Seed Endophytes: Biology and Biotechnology [1st ed.] 978-3-030-10503-7;978-3-030-10504-4

Table of contents :
Front Matter ....Pages i-ix
Front Matter ....Pages 1-1
Seed-Vectored Microbes: Their Roles in Improving Seedling Fitness and Competitor Plant Suppression (James Francis White Jr, Kathryn L. Kingsley, Susan Butterworth, Lara Brindisi, Judy W. Gatei, Matthew T. Elmore et al.)....Pages 3-20
Thinking About PPFM Bacteria as a Model of Seed Endophytes: Who Are They? Where Did They Come from? What Are They Doing for the Plant? What Can They Do for Us? (Mark A. Holland)....Pages 21-34
Seed Endophytes and Their Potential Applications (Haiyan Li, Shobhika Parmar, Vijay K. Sharma, James Francis White Jr)....Pages 35-54
Exploring Endophytic Communities of Plants: Methods for Assessing Diversity, Effects on Host Development and Potential Biotechnological Applications (Satish K. Verma, Ravindra N. Kharwar, Surendra K. Gond, Kathryn L. Kingsley, James Francis White Jr)....Pages 55-82
Understanding the Indigenous Seed Microbiota to Design Bacterial Seed Treatments (Birgit Wassermann, Eveline Adam, Tomislav Cernava, Gabriele Berg)....Pages 83-99
Front Matter ....Pages 101-101
The Ecology of Seed Microbiota (Pablo Hardoim)....Pages 103-125
Programming Plants for Climate Resilience Through Symbiogenics (Rusty Rodriguez, Alec Baird, Sang Cho, Zachery Gray, Evan Groover, Roman Harto et al.)....Pages 127-137
Agave Seed Endophytes: Ecology and Impacts on Root Architecture, Nutrient Acquisition, and Cold Stress Tolerance (America Martinez-Rodriguez, Gloria Macedo-Raygoza, Aurora X. Huerta-Robles, Ileana Reyes-Sepulveda, Jhovana Lozano-Lopez, Evelyn Y. García-Ochoa et al.)....Pages 139-170
Chemical Warfare in the Plant Microbiome Leads to a Balance of Antagonisms and a Healthy Plant (Barbara Joan Schulz, Laura Rabsch, Corina Junker)....Pages 171-189
Fungal and Bacterial Maize Kernel Interactions with the Vertically Transmitted Endophytic State of Fusarium verticillioides (Charles W. Bacon, Dorothy M. Hinton)....Pages 191-209
Front Matter ....Pages 211-211
Functional Roles of Seed-Inhabiting Endophytes of Rice (Gaurav Pal, Kanchan Kumar, Anand Verma, James Francis White Jr, Satish K. Verma)....Pages 213-236
Mechanism of Interaction of Endophytic Microbes with Plants (Neethu Sahadevan, E. K. Radhakrishnan, Jyothis Mathew)....Pages 237-257
Fitness Attributes of Bacterial and Fungal Seed Endophytes of Tall Fescue (Elizabeth Lewis Roberts, Brendan Mormile, Christopher Adamchek)....Pages 259-271
Role of the Plant Root Microbiome in Abiotic Stress Tolerance (Daniel F. Caddell, Siwen Deng, Devin Coleman-Derr)....Pages 273-311
Endophytic Microbes: Prospects and Their Application in Abiotic Stress Management and Phytoremediation (Divya Singh, Vipin Kumar Singh, Amit Kishore Singh)....Pages 313-333
Pine Seeds Carry Symbionts: Endophyte Transmission Re-examined (Ron J. Deckert, Catherine A. Gehring, Adair Patterson)....Pages 335-361
Front Matter ....Pages 363-363
Seed Endophytes of Jasione montana: Arsenic Detoxification Workers in an Eco-friendly Factory (María del Carmen Molina, James Francis White Jr, Kathryn L. Kingsley, Natalia González-Benítez)....Pages 365-384
Agricultural Applications of Endophytic Microflora (John Reshma, Chandran Vinaya, Mathew Linu)....Pages 385-403
Rhizome Endophytes: Roles and Applications in Sustainable Agriculture (Akanksha Gupta, Hariom Verma, Prem Pratap Singh, Pardeep Singh, Monika Singh, Virendra Mishra et al.)....Pages 405-421
Agriculturally Important Biosynthetic Features of Endophytic Microorganisms (S. Sreejith, R. Aswani, E. K. Radhakrishnan)....Pages 423-447
Microbial Endophytes of Maize Seeds and Their Application in Crop Improvements (Sandip Chowdhury, Rusi Lata, Ravindra N. Kharwar, Surendra K. Gond)....Pages 449-463
Colonization of Seeds by Soilborne Fungi: Linking Seed Dormancy-Defense Syndromes, Evolutionary Constraints, and Fungal Traits (Simon Maccracken Stump, Carolina Sarmiento, Paul-Camilo Zalamea, James W. Dalling, Adam S. Davis, Justin P. Shaffer et al.)....Pages 465-481
Seed Endophytes in Crop Plants: Metagenomic Approaches to Study the Functional Roles and Interactions (Priyanka Verma)....Pages 483-507

Citation preview

Satish Kumar Verma  James Francis  White, Jr Editors

Seed Endophytes Biology and Biotechnology

Seed Endophytes

Satish Kumar Verma • James Francis White, Jr Editors

Seed Endophytes Biology and Biotechnology

Editors Satish Kumar Verma Department of Botany Institute of Science, Banaras Hindu University Varanasi, India

James Francis White, Jr Department of Plant Biology Rutgers University New Brunswick, NJ, USA

ISBN 978-3-030-10503-7 ISBN 978-3-030-10504-4 https://doi.org/10.1007/978-3-030-10504-4

(eBook)

Library of Congress Control Number: 2019934965 # Springer Nature Switzerland AG 2019 This work is subject to copyright. All rights are reserved 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, express 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 Switzerland AG. The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Prologue

What Are Endophytes? Endophytes are those bacteria and fungi that exist within the tissues of plants without negative effects on host plants. They have been reported from all the plants examined to date. Some endophytes are vectored on or within seeds of plants. These microbes are acquired by plant seeds and may be important to seedling development. Endophytic microbes support plants through mobilization of nutrients from soils, production of phytohormones, and further protecting plants from biotic (fungal pathogens and insects) and abiotic stresses. Further, seed microbes move into the plant body, protecting the plant from disease and improving the nutritional status of the entire plant. Endophytic bacteria stimulate the expression of host genes relating to nitrogen metabolism and hormone synthesis. The presence of endophytes also induces host defense genes in plants and makes them less susceptible to disease. Endophytic microbes produce a large number of agriculturally important metabolites. Seed-associated microbes are believed to be more adapted to plants and have ecological significance. Application of the seed microbiome in agriculture is needed at this time. We believe that seed and seedling microbes will ultimately replace the use of chemical fertilizers and pesticides that degrade the environment.

A New Definition for Seeds: A Miniature “Noah’s Ark” for Plant Colonization A seed may be defined as a dormant embryonic plant supplied with nutrients enough to fuel early seedling growth and contained in a hardened protective coating. However, it is becoming increasingly clear that this definition is not complete; seeds also carry a community of symbiotic microbes that provide multiple critical functions for developing seedlings—and without these symbiotic microbes seedlings are less likely to survive. In this respect, a seed is like “Noah’s ark” containing the plant and the microbes that are needed for the growth and survival of the seeding as it colonizes in a new place. v

vi

Prologue

The Intention of This Book With this book we intend to begin to complete the definition of a seed by revealing the many microbes transmitted in and on seeds—and their numerous functions in cultivated and non-cultivated species. We also intend to show the importance of the microbial components of seeds and the adaptations on/in seeds for vectoring microbes. Finally, we intend to show how seed-vectored microbes can be used in biotechnologies to enhance cultivation of crop plants. This book contains chapters describing biology of endophyte communities, endophyte functions in plants, and applications in agriculture and biotechnology. This book will be useful to students, teachers, and scientist working in the areas of plant–microbe interactions and biocontrol. Since interactions of endophytic microbes with plants are relatively underexplored, people interested to work in this area will find this book useful. Varanasi, India New Brunswick, NJ May 31, 2018

Satish Kumar Verma James Francis White, Jr

Contents

Part I 1

2

Seed Endophytes: Introduction, and Methods for Assessment and Management

Seed-Vectored Microbes: Their Roles in Improving Seedling Fitness and Competitor Plant Suppression . . . . . . . . . . . . . . . . . . . James Francis White, Jr, Kathryn L. Kingsley, Susan Butterworth, Lara Brindisi, Judy W. Gatei, Matthew T. Elmore, Satish Kumar Verma, Xiang Yao, and Kurt P. Kowalski Thinking About PPFM Bacteria as a Model of Seed Endophytes: Who Are They? Where Did They Come from? What Are They Doing for the Plant? What Can They Do for Us? . . . . . . . . . . . . . . Mark A. Holland

3

Seed Endophytes and Their Potential Applications . . . . . . . . . . . . . Haiyan Li, Shobhika Parmar, Vijay K. Sharma, and James Francis White, Jr

4

Exploring Endophytic Communities of Plants: Methods for Assessing Diversity, Effects on Host Development and Potential Biotechnological Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Satish K. Verma, Ravindra N. Kharwar, Surendra K. Gond, Kathryn L. Kingsley, and James Francis White, Jr

5

Understanding the Indigenous Seed Microbiota to Design Bacterial Seed Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Birgit Wassermann, Eveline Adam, Tomislav Cernava, and Gabriele Berg

Part II 6

3

21 35

55

83

Seed Endophytes: Ecology, Transmission and Adaptations

The Ecology of Seed Microbiota . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Pablo Hardoim

vii

viii

Contents

7

Programming Plants for Climate Resilience Through Symbiogenics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 Rusty Rodriguez, Alec Baird, Sang Cho, Zachery Gray, Evan Groover, Roman Harto, Marian Hsieh, Katie Malmberg, Ryan Manglona, Malia Mercer, Natalie Nasman, Tia Nicklason, Melissa Rienstra, Alex Van Inwegen, Andy VanHooser, and Regina Redman

