp. SLS18 Burkholderia vietnamiensis, Enterobacter sp., Rhanella sp., Herbaspirillum sp., Pseudomonas graminis, Acinetobacter sp., Sphingomonas yanoikuyae, Pseudomonas putida, Sphingomonas, Sphingomonas yanoikuyae Achromobacter sp., and Acinetobacter sp. Pseudomonas fluorescens

(Luo et al. 2012) (Knoth et al. 2014)

Pseudomonas sp., Paentbacillus sp., Sphingomonas azotifigens Rhanella aquatilis Bacillus subtilis Burkholderia vietnamiensis, Rhanella sp., Acinetobacter sp., Herbaspirillum sp., Pseudomonas putida, Sphingomonas sp. Herbaspirillum seropedicae

(Castanheira et al. 2017)

Carrot Scots pine Grass Miscanthus sinensis Germander (medicinal plant) Sweet sorghum Poplar and willow

Wheat Black nightshade and tobacco Ryegrass Sweet potato Mulberry Sweet corn

(Olivares et al. 1996) (Coombs and Franco 2003) (Puri et al. 2019) (Engelhard et al. 2000; Reinhold-Hurek et al. 1993) (Sturz and Kimpinski 2004) (Ingham et al. 2005) (Costa et al. 2012) (Kuklinsky-Sobral et al. 2004) (Kavino et al. 2007)

(Patel and Archana 2017) (Long et al. 2008)

(Khan and Doty 2009) (Ji et al. 2008) (Knoth et al. 2013)

(Roncato-Maccari et al. 2003) (continued)

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Table 12.1 (continued) Plant Maize, wheat, rice and sorghum Alfalfa, arabidopsis, wheat and rice Douglas-fir

Kentucky bluegrass White lupin and maize Grapevine Wheat and sorghum Pea Rice

Bacterial Species

Citations

Klebsiella pneumonia

(Dong et al. 2003)

Rhizobium tropici bv. populus, Acinetobacter calcoaceticus, Rhanella sp., Burkholderia sp., Sphingomonas sp. Burkholderia vietnamiensis

(Khan et al. 2015b)

Burkholderia phytofirmans

(Kost et al. 2014)

Burkholderia phytofirmans Gluconacetobacter diazotrophicus

(Compant et al. 2005) (Luna et al. 2010)

Pseudomonas fluorescens Burkholderia vietnamiensis, Rhizobium tropici, Acinetobacter calcoaceticus, Rhanella sp., Burkholderia sp., Sphingomonas yanoikuyae, Pseudomonas sp., Sphingomonas sp.

(Oteino et al. 2015) (Kandel et al. 2015)

(Xin et al. 2009)

et al. 2011). Shreds of evidence also suggest that the population of endophytic organisms vary from organ to organ of plants (Thongsandee et al. 2012). It is not always possible to culture bacteria naturally; likewise, endophytes are also not always cultivable and require modern techniques like molecular fingerprinting for studying its diversity (Singh et al. 2017). So the advanced techniques surpass the traditional methods for the isolation and characterization of these endophytes. In the last few years, extensive research is done using modern technologies such as 16S rRNA pyrosequencing, length-heterogeneity PCR and taxon-specific-RT PCR and the power of these tools in analyzing the endophytes population is revealed (Romero et al. 2014). In a study on endophytes of wheat, it was seen that a more diverse community of actinobacteria were observed using molecular tools than using the culture techniques (Hallmann et al. 1997). In Solanum tuberosum, the diversity of endophytes was observed by using techniques like PCR-Denaturing gradient gel electrophoresis (DGGE) using DNA from sterilized plant tissues and found 103–105 CFU g 1 of organisms in potato’s stem as well as in root (Garbeva et al. 2001). In another work on citrus, both culture-dependent (plating techniques) and culture-independent (DGGE) methods were used to study endophyte and dominant organisms observed were Curtobacterium flaccumfaciens, Methylobacterium sp., Bacillus pumilus, Nocardia sp., and Enterobacter cloacae. In the grape plant, it was found that the seasonal changes have influenced the population and diversity of endophytes (Bulgari et al. 2011). The different species of Proteobacteria followed by Actinobacteria and Firmicutes are known to be the dominant endophytes currently

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reported in many studies. As discussed in the introduction section, other commonly reported bacterial genera are Bacillus, Mycobacterium and Micrococcus (Smith et al. 2008; Romero et al. 2014). With the advancements in the molecular techniques, more insights into the lifestyle of endophytes are revealed, which has widened the horizons of utilization of these microorganisms in the improvement of crop growth promotion and horticulture studies.