8

Agave Seed Endophytes: Ecology and Impacts on Root Architecture, Nutrient Acquisition, and Cold Stress Tolerance . . . . 139 America Martinez-Rodriguez, Gloria Macedo-Raygoza, Aurora X. Huerta-Robles, Ileana Reyes-Sepulveda, Jhovana Lozano-Lopez, Evelyn Y. García-Ochoa, Luis Fierro-Kong, Marisa H. G. Medeiros, Paolo Di Mascio, James Francis White, Jr, and Miguel J. Beltran-Garcia

9

Chemical Warfare in the Plant Microbiome Leads to a Balance of Antagonisms and a Healthy Plant . . . . . . . . . . . . . . . . . . . . . . . . 171 Barbara Joan Schulz, Laura Rabsch, and Corina Junker

10

Fungal and Bacterial Maize Kernel Interactions with the Vertically Transmitted Endophytic State of Fusarium verticillioides . . . . . . . . 191 Charles W. Bacon and Dorothy M. Hinton

Part III

Seed Endophytes: Biology and Functional Roles in Plant Development

11

Functional Roles of Seed-Inhabiting Endophytes of Rice . . . . . . . . . 213 Gaurav Pal, Kanchan Kumar, Anand Verma, James Francis White, Jr, and Satish K. Verma

12

Mechanism of Interaction of Endophytic Microbes with Plants . . . . 237 Neethu Sahadevan, E. K. Radhakrishnan, and Jyothis Mathew

13

Fitness Attributes of Bacterial and Fungal Seed Endophytes of Tall Fescue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 Elizabeth Lewis Roberts, Brendan Mormile, and Christopher Adamchek

14

Role of the Plant Root Microbiome in Abiotic Stress Tolerance . . . 273 Daniel F. Caddell, Siwen Deng, and Devin Coleman-Derr

15

Endophytic Microbes: Prospects and Their Application in Abiotic Stress Management and Phytoremediation . . . . . . . . . . . . . . . . . . . 313 Divya Singh, Vipin Kumar Singh, and Amit Kishore Singh

Contents

16

ix

Pine Seeds Carry Symbionts: Endophyte Transmission Re-examined . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 Ron J. Deckert, Catherine A. Gehring, and Adair Patterson

Part IV

Seed Endophytes: Agricultural Applications and Biotechnology

17

Seed Endophytes of Jasione montana: Arsenic Detoxification Workers in an Eco-friendly Factory . . . . . . . . . . . . . . . . . . . . . . . . 365 María del Carmen Molina, James Francis White, Jr, Kathryn L. Kingsley, and Natalia González-Benítez

18

Agricultural Applications of Endophytic Microflora . . . . . . . . . . . . 385 John Reshma, Chandran Vinaya, and Mathew Linu

19

Rhizome Endophytes: Roles and Applications in Sustainable Agriculture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405 Akanksha Gupta, Hariom Verma, Prem Pratap Singh, Pardeep Singh, Monika Singh, Virendra Mishra, and Ajay Kumar

20

Agriculturally Important Biosynthetic Features of Endophytic Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423 S. Sreejith, R. Aswani, and E. K. Radhakrishnan

21

Microbial Endophytes of Maize Seeds and Their Application in Crop Improvements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449 Sandip Chowdhury, Rusi Lata, Ravindra N. Kharwar, and Surendra K. Gond

22

Colonization of Seeds by Soilborne Fungi: Linking Seed Dormancy-Defense Syndromes, Evolutionary Constraints, and Fungal Traits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465 Simon Maccracken Stump, Carolina Sarmiento, Paul-Camilo Zalamea, James W. Dalling, Adam S. Davis, Justin P. Shaffer, and A. Elizabeth Arnold

23

Seed Endophytes in Crop Plants: Metagenomic Approaches to Study the Functional Roles and Interactions . . . . . . . . . . . . . . . . 483 Priyanka Verma

Part I Seed Endophytes: Introduction, and Methods for Assessment and Management

1

Seed-Vectored Microbes: Their Roles in Improving Seedling Fitness and Competitor Plant Suppression James Francis White, Jr, Kathryn L. Kingsley, Susan Butterworth, Lara Brindisi, Judy W. Gatei, Matthew T. Elmore, Satish Kumar Verma, Xiang Yao, and Kurt P. Kowalski

Contents 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 1.10

The Seed Microbiome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adaptations of Seeds to Carry Symbiotic Microbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Roles of Seed-Vectored Microbes in Plant Seedlings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . What Happens to Seed-Vectored Microbes? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signal Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endobiome Interference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mode of Entry of Micrococcus luteus into Root Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intracellular Phases of Aureobasidium pullulans and Rhodotorula sp. . . . . . . . . . . . . . . . . . . Does Endobiome Interference Affect Plant-Plant Interactions? . . . . . . . . . . . . . . . . . . . . . . . . . . Potential Applications of Endobiome Interference to Control Invasive or Weedy Plant Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.11 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4 4 5 6 8 9 14 14 15 15 16 16

Abstract

This chapter discusses the roles of seed-vectored microbes in modulating seedling development and increasing fitness of plants in terms of increased biotic and J. F. White, Jr (*) · K. L. Kingsley · S. Butterworth · L. Brindisi · J. W. Gatei · M. T. Elmore Department of Plant Biology, Rutgers University, New Brunswick, NJ, USA S. K. Verma Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi, UP, India X. Yao Department of Plant Biology, Rutgers University, New Brunswick, NJ, USA State Key Laboratory of Grassland Agro-ecosystems, Key Laboratory of Grassland Livestock Industry Innovation, Ministry of Agriculture and Rural Affairs, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou, China K. P. Kowalski U.S. Geological Survey, Great Lakes Science Center, Ann Arbor, MI, USA e-mail: [email protected] # Springer Nature Switzerland AG 2019 S. K. Verma, J. F. White, Jr (eds.), Seed Endophytes, https://doi.org/10.1007/978-3-030-10504-4_1

3

4

J. F. White et al.

abiotic stress tolerance. Particular emphasis is placed on microbes that function in the rhizophagy cycle. These microbes have been shown to enter into root cells and stimulate root growth. In some cases microbe entry into root cells results in root growth repression. The term ‘endobiome interference’ has been applied to the phenomenon of plant growth repression due to intracellular microbes. The potential application of endobiome interference to produce bioherbicides that selectively enhance growth of target crops but inhibit competitor weeds is discussed. Keywords

Bioherbicide · Endophyte · Endobiome interference · Growth promotion · Rhizophagy cycle

1.1

The Seed Microbiome

Plant seeds carry embryonic plants and nutrients for early stages of seedling growth; in some plants seeds also carry small communities of symbiotic microbes (primarily bacteria and fungi) that are needed for defense from pathogens, modulation of plant development, and nutrient acquisition in seedlings (Doty 2017; Gond et al. 2015; Hardoim et al. 2015; Hurek et al. 2002; Clay et al. 2016; Irizarry and White 2017; Johnston-Monje and Raizada 2011; Rodríguez et al. 2017; Sherin et al. 2018; Soares et al. 2015, 2016; Puente et al. 2009; Verma et al. 2017a, b, 2018; White et al. 2012, 2015). Seed-vectored symbiotic microbes are adapted to their host plants and may enable seedlings to survive and thrive (Compant et al. 2010; Kandel et al. 2017). Without symbiotic microbes, many seedlings do not develop properly. They often lack normal root gravitropic response where roots do not grow downward into the soil or other substrates, sometimes growing upward, not producing root hairs, or producing hairs that are sparse or short (Holland 1997; Verma et al. 2017a, b; White et al. 2012). Seedlings without their microbes are more susceptible to abiotic and biotic stress: diseases, herbivory, oxidative stresses, drought, and heavy metals (Rodriguez et al. 2009; Torres et al. 2012; Waller et al. 2005; White and Torres 2010).

1.2

Adaptations of Seeds to Carry Symbiotic Microbes

In many grasses of subfamily Pooidae, fungal Epichloë endophytes colonize the ovules of the maternal plant and grow into the embryo inside caryopsis—thus germinating seedlings already contain the fungal endophyte (White and Cole 1986). We have found that some seed-associated tissues appear to function to vector microbes on seeds. Dried paleas and lemmas that adhere closely to grass seed coat (or caryopsis testa) vector bacteria and sometimes fungi that colonize roots and shoots of the germinating seedlings as they emerge from the seeds (White et al. 2012). The characteristically winged seeds of species in the plant family Polygonaceae, where wings are thought to function in dispersal, also carry bacteria that colonize germinating seedlings. In cotton (Gossypium spp.; Malvaceae),

1

Seed-Vectored Microbes: Their Roles in Improving Seedling Fitness and. . .

5

elongated trichomes (cotton fibers) carry bacteria that may stimulate seedling growth and protect cotton plants from diseases. Removal of the cotton fibers by acid delinting as is commonly done makes seeds easier to process in mechanical planters but also removes symbiotic bacteria from cotton seeds, leaving the seedlings defenseless from pathogens and insect pests and compromised developmentally (Irizarry and White 2017, 2018). As a consequence, cotton is often considered to be “the world’s dirtiest crop” due to the amount of agrochemicals frequently used in its cultivation (Environmental Justice Foundation 2007).

1.3

Roles of Seed-Vectored Microbes in Plant Seedlings

Considerable experimental evidence has been accumulated that supports the diseasesuppressive role of seed-vectored microbes (Verma et al. 2018). These microbes control disease in multiple ways: (1) by direct colonization of potentially pathogenic soilborne fungi and suppression of their growth and virulence, (2) colonization of seedlings resulting in upregulation of defense-related genes that makes plants more resistant to disease (Gond et al. 2015; Irizarry and White 2018), and (3) excluding pathogenic microbes by monopolizing space and/or production of antibiotics or toxins. Bacteria and fungi associated with seed tissue also influence development of seedlings. This process is not well understood, but microbes colonize the seedlings and increase gravitropic response, root elongation rate, root branching, and root hair elongation (Irizarry and White 2017; Verma et al. 2017a, b; White et al. 2012, 2017). It has been hypothesized that the capacity of microbes to alter hormone levels in plants may account for the capacity of microbes to modulate plant development (Bacon and White 2015). For example, the gravitropic response in seedlings may be suppressed when seedlings contain high levels of ethylene (Buer et al. 2006). Microbes that possess an enzyme (ACC deaminase) to remove a precursor to ethylene may remove ethylene that inhibits the gravitropic response in roots, and this could account for the gravitropic response modulation effect (Bacon and White 2015). Microbes also produce indole acetic acid (IAA); this hormone could explain other aspects of modulation of root growth—including branching and root hair elongation (Bacon and White 2015). In one experiment (White et al. 2012) using grass seedlings with and without seed-vectored bacteria, it was shown that both gravitropism and root hair formation could be restored in axenic seedlings by seed germination and seedling growth on agarose that contained low concentrations of proteins, certain amino acids, or the vitamin thiamine. In this study, it was suggested that microbes modulate plant root development by supplying organic nitrogen or vitamins. Seed-vectored bacteria and fungi also modify the physiological readiness of the seedling to tolerate oxidative stresses—either biotic or abiotic in origin. Many stresses of biotic and abiotic origin affect plants negatively by increasing internally generated reactive oxygen species (ROS)—which leads to increased internal oxidative damage in plants to membranes, proteins, and nucleic acids—and eventually to cell death (Cabiscol et al. 2000; Hamilton et al. 2012; White and Torres 2010). Seed-

6

J. F. White et al.

vectored microbes colonize seedlings and elicit a reactive oxygen defense response in plants that causes seedlings to upregulate stress resistance and antioxidant genes—resulting in seedlings that are more tolerant to oxidative stresses than seedlings without the microbes (Hamilton et al. 2012; Irizarry and White 2018; Kuldau and Bacon 2008; White and Torres 2010). Some seed-vectored microbes have been found to produce secondary metabolites that directly impact herbivores of the plant and may deter feeding by herbivores (Clay 1988; Clay et al. 2005). Fungal endophytes in genus Epichloë (Clavicipitaceae; Ascomycota) produce alkaloids that may intoxicate herbivores and repel them from infected seedlings (Schardl et al. 2013). In the toxic locoweeds (Oxytropis and Astragalus spp.; Fabaceae) and other plant species, endophytic fungi (e.g., Undifilum spp.; Pleosporaceae; Ascomycota) produce the toxic alkaloid swainsonine that intoxicates animals and deters many herbivores from consuming the plant (Cook et al. 2014, 2017). Similarly, seedvectored fungi of genus Periglandula (Clavicipitaceae; Ascomycota) in morning glories (Ipomoea spp.; Convolvulaceae) produce ergot alkaloids that render the plant toxic to animals and deter herbivory (Steiner et al. 2011).

1.4

What Happens to Seed-Vectored Microbes?

Some of the microbes that associate with plants are to be found primarily in the rhizosphere and function in the soil. Other microbes colonize surfaces of the plant and function in the rhizoplane (root surface)—or phylloplane (leaf surface). However, some of the seed-vectored microbes show the capacity to colonize seedlings internally and enter into tissues of the plant either intercellularly or intracellularly (BeltránGarcía et al. 2014; Stone et al. 2000; White et al. 2014). This internal niche has been referred to as the “endosphere” (Hardoim et al. 2015; Kandel et al. 2017). The microbes that inhabit the endosphere as endophytes form the “endobiome” (Kaul et al. 2017). A diversity of microbes (prokaryotic and eukaryotic) constitute the endobiome. A subset of endobiome microbes internally colonize plant cells—typically locating in the periplasmic spaces between the plasma membrane and plant cell wall (Thomas and Reddy 2013; Thomas and Soly 2009; White et al. 2017, 2018a, b). While microbes may colonize shoots and roots of plant seedlings upon germination, only those in roots play roles in acquiring nutrients from soils (Bowsher et al. 2016). Plants secrete exudates that contain sugars, amino acids, organic acids, and vitamins from roots that stimulate growth of microbes carried on or within seeds—and other microbes may be recruited from soils. It is generally believed that through secretion of exudates, plants alter the numbers and diversity of microbes on root surfaces and in the rhizosphere (Broeckling et al. 2008). Plants are known to increase secretion of exudates in nutrient-limiting soils, likely leading to increased microbial activity around roots and increased “microbial mining” for nutrients (Bowsher et al. 2016). Root exudates attract bacteria in particular that will grow in a biofilm in the root exudates (Funk-Jensen and Hockenhull 1984). In this sense, root exudates act as signal molecules that attract a diverse community of microbes to the exudate zone and biofilm around the root tip meristem (Rudrappa et al. 2008; Badri and Vivanco

1

Seed-Vectored Microbes: Their Roles in Improving Seedling Fitness and. . .

7

2009). Through the continued secretion of root exudates, plants are cultivating microbes, and when nutrients are scarce, plants increase cultivation of microbes by producing more exudates (Bowsher et al. 2016). The response of plants to increase the density and diversity of the microbial community around roots by secreting more root exudates in nutrient-limiting situations is consistent with the hypothesis that the rootassociated microbes function in nutrient acquisition. The microbes function to make available to plant roots macronutrients and micronutrients needed by plants. White et al. (2015) conducted an isotope tracking experiment where 15N-labeled protein was incorporated into agarose—then tall fescue (Lolium arundinaceum) seedlings, both with and without seed-surface microbes, were grown on the agarose. After analysis of the 15N content of seedling shoots, it was found that seedlings with seed-vectored microbes contained 30% more of the 15N than seedlings without microbes. This 30% increase in nitrogen absorption into plants with seed-vectored microbes may reflect “nutrient mining” by the seed microbes. The superior capacity of microbes to move around in the rhizosphere either by mycelial growth for fungi or by flagellar motility for bacteria enables them to access pools of nutrients that the plants may be unable to obtain alone. The possession of siderophores by bacteria and fungi (Johnstone and Nolan 2015) further enables them to acquire nutrients (like metals iron, zinc, copper, and magnesium) and transport these nutrients back to plants where they may be transferred to plant roots. A subset of the seed-vectored bacteria and yeasts colonize the exudate zone around the root tip meristem. These microbes penetrate into the outer layers of recently formed root parenchyma cells around the root meristem. These microbes then become situated in the periplasmic spaces of root parenchyma (Verma et al. 2018; White et al. 2019). Initially, intracellular bacteria retain their cell walls and cell shapes—but as root cells differentiate, bacteria are exposed to secreted ROS produced by NADPH oxidases (NOX) present on root cell plasma membranes (White et al. 2014). Intracellular bacteria lose their cell walls to form wall-less L-forms (Errington et al. 2016). Intracellular L-forms proliferate in the periplasmic space by a continuous budding process—referred to as “blebbing” (Errington et al. 2016; Beran et al. 2006). L-forms are exposed to ROS, the most potent of which is likely superoxide produced by NOX enzymes (White et al. 2012, 2014). ROS oxidizes bacterial L-form plasma membranes—and penetrates into the bacterial cytoplasm resulting in oxidative damage internally (Cabiscol et al. 2000; Lamb and Dixon 1997). The net effect of the continuous bombardment of bacterial L-forms by ROS is that bacterial membranes become leaky and cell contents including electrolytes are lost and some of the bacterial cells are entirely degraded (Paungfoo-Lonhienne et al. 2010, 2013; White et al. 2012, 2017). Surviving bacteria trigger elongation of root hairs, and as hairs elongate, bacteria exit the hair at the elongating hair tip where cell walls are incompletely formed (White et al. 2017, 2018a, b); as bacteria exit, they reform cell walls and reenter the rhizosphere where they may acquire additional nutrients. The process of degradation of microbes within roots has been termed “rhizophagy” (meaning “root eating”) (Paungfoo-Lonhienne et al. 2013). The cyclic process where symbiotic bacteria alternate between a free-living soil phase and an intracellular endophytic phase has been termed “rhizophagy cycle” or “rhizophagy

8

J. F. White et al.

symbiosis” (Verma and White 2018; White et al. 2018a, b). It seems reasonable that the primary function of the rhizophagy cycle is the transport of nutrients via microbes from the rhizosphere to the plant root where nutrients are extracted from microbes (Hill et al. 2011; Beltrán-García et al. 2014; Prieto et al. 2017; White et al. 2018b). It is also logical that microbes that are symbiotic with plants and function in the rhizophagy cycle are adapted to the host plant and show the following features: (1) possess the capacity to enter plant cell walls at the root tip meristem; (2) release electrolytes to plant cells on exposure to ROS secreted by root cell plasma membranes; (3) possess the ability to survive ROS exposure in its host; (4) trigger root hair elongation to exit the hair as it elongates; and (5) are attracted back to the root exudate zone at root tip meristems.