12.3

Isolation, Identification and Colonization of Endophytes

Sterilizing the surface of plant tissues that are non-pathogenic and then isolating bacterial endophytes happens to be the most common technique of isolating bacterial endophytes from different parts of plants like root, stem, nodules and the leaves (Jackson et al. 2013; Kandel et al. 2017). Earlier, the identification of the bacterial endophytes was made using the traditional approach of biochemical characterization, but nowadays, molecular techniques such as ITS and ribosomal DNA are used as explained earlier in the chapter. Table 12.1, highlights the isolation of different bacterial endophytic species from different plants. For cultivation technique medium used for the growth is either synthetic or semi-synthetic medium (Rouws et al. 2010; Castanheira et al. 2017). As discussed before, many bacterial isolates are omitted in the cultivation technique that may be identified through molecular techniques. Different microscopic techniques such as the confocal microscopy, scanning electron microscopy and transmission electron microscopy can record the pattern of colonization of endophytes in the plant tissues (Tables 12.2 and 12.3; Castanheira et al. 2017; Chen et al. 2020). Colonization of bacteria generally happens intracellular, and the majority get colonizes through passive invasion in the plant’s vascular tissues, the initial entry point of colonization in the plant is the root surface called rhizoplane and invasion through this first entry point is sufficient for the proper colonization (Surjit and Rupa 2014; War Nongkhlaw and Joshi 2017). The process of colonization has its complexities like recognizing the host and penetrating its tissues, and for this, the endophytes must have a well-defined enzymatic system (cellulases, xylanases etc.) to break the external cell walls of plants (Reinhold-Hurek et al. 2006). One of the important criteria for the colonization of endophytes is the genotype of plants. The plant receptors play a crucial role in the colonization process along with the hormones such as salicylic acid, auxin and jasmonic acid. The endophytes swim towards the plants through the root exudates using chemotactic movement. To ease the initial step for the colonization process, some of the endophytic bacteria, i.e., Gluconacetobacter diazotrophicus Pal5, secrete exopolysaccharides (EPS) (Meneses et al. 2011) and for the attachment onto the plant surfaces, endophytic appendages such as fimbriae or flagella are also responsible. Endophytes generally invade the intercellular spaces of plant tissues because of the abundance of inorganic nutrients, carbohydrates and amino acids (Hardoim et al. 2015). The mechanism of attachment of endophytes differs from plant to plant and is comparatively unexplored.

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Table 12.2 Colonization of different root endophytes Origin point of host Pennisetum

Application towards other plant Triticum sp.

Azoarcus sp.

Laptochloa sp.

Oryza sativa

Bacillus megaterium, Bacillus pumilus, Rhizobium sp.

Zea mays, Oryza sativa, Triticum sp.

Zea mays, Oryza sativa, Triticum sp.

Healthy proliferation, maturation and development

Burkholderia phytofirmans

Allium cepa

Vitis sp., Panicum virgatum, Arabidopsis thaliana, Lupinus albus, and Zeamays

Healthy proliferation, maturation and development

Burkholderia vietnamiensis

Poa pratensis, Poa sp., Oryza sativa, Saccharum officinarum Oryza sativa

Healthy proliferation, maturation and development

Corynebacterium flavescens,

Populus sp., Oryza sativa, Saccharum officinarum Oryza sativa

Enterobacter sp.

Zea mays

Zea mays

Herbaspirillum seropedicae

Oryza sativa

Oryza sativa, Triticum sp.

Name of endophytes Achromobacter sp. and Acinetobacter sp.