1.5

Signal Molecules

It has been hypothesized that plants signal microbes to come to the roots by the composition of root exudates (Badri and Vivanco 2009; Clarkson and Marshner 1995); thus the exudates themselves represent signals to symbiotic microbes. We have evidence (White et al. 2018b) that plants detect the presence of some bacteria in the exudate zone by detecting fermentation products of the bacteria—one of which is butyric acid. Butyric acid is an anaerobic fermentation product of carbohydrates by some bacteria. Butyric acid is absorbed in root tip meristems (Lanzagorta et al. 1988; Tramontano and Scanlon 1996)—and its removal from the bacterial biofilm in the exudate zone around the meristem causes some bacteria to upregulate virulence genes (Cox et al. 1994; Sun and O’Riordan 2013) and infect plant root cells around the meristem. A similar mechanism is seen in the intestinal tracts of animals. In animal intestines, bacteria produce butyric acid. As long as butyric acid remains in elevated concentration of the biofilm in which bacteria grow, bacteria remain in the biofilm; however if levels of butyric acid fall due to dysbiosis, gut bacteria like Salmonella spp. become virulent and infect gut epithelial tissues (Sun and O’Riordan 2013). In experiments using grass seedlings (Poa annua) inoculated with Pseudomonas spp. and germinated and grown on agarose amended with 0–10 mM butyric acid, we demonstrated that butyric acid at approximately 5 mM concentration suppressed entry of bacteria into root tip meristem cells. In this experiment (White et al. 2018b), the root tip meristem was unable to remove butyric acid from the bacterial biofilm around the meristem, and as a consequence, bacteria did not penetrate into the root meristem cells. Suppression of bacterial entry into meristem cells resulted in the loss of gravitropic response and root hair formation in seedling roots (White et al. 2018b). Another fermentation product that acts as a signal molecule for plants and bacteria is propionic acid. When propionic acid is present at a sufficient level in the bacterial biofilm around the root tip meristem, bacteria do not penetrate into meristem cells (Unpublished data). Propionic acid is also absorbed by root tip meristems (Lanzagorta et al. 1988; Tramontano and Scanlon 1996), and its removal from bacterial biofilms along with butyrate results in internal colonization of root tip meristem cells.

1

Seed-Vectored Microbes: Their Roles in Improving Seedling Fitness and. . .

1.6

9

Endobiome Interference

We hypothesize that microbes of the endobiome of a particular plant are adapted to the internal conditions of that plant and that the conditions in the endospheres of plants may differ between species of plants. The removal of endobiome microbes from hosts to which they are adapted, and transference to seedling hosts to which they are not adapted, could result in (1) internal colonization, (2) interference with the functioning of other microbes of the endobiome, (3) interference with plant development, or (4) increases in seedling mortality. Perturbations in seedling development or increases in seedling mortality as a result of colonization by nonadapted microbes may result from “endobiome interference.” To evaluate whether “endobiome interference” occurs, we conducted a series of experiments where we removed microbes (bacteria and yeasts) from seeds of plants, including species rosary pea (Abrus precatorius), snakecotton (Froelichia gracilis), tomato (Lycopersicum esculentum), and annual bluegrass (Poa annua), and inoculated them onto axenic seedlings in agarose. We then assessed internal colonization of seedling roots, root growth, and seedling mortality (Table 1.1; Figs. 1.1, 1.2, and 1.3). Test seedlings included dandelion (Taraxacum officinale), curly dock (Rumex crispus), and clover (Trifolium repens). Some experiments were also done where microbes were inoculated onto seedlings of prince’s-feather (Amaranthus hypochondriacus) and green amaranth (Amaranthus viridis). Microbes included yeasts Rhodotorula sp. (strain Abrus#1) and Aureobasidium pullulans (strain Froelichia#2) and bacteria Sphingomonas sp. (strain Abrus#3), Rhodococcus sp. (strain AbrusR), Micrococcus luteus (strain Lycopersicon#1), Curtobacterium sp. (strain Froelichia#4), and Paenibacillus sp. (strain PA-NA-2B1). None of these microbes appeared to be pathogenic or inhibitory of root growth in their original hosts based on growth of seedlings containing microbes on agarose media. In fact, Micrococcus luteus was found to be growth promotional in tomato seedlings, resulting in intracellular colonization and increased root hair length. All of the microbes were found to become intracellular in seedling root cells when inoculated onto germinating seeds (Figs. 1.1c, e, f, 1.2, and 1.3). The occurrence of effects that we consider to constitute endobiome interference depended on the microbe, and the host seedling the microbe was inoculated into. The two most potent microbes in terms of increased mortality in seedlings after 3 weeks included the yeast Aureobasidium pullulans (Froelichia#2) and bacterium Micrococcus luteus (Lycopersicum#1). In terms of inhibition of root growth, Rhodotorula sp. (strain Abrus#1), Sphingomonas sp. (strain Abrus#3), and Micrococcus luteus (Lycopersicum#1) were more inhibitory. Curtobacterium (strain Froelichia#4) was growth promotional in all three species, increasing root growth and reducing seedling mortality in test seedlings. The mechanisms of inhibition of root growth or increase in seedling root growth are not clear. However, two factors seem relevant to the longevity of microbes, including (1) entry of microbes into root cells and (2) resistance of microbes to ROS secreted by the host. Microbes that are highly resistant to ROS secreted by root cells may be difficult to control once they are in the endosphere—and especially when they become intracellular. Microbes that enter root cells and situate in close contact

10

J. F. White et al.

Table 1.1 Endobiome interference experimental result summary Microbe Rhodotorula Rhodotorula

Host origin A. precatorius A. precatorius

Rhodotorula Sphingomonas Sphingomonas

A. precatorius A. precatorius A. precatorius

Sphingomonas Rhodococcus Rhodococcus

A. precatorius A. precatorius A. precatorius

Rhodococcus Aureobasidium Aureobasidium

A. precatorius F. gracilis F. gracilis

Aureobasidium Curtobacterium Curtobacterium

F. gracilis F. gracilis F. gracilis

Curtobacterium Micrococcus Micrococcus

F. gracilis L. esculentum L. esculentum

Micrococcus Paenibacillus Paenibacillus

L. esculentum P. annua P. annua

Paenibacillus

P. annua

Target host Dandelion Curly dock Clover Dandelion Curly dock Clover Dandelion Curly dock Clover Dandelion Curly dock Clover Dandelion Curly dock Clover Dandelion Curly dock Clover Dandelion Curly dock Clover

Intracellular Yes Yes

Δ Root length (%)a
40
45

Δ Mortality (%) +18
27

Yes Yes Yes


64
28
19

+24 +39 0

Yes Yes Yes


40
7
23


6 +50
3

Yes Yes Yes


13 +46
17

0 +44 +57

Yes Yes Yes

+2 +15 +10

+10
13
13

Yes Yes Yes

+7
60
30


7 +80
14

Yes Yes Yes


27
12
30

+31 +51
14

Yes

–

–

a

Percentages are from means of 40 seeds/seedlings; mortality includes germination suppression and seedling death after 3 weeks on agarose; Δ designates change (Δ Root length ¼ change in root length compared to control, and Δ Mortality ¼ change in mortality compared to control)

with root cell plasma membranes may be able to extract more nutrients from plant cells and may more frequently trigger cell death. Micrococcus luteus and Aureobasidium pullulans are good examples where this may be occurring. Both microbes are resistant to ROS due to production of antioxidants (White et al. 2018b). Micrococcus luteus produces antioxidant carotenoids, catalases, peroxidases, and other antioxidant enzymes that reduce the negative effects of host-secreted ROS (Mohanna et al. 2013). Similarly, Aureobasidium pullulans possesses antioxidant cell wall components mannans and glucans (Machova and Bystricky 2013), and because they are eukaryotic, their plasma membranes are reinforced with ergosterol to stabilize the membrane and prevent passage of ROS into the cytoplasm (White et al. 2018b). This oxidative resistance may enable Micrococcus luteus and

1

Seed-Vectored Microbes: Their Roles in Improving Seedling Fitness and. . .

11

Fig. 1.1 Micrococcus luteus. (a) Micrococcus luteus in the seedling of carrots (arrows) show white accumulations of bacteria around seedlings on agarose after 2 weeks. (b) Tetrads of Micrococcus luteus (arrows) from colonies on yeast extract-sucrose agar (bar ¼ 10 μm). (c) Cells around root tip meristem of a seedling of Rumex crispus showing tetrads of Micrococcus luteus (arrows) in the periplasmic space of cells (bar ¼ 25 μm). (d) Cells around the root tip meristem of a seedling of Rumex crispus that had not been inoculated with Micrococcus luteus, showing that bacteria are not visible in cells (bar ¼ 25 μm). (e) and (f) Parenchyma cells of Rumex crispus showing spherical bacterial L-forms of Micrococcus luteus (arrows) in the periplasmic space of cells (bar ¼ 25 μm)

Aureobasidium pullulans to proliferate within root cells in an unregulated manner. Overgrowth of these microbes within root cells and tissues results in diversion of seedling nutrients from support of seedling growth to microbe replication—resulting in seedling growth suppression. This is especially evident in the case of Micrococcus luteus where inoculated seedlings on agarose were found to have repressed root growth with bacteria accumulating en masse around seedling roots.

12

J. F. White et al.

Fig. 1.2 Micrococcus luteus in host tissues (stained with 3,3-diaminobenzidine for 15 h followed by aniline blue). (a) L-forms of Micrococcus luteus (arrows) in root hair of Rumex crispus (bar ¼ 25 μm). (b) Root hair initial of Rumex crispus seedling showing spherical L-forms in the periplasmic space (white arrow) and blue tetrads of Micrococcus luteus reforming as bacteria (black arrow) exit through the cell wall and spill off the side of the root hair initial (bar ¼ 25 μm). (c) Root hair of Rumex crispus seedling showing Micrococcus luteus exiting the root hair at the hair tip and reforming tetrads (arrows; bar ¼ 25 μm). (d) Root hairs of carrot (Daucus carota) seedling showing exiting of Micrococcus luteus from the tips of hairs (arrows; bar ¼ 20 μm). (e) Root hair initial of carrot seedling showing Micrococcus luteus emerging from the hair initial (arrows; bar ¼ 20 μm). (f) Root hair tip of Rumex crispus seedling showing spherical L-forms (white arrow) in the periplasmic space and tetrads (black arrow) of Micrococcus luteus just outside the cell wall; exit channels are visible passing through the cell wall (bar ¼ 5 μm)

1

Seed-Vectored Microbes: Their Roles in Improving Seedling Fitness and. . .