Impact of interaction on plant health Healthy proliferation, maturation and development Healthy proliferation, maturation and development

Healthy proliferation, maturation and development

Healthy proliferation, maturation and development, increased drought tolerance High extension of root, modify the gene expression, healthy proliferation,

References (Patel and Archana 2017; Kandel et al. 2015) (Hurek et al. 1994) (ReinholdHurek et al. 2006) (Liu et al. 2006; BacilioJiménez et al. 2001; Ji et al. 2008) (Compant et al. 2005) (Kim et al. 2012) (Kost et al. 2014) (Zúñiga et al. 2013) (Kandel et al. 2015) (Govindarajan et al. 2008) (Govindarajan et al. 2006) (BacilioJiménez et al. 2001; Gyaneshwar et al. 2001) (Kandel et al. 2017) (Riggs et al. 2001; Kandel et al. 2017)

(Pankievicz et al. 2016) (James et al. 2002) (Roncato(continued)

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Table 12.2 (continued) Name of endophytes

Microbacterium sp., Pseudomonas fluorescens

12.4

Origin point of host

Brassica napus

Application towards other plant

Brassica napus

Impact of interaction on plant health maturation and development, enhancement of biological N-fixation, amendment in metabolic profile Healthy proliferation, maturation and development, inflation of Pb absorption, root propagation

References Maccari et al. 2003) (BrusamarelloSantos et al. 2017)

(Luo et al. 2012; Long et al. 2008) (Duijff et al. 1997)

Strategies Employed by Endophytes for Plant Growth Promotion

Endophytes may benefit plants by promoting their health and growth using direct as well as indirect mechanisms (Fig. 12.1) and commonly entitled as plant growthpromoting endophytes (Hassan 2017). The direct as well as indirect strategies employed by endophytes are well known and studied (Glick 2012). Increasing exchange of nutrients, transport insoluble phosphate, modulating the hormones, lowering the levels of ethylene and delivering nitrogen are some of the beneficial aspects for the plants when microorganisms niche inside plant tissue system (Matsuoka et al. 2013; Khan et al. 2015a). Besides this, they also compete against the pathogenic bacteria that share the same niche as endophytes do and produce several biological compounds (commonly called as plant growth promoting agents) having diverse activities. Aceotin and 2–3 butanediol are such volatile compounds secreted by microorganisms that help plant growth (Ryu et al. 2003). In Pine plant, it was seen that the browning of tissues happened because of the production of adenine ribosides by endophytes (Pirttila et al. 2004). As discussed before, it is well established that the systemic mechanisms employed by bacterial endophytes and rhizospheric microorganism are almost similar, but for the phytoremediation, crop improvement and silviculture endophytes are always preferred. It is also important to indicate that there is no specific scientific differentiation method available which can be used as a basis of preference of PGPB by researchers around the world. The selection of PGPB is based on the choice of the individual research group wants to study a specific endophyte or is familiar with the application of these endophytes. The real-world applications of these organisms in

Solanum tuberosum, Populus sp.

Oryza sativa

Pseudomonas putida; Pseudomonas spp.

Serratia marcescens

Oryza sativa

Solanum tuberosum, Pisum sativum; Populus sp.

Zea mays, Triticum sp., Oryza sativa, Sorghum bicolor

Sorghum bicolor, Triticum spp.

Saccharum officinarum

Zea mays

Zea mays Morus spp. Vitis spp.; Zea mays

Application towards other plant species Saccharum officinarum

Original host Saccharum officinarum Zea mays Morus spp. Allium cepa

Herbaspirillum seropedicae

Name of isolates Acetobacter diazotrophicus Bacillus megaterium Bacillus subtilis Burkholderia phytofirmans, Enterobacter sp Gluconacetobacter diazotrophicus

Table 12.3 Colonization of different Stem endophytes

Healthy proliferation, maturation and development, Phytophthora infestans suppression Enhance sustainability in high 2,4-dichlorophenoxyacetic acid Healthy proliferation, maturation and development

NA

Na Decrease many microbial wilt disease Healthy proliferation, maturation and development, increased drought tolerance

Impact on plant health NA

(Gyaneshwar et al. 2001) (Kandel et al. 2017)

References (Dong et al. 1994; Kandel et al. 2017) (Liu and Xu 2008) (Ji et al. 2008) (Kost et al. 2014) (Zúñiga et al. 2013) (Riggs et al. 2001) (Meneses et al. 2011) (Luna et al. 2010) (Rouws et al. 2010) (do Amaral et al. 2014) (Roncato-Maccari et al. 2003) (Kandel et al. 2017) (Long et al. 2008) (Khan et al. 2014) (Andreote et al. 2009; Kandel et al. 2017)

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Fig. 12.1 Plant growth promotion by bacterial endophytes through direct (eg: doing nitrogen fixation, phosphate solubilisation, modulating hormones etc.) and indirect mechanisms (secretion of lytic enzymes, antibiotics etc.)

horticulture, agriculture industries and the medicine are already widespread in the public domain.

12.5

Applications of Bacterial Endophytes

Bacterial endophytes are well known for their plant growth-promoting activity either by producing growth hormones or by solubilizing nutrient required for the plant growth. Endophytes also help in suppressing plant diseases, inactivating environmental pollutant and alleviating environmental stresses. Some endophytes produce nanomaterials which may have several applications in cosmetics and biomedical field. In this chapter we will discuss the major applications of the bacterial endophytes and also other aspects associated with the commercialization of these microbes.

12.5.1 Bacterial Endophytes in Plant Growth Promotion The applications of bacterial endophytes have shown promising results in phytoremediation, pollution control, plant growth promotion and the manufacturing of industrial and medical appliances (Figs. 12.2 and 12.3). For the suppression of

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Fig. 12.2 Bacterial endophyte applications in the production of secondary metabolites such as alkaloids, terpenoids, phenol, nanoparticles and other cancerous compounds

plant diseases, endophytes activate the phenomenon of SAR (systemic acquired resistance) and ISR (induced systemic resistance) (Kloepper and Ryu 2006). Endophytes are the source of natural products and secondary metabolites like antibiotics (Ecomycins B and C), antivirals (Cytonic acids A and D), antifungal (Fusaricidin A–D), anti-cancerous (Taxol) and volatile compounds that are of low molecular weight and are active in small concentration in animals as well as humans (Miller et al. 1998; Guo et al. 2000; Strobel et al. 2004; Li et al. 2007). Populus deltoids has Methylobacterium that can biodegrade nitro-aromatic compounds like 2,4,6-trinitrotoluene (Van Aken et al. 2004). Pseudomonas from pea also had the capability of degrading 2,4-dichlorophenoxyacetic acid (2,4-D) an organochlorine herbicide (Germaine et al. 2006).

12.5.2 Endophytes-Mediated Biodegradation of Soil Contaminants Endophytes have an amazing capacity to deteriorate complex toxic mixes through bioremediation strategy, which depends on the metabolic activity of microorganisms and their interaction with the surrounding environment to breakdown these squanders to make the soil healthy. Expulsion of contaminations from the soil by utilization of endophytic bacteria is a widely accepted phenomena, and this is made conceivable because of the incredible microbial diversity. A recent study showed

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Fig. 12.3 Applications of bacterial endophytes (whether culturable or unculturable) in the field of phytoremediation, pollution control, promotion of plant growth, Bio control and in the manufacture of industrial and medical appliances have found promising results

that the endophytic microbes facilitate the removal of soil pollutants in interaction with cultivated tobacco plants (Nair and Padmavathy 2014). On high cadmium contamination zone potential use of endophytic bacteria brought a high improvement on the production of biomass on tobacco cultivation; however, the amount of Cd contamination was found maximum in untreated tobacco plants (Mastretta et al. 2009). Hence, these outcomes exhibited beneficial effects of seed endophytes during heavy metal stress (Pattnaik et al. 2020). Similarly, some varieties of bacterial isolates have proficiency towards the deterioration of petroleum-derived polymers. The family of Pestalotiopsis was also observed; a few strains belonging to this genus use Polyurethane (PUR) for their primary nutrient source in obligatory anaerobic environment. Some reports stated that endophytic bacteria that are present in the rhizosphere of the plant have the capacity to accumulate polyhydroxyalkanoates, and the studies revealed that these endophytes have the capacity to activate plant growth and degrade the contaminants, especially the heavy metals, from the soil to make the soil environment healthy (Singh Saharan et al. 2014). These endophytic microbes also help in root nodulation, and nitrogen fixation makes the soil rich with a profitable microbial diversity. Molecular characterization depicted serine hydrolase secreted from some of the entophytic strain can degrade the PUR and thus increase soil health (Nair and Padmavathy 2014).