13

Fig. 1.3 Aureobasidium pullulans in seedling cells (stained with 3,3-diaminobenzidine for 15 h, followed by aniline blue). (a) Root hair of Amaranthus hypochondriacus seedling inoculated with Aureobasidium pullulans, showing intracellular, brown-staining, and collapsed walled yeast cells (white arrow) and extracellular, blue-staining yeast cells (black arrows; bar ¼ 20 μm). (b) Amaranthus viridis seedling root parenchyma cell showing intracellular walled hypha (arrow) of Aureobasidium pullulans (bar ¼ 25 μm). (c) Froelichia gracilis seedling root hair showing intracellular yeast mycosomes (arrows; bar ¼ 20 μm). (d) Amaranthus viridis root parenchyma cells showing abundance of intracellular brown-staining yeast mycosomes (bar ¼ 25 μm). (e) Froelichia gracilis seedling root hair with intracellular yeast mycosomes (arrows; bar ¼ 20 μm). (f) Amaranthus viridis root hair without intracellular yeasts (bar ¼ 20 μm)

14

1.7

J. F. White et al.

Mode of Entry of Micrococcus luteus into Root Cells

We tracked Micrococcus luteus through seedling tissues and cells in the previously described “endobiome interference” experiments. Micrococcus luteus initially infected root meristem cells—entering periplasmic spaces of outer layers of root tip meristem cells as walled tetrads (Fig. 1.1b, c). As root cells matured, the tetrads converted to unicellular cells—likely L-forms. Spherical cells (wall-less L-forms) were visible in periplasmic spaces of root epidermal cells and root hairs when they formed (Figs. 1.1e, f and 1.2a, b, f). The spherical cells did not swell or lose capacity to stain with aniline blue, suggesting that plant ROS was not degrading the intracellular bacterial cells. This is an indication that Micrococcus luteus is resistant to ROS produced by NOX on the root cell plasma membranes. When less oxidatively resistant pseudomonads are used, L-forms in periplasmic spaces swell and lose interior staining with aniline blue due to loss of cell contents by the L-forms (White et al. 2014, 2017). The spherical bacteria of Micrococcus luteus in primordial root hairs were seen to exit root hair tips through channels in the plant cell walls. Once outside, bacteria reformed walls and tetrad shapes (Fig. 1.2b–f). This route of bacteria through root tissues is what has been observed for bacteria in the rhizophagy cycle. It seems evident that microbes that show endobiome interference are partially compatible with host plants but they are not adapted to the host like native symbiotic microbes—and rather than increase the growth and survival of seedlings, they reduce growth and/or increase seedling mortality.

1.8

Intracellular Phases of Aureobasidium pullulans and Rhodotorula sp.

Atsatt and Whiteside (2014) demonstrated that Aureobasidium pullulans and Rhodotorula pinicola develop an intracellular phase in plants. The intracellular phase includes cells that retain cell walls and those forms that lack cell walls termed “mycosomes.” Our experiments with various Amaranthaceae suggest that Aureobasidium pullulans may be a frequent endophyte in this family of plants—although much more work is needed to evaluate its functional role in these plants. In Abrus precatorius, Rhodotorula sp. may be a common seed-vectored endophyte. Mycosomes appear to behave like bacterial L-forms in that they bud or “bleb” sequentially to form chains (Atsatt and Whiteside 2014). The wall-less mycosome phase may also be a response to plant-produced reactive oxygen or to particular nutrients to which fungi are exposed to in plant tissues. Mycosomes have also been reported to spontaneously revert to the walled cell phase (Atsatt and Whiteside 2014). Intracellular walled Aureobasidium cells are visible in Fig. 1.3a, b, while mycosomes are seen in Fig. 1.3c–e. It is unclear whether these fungal endophytes may be degraded in plant cells or whether they can function in the rhizophagy cycle to provide nutrients to the host plant. Yeasts may be entering root cells at the root tip meristems just as do bacteria based on the presence of mycosomes

1

Seed-Vectored Microbes: Their Roles in Improving Seedling Fitness and. . .

15

in meristematic cells in our inoculation experiments. Much additional work is needed to evaluate the function of the intracellular phases of fungi in plant tissues.

1.9

Does Endobiome Interference Affect Plant-Plant Interactions?

It is possible that some plants may maintain microbial symbionts and nourish them within tissues and cells as defensive or offensive weapons that may be employed against competitor plant species. The plants from which we obtained seed-vectored microbes in the endobiome interference experiment are generally aggressive weedy species. However tomato plants are known to have allelopathic properties—where tomato plants may suppress growth of some other plant species. Abrus precatorius, Froelichia gracilis, and Poa annua are competitive and may be invasive. It is entirely possible that these species use their endobiomes against competitor plant species—where microbes may colonize competitor seedlings and reduce their growth and persistence. This possibility seems likely when it is considered that microbes in the rhizophagy cycle alternate between an endophytic/intracellular phase and a free-living soil phase (Prieto et al. 2017; Verma et al. 2018; White et al. 2018b). These microbes may move out from the plant, forming a zone around plants where certain vulnerable competitor species cannot grow. Seedlings of competitor species that begin to grow in that zone could be colonized and their nutrients used by the microbes for reproduction; return of bacteria to the original host plant may deliver nutrients extracted from the competitor plant species. In a previous study (White et al. 2017) of seed-vectored pseudomonads from invasive Phragmites australis, pseudomonads were seen to promote the growth of grass seedlings but were seen to inhibit growth of competitor dicot species (Taraxacum officinale and Rumex crispus). It is conceivable that plant species that share endobiome microbes, where those microbes are growth promotional in both plant species, may grow together, while endobiome interference may force plant species apart. Additional experiments are needed to determine whether endobiome interference is a factor in plant-plant interactions in natural plant communities.

1.10

Potential Applications of Endobiome Interference to Control Invasive or Weedy Plant Species

Invasive and weedy plant control generally employs the use of chemical herbicides or mechanical removal of plants (Kowalski et al. 2015). Endobiome interference could offer an alternative means whereby particular weeds could be controlled without herbicides. It may be possible to enhance growth of crop species and simultaneously repress growth of weedy competitor species through applications of microbes that are growth promotional in crops—but produce endobiome interference in competitor plants. Such an approach could reduce applications of agrochemicals in crops with economic and environmental benefits.

16

1.11

J. F. White et al.

Conclusions

Seed-vectored microbes play roles in modulation of seedling development, defense from abiotic and biotic stresses, defense from pathogens and herbivores, and nutrient acquisition. It is also possible that through endobiome interference, symbiotic microbes of one plant may suppress growth of competitor plant species by reducing seedling growth and increasing seedling mortality. The mechanisms of endobiome interference are not well understood; however, oxidative resistance of the microbe may reduce the capacity of host cells to control intracellular microbes using ROS produced by NOX enzymes on root cell plasma membranes. We hypothesize that endobiome interference is a factor in plant-plant interactions in natural plant communities. If the hypotheses expressed in this chapter are proven, endobiome interference could be a strategy that may be developed to control invasive or weedy plant species. Acknowledgments The authors acknowledge the Department of Plant Biology, Rutgers University, NJ, for research facilities and financial support. SKV is thankful to UGC, India, for providing a Raman Post Doctoral fellowship (No.-F 5-11/2016 IC) for the year 2016–2017 to conduct research in the USA. SKV is grateful to the Head and Coordinator CAS, FIST of Botany, B.H.U., Varanasi, India, for providing the leave to pursue research on endophytes. The authors are also grateful for support from USDA-NIFA Multistate Project W3147 and the New Jersey Agricultural Experiment Station. Funds for much of this work were from Cooperative Ecosystems Studies Unit CESU G16AC00433 between Rutgers University and the US Geological Survey for control of invasive Phragmites australis. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the US Government.

References Atsatt PR, Whiteside MD (2014) Novel symbiotic protoplasts formed by endophytic fungi explain their hidden existence, lifestyle switching, and diversity within the plant kingdom. PLoS One 9 (4):e95266. https://doi.org/10.1371/journal.pone.0095266 Bacon C, White JF (2015) Functions, mechanisms and regulation of endophytic and epiphytic microbial communities of plants. Symbiosis 68(1–3):87–98 Badri DV, Vivanco JM (2009) Regulation and function of root exudates. Plant Cell Environ 32:666–681 Beltrán-García MJ, White JF, Prado FM, Prieto KR, Yamaguchi LF, Torres MS, Kato MJ, Medeiros MHG, Di Mascio P (2014) Nitrogen acquisition in Agave tequilana from degradation of endophytic bacteria. Sci Rep 4:6938 Beran V, Havelkova M, Kaustova J, Dvorska L, Pavlik I (2006) Cell wall deficient forms of mycobacteria: a review. Vet Med 51:365–389 Bowsher AW, Ali R, Harding SA, Tsai C-J, Donovan LA (2016) Evolutionary divergences in root exudate composition among ecologically-contrasting Helianthus species. PLoS One 11(1): e0148280. https://doi.org/10.1371/journal.pone.0148280 Broeckling CD, Broz AK, Bergelson J, Manter DK, Vivanco JM (2008) Root exudates regulate soil fungal community composition and diversity. Appl Environ Microbiol 74:738–744 Buer CS, Sukumar P, Muday GK (2006) Ethylene modulates flavonoid accumulation and gravitropic responses in roots of Arabidopsis. Plant Physiol 140:1384–1396. https://doi.org/10.1104/pp.105. 075671

1

Seed-Vectored Microbes: Their Roles in Improving Seedling Fitness and. . .