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12.5.3 The Bacterial Endophytes Towards Inhibition of Plant Pathogens There are numerous ways for the endophytes to enhance plant and soil health; the most common way is by stifling the presence of the pathogen and its metabolism. This includes certain mechanistic approaches like antagonism or competition among the endophytes and the pathogens for their nutrients and survival. Hence the endophytes produce certain molecules like antimicrobial agents that either degrade the pathogens or enhance the resistance of the plant to fight against the pathogens via upregulating the plant’s defence network. Many studies proved that endophytes such as bacterial isolates protect the plants from the attack of diversified pest and pathogens and different irritation starting from sprouting time to growth and development stage of cultivation. As an example: Some gram-negative bacterial isolates belonging to genus Pseudomonas secrete antifungal mixes, including phenazinecarboxylic acid, DAPG, pyrrolnitrin and pyoleutirin, and volatiles like hydrogen cyanide molecules altogether restrain growth and development of contagious agents in the host plant (Morrison et al. 2017; Olanrewaju et al. 2017). Similarly, the family of Bacillus are having a significant contribution towards the regulation of the pathogens, as these isolates synthesize an enormous amount of biologically active compounds having the capacity to eradicate the plant pathogens. Recent research has proven an assortment of secreted lipopeptides from the endophytes when comes in contact with the parasitic fungus significantly decreases the toxicity of phytopathogens and most of the time pathogen stays avirulent instead of causing infection (Radhakrishnan et al. 2017; Khare et al. 2018). A large number of the antifungal mixes secreted by bacterial endophytes target to disrupt a different portion of the fungus, initiating spillage of supplement, diminishing the harmful effect of the pathogen on the plant growths. Endophytic symbionts improve plant immunity and protect plants from an expansive range of disease causing agents, especially due to secretion of induced systemic defence (ISR), salicylic acid (SA) and jasmonate (JA) pathways and ethylene or PR proteins (White et al. 2019).

12.5.4 Endophytes Maintain the Free Radicals in the Plant Tissue Environmental stress deregulates certain genes in plant cells for the production of free radicals which are mostly derived from oxygen known as reactive oxygen species. These secreted free radicals damage cellular enzymes, polypeptides, nucleic acids, cell wall and cell membrane. Quantitative PCR examination indicated that endophytic microorganisms induce the upregulation of ROS-detoxifying genes such as SOD1 and GR that translate to superoxide dismutase and glutathione reductase (Alquéres et al. 2013). These enzymes additionally diminish oxidative harm that occurs due to the attack of other infectious microbial agents in plants. Festuca arundinacea tissues contaminated with the endophytic bacteria possess significant amount of osmoprotectants and antifungal agent engaged with phytoprotection

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under oxidative pressure (Lata et al. 2018). Nevertheless, the presence of endophytes upregulates DREB2A, CBL1, RD29A and ANAC072 genes that help in the protection of plants from drought conditions (Waller et al. 2005; Sun et al. 2010).

12.6

Concluding Remarks and Future Perspectives

Current culture-independent and molecular techniques have helped in understanding the diversity of plant bacterial endophytes. The interactions between plants and endophytes that occurs naturally and also when exploited, genetically plays a vital role in the promotion of plant growth under biotic as well as abiotic stress condition. The mechanisms for tolerating stress include the accumulation of secondary metabolites and activation of ROS scavenging pathways. The endophytes are being used in the fields in many developing countries for the improvement of agro products. They are used as a natural mode of biofertilizer, which is non-toxic to plants and makes them significant at scientific and economic front. It is also believed that plants are in continuous communication with endophytes for their specific functions. More in-depth knowledge on this plant–endophyte interaction is yet untouched that can be investigated by using modern molecular biology techniques. For sustaining agro needs in this growing population era, the sudden need is to touch the untouched horizon of the plant–endophyte interactions and the molecular mechanisms involved in the process. It has been found that sometimes the rhizospheric bacteria convert themselves into endophytes, though the mechanism behind this transformation is a missing link, which needs further exploration with the help of molecular genetic techniques available. The rhizospheres are studied more than the endophytes, but with the development of technologies like NGS, more information on the phenotypic and genotypic nature of these endophytes are considered. So using the molecular techniques such as metagenomics and metaproteomics the novel genes of these endophytes will be explored, which may contribute to increase in crop yield, and also it will provide more information that will unfold this symbiotic plant–microbe interaction. Furthermore, more focus is required on exploring biotechnological and agro applications of endophytes and also the commercialization of these microbiome.

References Ali S, Charles TC, Glick BR (2012) Delay of flower senescence by bacterial endophytes expressing 1-aminocyclopropane-1-carboxylate deaminase. J Appl Microbiol 113:1139–1144. https://doi. org/10.1111/j.1365-2672.2012.05409.x Alquéres S, Meneses C, Rouws L et al (2013) The bacterial superoxide dismutase and glutathione reductase are crucial for endophytic colonization of rice roots by Gluconacetobacter diazotrophicus PAL5. Mol Plant-Microbe Interact 26:937–945. https://doi.org/10.1094/ MPMI-12-12-0286-R Andreote FD, de Araújo WL, de Azevedo JL et al (2009) Endophytic colonization of potato (Solanum tuberosum L.) by a novel competent bacterial endophyte, Pseudomonas putida Strain

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Bacterial Endophytes and Abiotic Stress Mitigation

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Sonali Jaiswal, Anupama Ojha, and Sarad Kumar Mishra

Abstract

Abiotic stresses are major constraints for worldwide agricultural productivity. Several adaptations and mitigations are required by plants to combat abiotic stresses. Plant develops physiological, biochemical, and morphological responses to overcome the adverse effects of these stresses. Higher forms of flora and fauna maintain crucial interactions with microorganisms. The theme of this chapter prioritizes endophytic bacteria and their underlying mechanisms, which help in increasing plant stress tolerance through various direct and indirect processes. We highlight the functional aspects of endophytic bacterial species to withstand unfavourable effects of abiotic stressors. Keywords

Biotic stress · Abiotic stress · Bioremediation · Plant growth and promotion · Plant health

13.1

Introduction

According to an estimate, 50% of the loss in yield of agricultural crops is due to abiotic stressors globally (Wang et al. 2007). India is challenged with many abiotic stresses. Abiotic stress comprises all non-living components that cause unpropitious effects on plant growth, development, and survival (Latef et al. 2016). It includes temperature, water, salinity, pH, heavy metals, nutrient deficiency, and starvation which reduce the viability of agricultural crops (Chaves and Oliveira 2004). S. Jaiswal · A. Ojha · S. K. Mishra (*) Department of Biotechnology, Deen Dayal Upadhyaya Gorakhpur University, Gorakhpur, Uttar Pradesh, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 A. K. Singh et al. (eds.), Bacterial Endophytes for Sustainable Agriculture and Environmental Management, https://doi.org/10.1007/978-981-16-4497-9_13

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Endophytes can act as a prerequisite tool to develop eco-friendly strategies and formulations which enhance soil fertility, improve yield potential, and plant performance under environmental stress. Nowadays, the role of endophytic species gains prominent function in counteracting abiotic stress. Endophytes are ubiquitous microbial mutualists that establish themselves inside plants, without causing any symptomatic characters of disease. Bacterial endophytes are beneficial, non-pathogenic, employ combined action of augmenting plant growth and protect them from pathogenic diseases and abiotic stress (Rosegrant et al. 2009; Rho et al. 2018). Therefore, endophytes have become an important ecologically innocuous element in burgeoning system of sustainable agriculture (Degrassi and Carpentieri-Pipolo 2020). Thus efforts must be extended to find indigenous microbes that contribute in harsh agro-ecologies. Bacterial endophytes show trophobiotic, mutualistic, symbiotic, and commensalistic associations within various tissues of host plants. Their interactions with the plants induce many local and systemic responses which boost the metabolic efficiency of the plants to thwart a wide array of abiotic stressors (Nguyen et al. 2016). They help in upgrading agricultural productive capacity by increasing plant growth and confer disease resistance (Phurailatpam and Mishra 2020). In recent studies, their potential roles have been found in the synthesis of nanoparticles, medicinal drugs, bioremediation, biofuel production, and as antimicrobial agents.