17

Cabiscol E, Tamarit J, Ros J (2000) Oxidative stress in bacteria and protein damage by reactive oxygen species. Int Microbiol 3:3–8 Clarkson D, Marschner H (1995) Mineral nutrition of higher plants, 2nd edn. Academic Press, London. 889 pp Clay K (1988) Fungal endophytes of grasses: a defensive mutualism between plants and fungi. Ecology 69:10–16 Clay K, Holah J, Rudgers JA (2005) Herbivores cause a rapid increase in hereditary symbiosis and alter plant community composition. PNAS 102:12465–12470 Clay K, Shearin ZRC, Bourke KA et al (2016) Diversity of fungal endophytes in non-native Phragmites australis in the Great Lakes. Biol Invasions 18:2703. https://doi.org/10.1007/ s10530-016-1137-y Compant S, Clement 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 Cook D, Gardner DR, Pfister JA (2014) Swainsonine-containing plants and their relationship to endophytic fungi. J Agric Food Chem 62:7326–7334. https://doi.org/10.1021/jf501674r Cook D, Donzelli BGG, Creamer R, Baucom DL, Gardner DR, Pan J, Schardl CL (2017) Swainsonine biosynthesis genes in diverse symbiotic and pathogenic fungi. G3 7:1791–1797. https://doi.org/10.1534/g3.117.041384 Cox NA, McHan F, Bailey JB, Shotts EB (1994) Effect of butyric or lactic acid on the In vivo colonization of Salmonella typhimurium. J Appl Poult Res 3:315–318 Doty SL (2017) Functional importance of the plant microbiome: implications for agriculture, forestry and bioenergy. In: Doty SL (ed) Functional importance of the plant microbiome. Springer, Amsterdam, pp 1–5 Environmental Justice Foundation (2007) The deadly chemicals in cotton. Environmental Justice Foundation in collaboration with Pesticide Action Network UK, London. ISBN No. 1-90452310-2 Errington J, Mickiewicz K, Kawai Y, Wu LJ (2016) L-form bacteria, chronic diseases and the origins of life. Philos Trans R Soc B 371:20150494. https://doi.org/10.1098/rstb.2015.0494 Funk-Jensen D, Hockenhull J (1984) Root exudation, rhizosphere microorganisms and disease control. Växtskyddsnotiser 48:49–54 Gond SK, Bergen M, Torres MS, White JF (2015) Effect of bacterial endophyte on expression of defense genes in Indian popcorn against Fusarium moniliforme. Symbiosis 66:133–140. https:// doi.org/10.1007/s13199-015-0348-9 Hamilton CE, Gundel PE, Helander M, Saikkonen K (2012) Endophytic mediation of reactive oxygen species and antioxidant activity in plants: a review. Fungal Divers 54:1–10 Hardoim PR, van Overbeek LS, Berg G, Pirttilä AM, Compant S, Campisano A, Döring M, Sessitsch A (2015) The hidden world within plants: ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiol Mol Biol Rev 79:293–320. https:// doi.org/10.1128/MMBR.00050-14 Hill PW, Quilliam RS, DeLuca TH, Farrar J, Farrell M et al (2011) Acquisition and assimilation of nitrogen as peptide-bound and D-enantiomers of amino acids by wheat. PLoS One 6(4):e19220. https://doi.org/10.1371/journal.pone.0019220 Holland MA (1997) Methylobacterium and plants. Recent Res Dev Plant Physiol 1:207–213 Hurek T, Handley LL, Reinhold-Hurek B, Piché Y (2002) Azoarcus grass endophytes contribute fixed nitrogen to the plant in an unculturable state. Mol Plant-Microbe Interact 15:233–242 Irizarry I, White JF (2017) Application of bacteria from non-cultivated plants to promote growth, alter root architecture and alleviate salt stress of cotton. J Appl Microbiol 122:1110–1120. https://doi.org/10.1111/jam.13414 Irizarry I, White JF (2018) Bacillus amyloliquefaciens alters gene expression, ROS production, and lignin synthesis in cotton seedling roots. J Appl Microbiol 124:1589–1603 Johnstone TC, Nolan EM (2015) Beyond iron: non-classical biological functions of bacterial siderophores. Daltan Trans 44:6320–6339. http://dxdoi.org/10.1039/c4dt03559c

18

J. F. White et al.

Johnston-Monje D, Raizada MN (2011) Conservation and diversity of seed associated endophytes in Zea across boundaries of evolution, ethnography and ecology. PLoS One 6(6):e20396. https://doi.org/10.1371/journal.pone.0020396 Kandel SL, Joubert PM, Doty LS (2017) Bacterial endophyte colonization and distribution within plants. Microorganisms 5:77. https://doi.org/10.3390/microorganisms5040077 Kaul S, Gupta S, Sharma S, Dhar MK (2017) The fungal endobiome of medicinal plants: a prospective source of bioactive metabolites. In: Agrawal D, Tsay HS, Shyur LF, Wu YC, Wang SY (eds) Medicinal plants and fungi: recent advances in research and development, Medicinal and aromatic plants of the world, vol 4. Springer, Singapore Kowalski KP, Bacon C, Bickford W, Braun H, Clay K, Leduc-Lapierre M, Lillard E, McCormick M, Nelson E, Torres M, White JF, Wilcox DA (2015) Advancing the science of microbial symbiosis to support invasive species management: a case study on Phragmites in the Great Lakes. Front Microbiol 6:95. https://doi.org/10.3389/fmicb.2015.00095 Kuldau G, Bacon CW (2008) Clavicipitaceous endophytes: their ability to enhance grass resistance to multiple stresses. Biol Control 46:57–71 Lamb C, Dixon RA (1997) The oxidative burst in plant disease resistance. Annu Rev Plant Physiol Mol Biol 48:251–275 Lanzagorta JMA, de la Torre C, Aller P (1988) The effect of butyrate on cell cycle progression in Allium cepa root meristems. Physiol Plant 72:775–781 Machova E, Bystricky S (2013) Antioxidant capacities of mannans and glucans are related to their susceptibility to free radical degradation. Int J Biol Macromol 61:308–311 Mohanna DC, Thippeswamy S, Abhishek RU (2013) Antioxidant, antibacterial, and ultraviolet protective properties of carotenoids isolated from Micrococcus spp. Radiat Prot Environ 36:168–174 Paungfoo-Lonhienne C, Rentsch D, Robatzrk S, Webb RI, Sagulenko E, Nasholm T, Schmidt S, Lonhienne TGA (2010) Turning the table: plants consume microbes as a source of nutrients. PLoS One 5(7):e11915. https://doi.org/10.1371/journal.pone.0011915 Paungfoo-Lonhienne C, Schmidt S, Webb R, Lonhienne T (2013) Rhizophagy – a new dimension of plant-microbe interactions. In: de Briujn FJ (ed) Molecular microbial ecology of the rhizosphere. Wiley-Blackwell, Hoboken, NJ Prieto KR, Echaide-Aquino F, Huerta-Robles A, Valerio HP, Macedo-Raygoza G, Prado FM, Medeiros M, Brito HF, da Silva I, Felinto MCF, White JF, Di Masci P, Beltran-Garcia M (2017) Endophytic bacteria and rare earth elements; Promising candidates for nutrient use efficiency in plants. In: Hossain M, Kamiya T, Burritt D, Tram L-SP, Fujiwara T (eds) Plant macronutrient use efficiency. Academic Press, Cambridge, MA, pp 285–302 Puente ME, Lib CY, Bashan Y (2009) Endophytic bacteria in cacti seeds can improve the development of cactus seedlings. Environ Exp Bot 66:402–408 Rodriguez RJ, Woodward C, Kim YO, Redman RS (2009) Habitat-adapted symbiosis as a defense against abiotic and biotic stresses. In: White JF Jr, Torres MS (eds) Defensive mutualism in microbial symbiosis. CRC Press, Boca Raton, FL, pp 335–346 Rodríguez CE, Mitter B, Barret M, Sessitsch A, Compant S (2017) Commentary: seed bacterial inhabitants and their routes of colonization. Plant Soil 422:129–134. https://doi.org/10.1007/ s11104-017-3368-9 Rudrappa T, Czymmek KJ, Paré PW, Bais HP (2008) Root-secreted malic acid recruits beneficial soil bacteria. Plant Physiol 148:1547–1556 Schardl CL, Young CA, Pan J, Florea S, Takach J, Panaccione DG, Farman ML, Webb JS, Jaromczyk J, Charlton ND, Nagabhyru P, Chen L, Shi C, Leuchtmann A (2013) Currencies of mutualisms: sources of alkaloid genes in vertically transmitted epichloae. Toxins 5:1064–1088 Shearin ZRC, Filipek M, Desai R et al (2018) Fungal endophytes from seeds of invasive, non-native Phragmites australis and their potential role in germination and seedling growth. Plant Soil 422:183. https://doi.org/10.1007/s11104-017-3241-x

1

Seed-Vectored Microbes: Their Roles in Improving Seedling Fitness and. . .