13.2

International and Indian Scenario of Various Abiotic Stress

As indicated in the report of World Atlas 2018, Morocco, Uganda, Somalia, Iran, Pakistan, China, Afghanistan, Eritrea, Sudan, and Ethiopia are most droughtaffected countries in the world. According to a study done by Teixeira et al. (2013), South Asia (Northern India), East Asia (North Eastern China), continental parts of Central Asia (Russian Federation and Kazakhstan), and North America, are the main hotspots of heat stress. A manual published by Department of Agriculture and Cooperation, India, 2009 showed that 68% of the country area is susceptible to drought with changing degrees of precipitation accounting arid (19.6%), semi-arid (37%), and sub-humid areas (21%) (https://nidm.gov.in/PDF/manuals/Drought_ Manual.pdf). According to an estimate, the latter half of the nineteenth century witnessed 25 major drought famines causing paucity of food grains in India, leading to carnage of 30–40 million people. Major drought-prone states are Maharashtra, Odisha, Telangana, Rajasthan, Madhya Pradesh, Jharkhand, Uttar Pradesh, Karnataka, Andhra Pradesh, and Chhattisgarh. The regions affected by excessive salinity levels across the world are Asia Pacific and Australia. Both cover a total agricultural area of 2016.63 million hectares, in which 27% (549.30 million hectares) is saline affected. Africa and America have 72.2 and 130.5 million ha of agricultural land having salinity, respectively (FAO 2019; World Population Prospects 2019). Salt-affected soil area in India is about 6.73 million hectares, which is found in the states of Gujarat (2.23 m ha), Uttar Pradesh (1.37 m ha), Maharashtra (0.61 m ha), West Bengal (0.44 m ha), and Rajasthan (0.38 m ha). It

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comprises almost 75% of saline and sodic soil of the country (Narayana and Babu 1983). Many regions of the world, such as China, USA, and Australia, are affected by frost in spring season (Xiao et al. 2018). In India, the states of Maharashtra, Rajasthan, Madhya Pradesh, Punjab, Haryana, Himachal Pradesh, and Uttar Pradesh (U.P.) have reported the damage of several crops due to cold stress (https://weather. com/en-IN/india/news/news/2019-01-10-frost-risk-rabi-crop). Heavy metal exposure is amplifying in many parts of world leading to higher concentration of these metals in their flora and fauna. For example, use of mercury for gold mining in areas of Latin America increases its toxicity in environment (Yadav 2010; https://www. scientificamerican.com/article/worlds-top-10-most-polluted-places/). Increase in the development of infrastructure and automobile industry leads to heavy metal toxicity in soil and water of several rivers. The rivers such as Ganga, Arkavathi, Orsang, Rapti, Sabarmati, and Saryu have been found heavily polluted with harmful metals. Water of these contaminated rivers affects grain and vegetable grown in that area (https://weather.com/en-IN/india/pollution/news/2018-05-16-heavy-metal-toxicityindia-rivers). According to report by INSA 2011, chromium, lead, arsenic, copper, and mercury heavy metal toxicity has been found in different parts of Tamil Nadu, Uttar Pradesh, Gujarat, Orissa, Madhya Pradesh, Andhra Pradesh, Chhattisgarh, West Bengal, and Jharkhand (http://www.insaindia.res.in/pdf/Hazardous_Metals. pdf).

13.3

Plant Responses to Abiotic Stress

13.3.1 Drought and Heat Stress Drought stress manifests when the humidity of soil and atmosphere decreases with the increase in air temperature. There is an asymmetric balance between the evapotranspiration flux and intake of water from the root zone (Lipiec et al. 2013). Heat stress takes place when there is profound increment in soil and air temperature above a threshold value that causes permanent harm to plant growth in minimal amount of time. The combination of both stresses produces more destructive effect on overall performance of plant (Dreesen et al. 2012). Drought and heat stress induces almost similar responses. A sequence of physiological and molecular incidents takes place in drought and heat stress, which lead to physiological dehydration (Vinocur and Altman 2005). It also affects the overall turgor which interferes with normal functions, changing morphological and physiological attributes in plants (Slater et al. 2008; Rahdari and Hoseini 2012). Drought affects leaf and soil water potential, and hinders the extraction of water by the root from the soil. Drought stress induces negative impact on transport of soil nutrients, like nitrate, sulfate, calcium, magnesium, and silicon (Selvakumar et al. 2012; Zandalinas et al. 2016). It causes reduction of photosynthetic efficiency and leads to genesis of reactive oxygen species (ROS). Increase of free radicals generate oxidative stress which are extremely reactive and toxic, responsible for apoptosis, cell/tissues damage or death (Zlatev and Lidon 2012; Das and Roychoudhury 2014). ROS results in

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peroxidation of lipids and distortion in the structure of proteins and DNA (Yu et al. 1998). Ethylene acts as a signalling molecule at low concentration. Drought and heat stress triggers ethylene biosynthesis which inhibits plant growth, maturation, and progression. During drought stress, there is an increase in the amount of abscisic acid (ABA). This acid regulates the process of root hydraulic conductance, and cellular dehydration tolerance (Daszkowska-Golec 2016). A cluster of low molecular mass compounds categorized as secondary metabolites like quinones, anthocyanins, phenolics, alkaloids, terpenoids, and lignans are produced by the plants to overcome drought stress (Wink 2003).

13.3.2 Salt Stress A soil in which electrical conductivity in root zone exceeds 4ds/m of the saturation extract (ECe), at 25
C, with sodium exchangeable up to 15% is called saline soil (Munns 2005). Salinity stress induces the acquisition of water-soluble salts to a level that adversely affects the economy, environment and agricultural productivity (Jones et al. 2012). Excess concentration of salt induces ionic and osmotic stress in plant cells (Saffan 2008). Salt stress induces ion-toxicity by removal of K+ by Na+ in biochemical reactions (Zhu 2002). Successful scavenging of ROS is a crucial step in the mitigation of drought and salt stress. Several studies reported that accumulation of ROS causes metabolic dysfunction and plant cell death (Yang and Guo 2018). Antioxidant (enzymatic and non-enzymatic) machinery works together as safeguard for cells against oxidative injury (Vardharajula et al. 2011). Hypertonic and osmotic stress cause metabolic imbalance, leading to oxidative damage (Shi-Ying et al. 2018). Plant cell responds in osmotic stress by production of ‘compatible solutes’ (osmolytes or osmoproctectants). It is divided into two broad classes: (1) sugar and sugar alcohols such as mannitol, sorbitol, pinitol, trehalose, and fructans and (2) zwitterionic compounds such as proline and quaternary ammonium compounds (glycine betaine) (Slater et al. 2008). High accumulation of sodium ions causes detrimental effect on diverse microbial communities around and within rhizospheric zone of plant, inhibit water conductance, porosity of soil and interfering proper aeration (Kumar et al. 2020). High concentration of Na+ inside the cells can reduce germination of seeds, flowering, fruiting, and growth of seedlings (Singh et al. 2015). Excessive salt stress causes inhibition of proton pumps, interferes in nutrients uptake, leads to iron deficiency, and causes chlorosis in plants (Masalha et al. 2000; Bano and Fatima 2009). Photosynthetic efficiency is adversely affected during salinity stress by decrease in the pigment (chlorophyll), leaf area, and inhibition of photosystem II activity. It obstructs crop growth and makes it more vulnerable to soil-borne diseases (Netondo et al. 2004; Kalaji et al. 2011; Hashem et al. 2019).

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13.3.3 Cold Stress The chemical and physical modifications in biological molecules caused by low temperature with chilling (