19

Soares MA, Li H, Bergen M, White JF (2015) Functional role of an endophytic Bacillus amyloliquefaciens in enhancing growth and disease protection of invasive English ivy (Hedera helix L.). Plant Soil 405:107–123. https://doi.org/10.1007/s11104-015-2638-7 Soares MA, Li H-Y, Kowalski KP, Bergen M, Torres MS, White JF (2016) Functional roles of bacteria from invasive Phragmites australis in promotion of host growth. Microb Ecol 72:407–417 Steiner U, Leibner S, Schardl CL, Leuchtmann A, Leistner E (2011) Periglandula, a new fungal genus within the Clavicipitaceae and its association with Convolvulaceae. Mycologia 103:1133–1145 Stone JK, Bacon CW, White JF (2000) An overview of endophytic microbes: endophytism defined. In: Bacon CW, White JF (eds) Microbial endophytes. Marcel-Dekker, New York, pp 3–30 Sun Y, O’Riordan M (2013) Regulation of bacterial pathogenesis by intestinal short-chain fatty acids. Adv Appl Microbiol 85:93–118 Thomas P, Reddy KM (2013) Microscopic elucidation of abundant endophytic bacteria colonizing the cell wall-plasma membrane peri-space in the shoot-tip tissue of banana. AOB Plants 5:plt011. https://doi.org/10.1093/aobpla/plt011 Thomas P, Soly TA (2009) Endophytic bacteria associated with growing shoot tips of banana (Musa sp.) cv. Grand Naine and the affinity of endophytes to the host. Microb Ecol 58:953–964 Torres MS, White JF, Zhang X, Hinton DM, Bacon CW (2012) Endophyte-mediated adjustments in host morphology and physiology and effects on host fitness traits in grasses. Fungal Ecol 5:322–330 Tramontano WA, Scanlon C (1996) Cell cycle inhibition by butyrate in legume root meristems. Phytochemistry 41:85–88 Verma SK, White JF (2018) Indigenous endophytic seed bacteria promote seedling development and defend against fungal disease in browntop millet (Urochloa ramosa L.). J Appl Microbiol 124:764–778. https://doi.org/10.1111/jam.13673 Verma SK, Kingsley K, Irizarry I, Bergen M, Kharwar RN, White JF (2017a) Seed vectored endophytic bacteria modulate development of rice seedlings. J Appl Microbiol 122:1680–1691 Verma SK, Kingsley K, Bergen M, English C, Elmore M, Kharwar RN, White JF (2017b) Bacterial endophytes from rice cut grass (Leersia oryzoides L.) increase growth, promote root gravitropic response, stimulate root hair formation, and protect rice seedlings from disease. Plant Soil 422:223–238. https://doi.org/10.1007/s11104-017-3339-1 Verma SK, Kingsley KL, Bergen MS, Kowalski KP, White JF (2018) Fungal disease protection in rice (Oryza sativa) seedlings by growth promoting seed-associated endophytic bacteria from invasive Phragmites australis. MDPI: Microorganisms. https://doi.org/10.3390/microorganisms6010021 Waller F, Achatz B, Baltruschat H, Fodor J, Becker K, Fisher M, Heier T, Huckelhoven R, Neumann C, Wettstein D, Franken P, Kogel KH (2005) The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield. PNAS 102:13386–13391 White JF, Cole GT (1986) Endophyte-host associations in forage grasses. IV. The endophyte of Festuca versuta. Mycologia 78:102–107 White JF, Torres MS (2010) Is endophyte-mediated defensive mutualism oxidative stress protection? Physiol Plant 138:440–446 White JF, Crawford H, Torres MS, Mattera R, Irizarry I, Bergen M (2012) A proposed mechanism for nitrogen acquisition by grass seedlings through oxidation of symbiotic bacteria. Symbiosis 57:161–171. https://doi.org/10.1007/s13199-012-0189-8 White JF, Torres MS, Somu MP, Johnson H, Irizarry I, Chen Q, Zhang N, Walsh E, Tadych M, Bergen M (2014) Hydrogen peroxide staining to visualize intracellular bacterial infections of seedling root cells. Microsc Res Tech 77:566–573. https://doi.org/10.1002/jemt.22375 White JF, Chen Q, Torres MS, Mattera R, Irizarry I, Tadych M, Bergen M (2015) Collaboration between grass seedlings and rhizobacteria to scavenge organic nitrogen in soils. AoB Plants 7: plu093. https://doi.org/10.1093/aobpla/plu093

20

J. F. White et al.

White JF, Kingsley KL, Kowalski KP, Irizarry I, Micci A, Soares MA, Bergen MS (2017) Disease protection and allelopathic interactions of seed-transmitted endophytic pseudomonads of invasive seed grass (Phragmites australis). Plant Soil 422:195–208. https://doi.org/10.1007/s11104-0163169-6 White JF, Kingsley K, Harper CJ, Verma SK, Brindisi L, Chen Q, Chang X, Micci A, Bergen M (2018a) Reactive oxygen defense against cellular endoparasites and the origin of eukaryotes. In: Krings M, Harper CJ, Cuneo NR, Rothwell GW (eds) Transformative paleobotany: Papers to commemorate the life and legacy of Thomas N. Taylor. Elsevier, Amsterdam White JF, Kingsley KL, Verma SK et al (2018b) Rhizophagy cycle: an oxidative process in plants for nutrient extraction from symbiotic microbes. Microorganisms 6(3):95. https://doi.org/10. 3390/microorganisms6030095 White JF, Torres MS, Verma SK, Elmore MT, Kowalski KP, Kingsley KL (2019) Evidence for widespread microbivory of endophytic bacteria in roots of vascular plants through oxidative degradation in root cell periplasmic spaces. Pages 167–193. In: Kumar A, Singh A, Singh V (eds) PGPR amelioration in sustainable agriculture: food security and environmental management. Elsevier

2

Thinking About PPFM Bacteria as a Model of Seed Endophytes: Who Are They? Where Did They Come from? What Are They Doing for the Plant? What Can They Do for Us? Mark A. Holland

Contents 2.1 Fooled by a Bacterium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Recognizing the Significant Role of Bacteria in Plant Metabolism . . . . . . . . . . . . . . . . . . . . . . . 2.3 Looking at Seed Endophytes Through a Pink-Pigmented Facultatively Methylotrophic Lens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1 So Where Does This Relationship Come from? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2 What Are They Doing for Seeds? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.3 What Are They Doing for the Plants? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.4 A Role for Seed Endophytes in Plant Improvement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.5 As an Aside: Endophytes Influence the Quality of Seeds as Food Items for Us . . . 2.4 Outstanding Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

22 22 23 23 25 25 28 29 29 30 31

Abstract

Bacteria in the genus Methylobacterium (PPFM bacteria) are distributed globally and are associated with algae, mosses, ferns, liverworts, gymnosperms and angiosperms as co-evolved symbionts. As such, they share significantly in plant metabolism, stimulating plant growth and development through the production of plant hormones and vitamins. This chapter discusses the PPFMs as model plant symbionts and considers how their symbiotic relationship with plants and be exploited to our benefit. Keywords

Methylobacterium spp. · PPFM bacteria · Plant/microbe symbiosis

M. A. Holland (*) Department of Biology, Salisbury University, Salisbury, MD, USA e-mail: [email protected] # Springer Nature Switzerland AG 2019 S. K. Verma, J. F. White, Jr (eds.), Seed Endophytes, https://doi.org/10.1007/978-3-030-10504-4_2

21

22

2.1

M. A. Holland

Fooled by a Bacterium

My interest in seed endophytes comes from a time when I was fooled by them. In the summer of 1988, I was a postdoctoral fellow studying the urease enzymes of soybean in Joe Polacco’s lab at the University of Missouri. The lab had identified two distinct urease isozymes by genetic means, and crosses were made to produce urease knockouts. Surprisingly, the knockouts retained a urease activity. Was this a third isozyme? The possibility was unlikely given the comprehensive screening for urease mutants that the lab had just carried out. We were still wondering about how we could have missed the third soy urease when we heard a talk by plant pathologist, John Dunleavy (1988). He described finding a methylotrophic bacterium on the leaf surfaces of corn and soybean plants distributed ubiquitously across Iowa. Of significance to us, he mentioned that the bacterium produced a urease enzyme. This was a true “aha” moment that left us wondering whether this bacterium could be the source of the third urease. The bacterium was Methylobacterium, known in the literature as a PPFM (pink-pigmented facultative methylotroph: Corpe and Basile 1982; Corpe and Rheem 1989). Returning to the lab, I ground up some soybeans and leaves and plated them out on a selective medium to look for the PPFMs. Of course, they were abundant on all of our plates and easily recognized by their bright pink color. It took a while for us to convince ourselves that the urease produced by these bacteria was the mysterious third urease in soy, not because the two activities were not biochemically identical; they were. What took time was convincing ourselves that this enzyme activity produced by a bacterium associated with the plant could be expressed at a level that allowed it to masquerade as a plant enzyme. We had been fooled by a bacterial endophyte (Holland and Polacco 1992).

2.2

Recognizing the Significant Role of Bacteria in Plant Metabolism

I grew up with the idea that seeds, fruits, and the internal tissues of plants were sterile. The notion that internal tissues of plants are sterile traces back to the experiments of Louis Pasteur, who demonstrated that grape juice drawn from the fruit with a sterile needle did not ferment like juice from crushed grapes. His conclusion was that there were no bacteria or fungi under the skin of the fruit. This idea ran counter to results obtained by Galippe, whose experiments concluded that bacteria from the soil were a source of bacteria on all fruits and vegetables (reviewed in Compant et al. 2012), but such was the reputation of Pasteur that his results influenced generations of botanists and botany texts, while Galippe’s results were buried. One of Pasteur’s students, Fernbach, even carried out experiments on a variety of fruits and vegetables to confirm his mentor’s results. Although he routinely found bacteria associated with the plants he examined, he passed them off as contaminants of what must otherwise have been sterile tissue (Fernbach 1888). Given the idea that bacteria are no more than insignificant contaminants on plants,

2

Thinking About PPFM Bacteria as a Model of Seed Endophytes: Who Are They?. . .

23

it is no wonder that it was difficult for us to accept the idea that a seed-transmitted bacterium could influence the metabolic profile of its soybean host.

2.3

Looking at Seed Endophytes Through a Pink-Pigmented Facultatively Methylotrophic Lens

Additional experiments with the PPFM bacteria led us to ask, “Is there more to plant physiology than just plant?” (Holland and Polacco 1994) and we have been trying to answer that question ever since. Of course, the PPFMs are not the only seed endophytes. One current review that shows the great diversity of microbes associated with seeds is Shahzad et al. (2018), but much of what we know comes from the work with PPFMs, and much of this chapter is devoted to them as model seed endophytes and plant symbionts.

2.3.1

So Where Does This Relationship Come from?

The PPFM bacteria are distributed in seeds of many (if not all) species of plants (Corpe and Basile 1982; Corpe 1985). This ubiquitous distribution in itself suggests that the relationship between plants and PPFMs must be a long-standing one. Bacteria must have been associated with plants from the start of their evolutionary lineage (discussed by Hassani et al. 2018). In fact, PPFMs are found in association with both freshwater and marine algae; they are abundant in freshwater and marine environments wherever algae are present (Fig. 2.1). Significantly, the PPFMs both produce and secrete vitamin B12, for which many algal species are auxotrophic (Basile et al. 1985; Croft et al. 2005; Helliwell et al. 2011; Grossman 2017), and this alone may be the basis for a symbiosis. The PPFMs also produce auxin (Doronina et al. 2002), cytokinin (Freyermuth et al. 1996; Koenig et al. 2002; Holland et al. 2002), and gibberellin (Siddikee et al. 2010) and inhibit the production of ethylene by producing an ACC deaminase (Madhaiyan et al. 2006), all of which influence algal growth. In my lab, co-cultivation of the algal feedstocks used in aquaculture with PPFMs resulted in faster growth of the cultures and improved nutritional quality (Kelly 2015). Cultures grown with the PPFMs produced 35% more protein and 100% more fatty acids. Early terrestrial plants also harbor PPFM populations. Basile et al. (1969) demonstrated a growth-promoting relationship between the PPFMs and a liverwort. The PPFMs also have been described in mosses as contributing to gametophore production (reviewed by Pohjanen et al. 2014). We have isolated them by “leaf printing” from mosses and ferns as well as from angiosperms (Holland et al. 2000). So it is easy to imagine that PPFM bacteria accompanied gymnosperm and angiosperm lines through evolutionary time. The contribution of symbiotic bacteria to plant evolution is discussed by Barrow et al. (2008). Johnston-Monje and Raizada (2011) consider the fate of seed endophytes in Zea over evolutionary time and in different environments.

24

M. A. Holland

Fig. 2.1 Top. Water from the Sinepuxent Bay, MD, USA, was filtered through a 0.2 micron filter (millipore) and the filter placed on ammonium mineral salts medium (ATCC # 784) to encourage the growth of PPFM bacteria. The bacteria are abundant in waters where microalgae are abundant. Bottom. PPFM bacteria growing on a “leaf print” of Fucus collected at Ocean City, MD, USA. The medium is ammonium mineral salts and the method is described in Holland et al. (2000)

The PPFMs, like some other seed endophytes, are vertically transmitted through seed residing under the seed coat (Mundt and Hinkle 1976; Holland 1997; Amer. Soc. For Micro. 2014; Truyens et al. 2015; Frank et al. 2017). This provides a reliable inoculum for the germinating seed, and the PPFM bacteria are maintained on plant surfaces as the plant grows. Such reliably inherited bacteria have been called “quasi-organelles” (White et al. 2012). Is it important that they be carried under the seed coat? At least in the angiosperms, seeds enclosed in a fleshy fruit, consumed by fructivores, might otherwise lose their bacteria as they pass through the animal gut. Packing them under the seed coat makes their survival more likely (Briand and Holland 1999). We also recognize that PPFM bacteria must cycle into the soil with dead plant material. They can be isolated readily from any soils that contain organic matter. From the soil, they can be washed into fresh and ultimately marine environments and might be horizontally transmitted to the phylloplane associated with airborne soil particles or aerosols (Omer et al. 2004).

2

Thinking About PPFM Bacteria as a Model of Seed Endophytes: Who Are They?. . .

2.3.2

25

What Are They Doing for Seeds?

To answer the question “What are they doing for the seeds?,” we cured the seeds of their PPFMs. Our standard lab treatment consists of heating the seeds in a dry oven at 50  C of 48 h (Rodriguez Periera et al. 1972). This reduces the PPFM population to about 3% of the untreated controls (Holland and Polacco 1992). Cured seeds show reduced germination and abnormal growth, but normal germination and growth are restored by inoculation with a culture of the PPFM bacteria (Fig. 2.2). These experiments suggest that sometimes, when seeds fail to germinate, it is only because their bacterial population has died. We tested this possibility by obtaining samples of fresh and aged seeds of the same genotype of safflower, Carthamus tinctorius, from the National Seed Storage Laboratory in Fort Collins, CO. The fresh seeds were germinating at 75%, while germination of the aged seed had fallen to 18%. When the seeds were inoculated with PPFMs, fresh seeds germinated at 87%, and the aged seeds germinated at 38%. We have since repeated this experiment numerous times using aged seeds with poor germination obtained from the Maryland Department of Agriculture Seed Testing Laboratory. Representative results of one such experiment are shown in Fig. 2.3. The application of PPFM bacteria to seed as a remedy for poor germination or as treatment to improve longevity of seeds in storage was the subject of US Patent #5,512,069 (Holland and Polacco 1996). Effects on seed germination mediated by bacterial endophytes have been demonstrated in many different plants by many different endophytes (Samova et al. 2001; Gunfel et al. 2007; Cruz et al. 2014; Sudre and Akiba 2015; Zhu et al. 2017). It is interesting, however, that the mechanism for the stimulatory effect is not clear. We recently made a list of all the compounds known to be secreted by the PPFMs, including phytohormones, vitamins, and amino acids, and tested to see whether any of them singly, in the absence of the bacteria, stimulated germination. A small effect was produced by each of them. When we tested them in combinations of two, the effect was much more pronounced. Interestingly, when we introduced PPFM bacteria into the mix, germination and growth were increased well beyond any of the other treatments (Fig. 2.4; Holland, unpublished). Obviously, we have not nailed down the magic stimulant; this work continues.

2.3.3

What Are They Doing for the Plants?

Phytohormones produced by the endophytes have a decided effect. One of my students demonstrated that the concentration of tissue-extractable cytokinin in soy seedlings is highly correlated with the size of the PPFM population on the plant (Butler 2001). Another showed that soybeans could be boosted to higher yields under conditions of drought and heat stress by a foliar application of the PPFMs during seed set (Munsanje et al. 1998; Munsanje 1999). Others have demonstrated antibacterial or antifungal properties of seed endophytes (Barret et al. 2016; Hererra et al. 2016; Khalaf and Raizada 2018), mitigation of stress on contaminated soils (Shuang et al. 2015; Sanchez-Lopez et al. 2018), amelioration of drought stress (Delshadi et al. 2017), and

26

M. A. Holland

Fig. 2.2 Top. Soybean seedling from seed “cured” of its bacteria by heating at 50  C in a dry oven for 48 h. Bottom. “Cured” soybean seeds germinating without PPFM bacteria (top center) compared to control (top left). Seeds germinating after having PPFM bacteria restored (bottom center)

2

Thinking About PPFM Bacteria as a Model of Seed Endophytes: Who Are They?. . .

27

Fig. 2.3 Top. Soybean seeds aged 3 years and germinating at 41%. Bottom. Soybean seeds, the same seed lot as above, aged 3 years but inoculated with PPFM bacteria and germinating at 93%

growth promotion at low temperature (Yarzabal et al. 2018). We have characterized the PPFMs as waste managers, first stimulating growth and then consuming methanol generated during cell division from the demethylation of cell wall pectin by pectin methylesterase and other waste products; there is also evidence that they play give and take in plant nitrogen metabolism (Holland 2011). Some PPFM species have been shown to nodulate their plant host and fix atmospheric nitrogen (Sy et al. 2001). There is little doubt that seed endophytes in general and the PPFMs in particular contribute to generalized growth promotion and plant health (Pitzschke 2016; Walitang et al. 2017; Rahman et al. 2018).

ä Fig. 2.2 (continued) compared to untreated seeds with additional PPFMs added (bottom left). Seeds to the far right (top and bottom) show the effect of extended heat treatment

28

M. A. Holland

Fig. 2.4 Top left. Germination of aged collard seeds, Brassica oleracea. Top right. Germination of collard seeds inoculated with B12-overproducing mutant PPFM bacteria. Bottom left. Germination of collard seeds inoculated with B12-overproducing mutant PPFM plus additional B12 (5 mL at 10 ng/L). Bottom right. Germination of collard seeds inoculated with B12-overproducing mutant PPFM plus additional cobalt (5 mL as CoCl2 at 160 μg/L)

2.3.4

A Role for Seed Endophytes in Plant Improvement

Morsy in 2015 and Gopal and Gupta in 2016 suggested that seed endophytes represent a potential “bio-boom” and that they might be part of a “next-generation plant breeding strategy.” Seed endophytes, as already mentioned, can be introduced by inoculation of seed with the bacterial culture or with a suspension of washed cells (Holland and Polacco 1992). Mitter et al. (2012, 2017) teach a method for introducing endophytes at flowering to progeny seeds. Examples of applications of seed endophytes to plant improvement are given in a review by Wu et al. (2009), in sunflower by Jalilian et al. (2012), in food production by Bokulich et al. (2016), in a variety of crop plants by O’Callaghan (2016), and in coffee by Vaughan et al. (2015). Technology developed in my lab involving the application of the PPFM bacteria to crop plants with the aim of improving growth and yield is the basis for

2

Thinking About PPFM Bacteria as a Model of Seed Endophytes: Who Are They?. . .

29

several US patents (Joshi and Holland 1999, 2001a, b). The use of transgenic microbes as seed endophytes to modify the enzyme profile of a plant, thus altering its metabolism, has also been suggested (Polacco and Holland 1993).

2.3.5

As an Aside: Endophytes Influence the Quality of Seeds as Food Items for Us

Metabolites produced by seed endophytes can enhance the nutritional quality of food crops. In barley, for example, the vitamin content, nutritional quality, and enzyme activity of the grain have been attributed to seed microbes (Yousaf et al. 2015, 2017). The B12 content of daikon has been elevated by absorption from the seeds (Sato et al. 2004). Seed endophytes also have been manipulated to improve the yield and antioxidant content of strawberry (Rahman et al. (2018) and the quality of other horticultural crops (Jimenez-Gomez et al. 2017). In my lab, the PPFM bacteria have been shown to influence cytokinin content of plant tissues (Butler 2001). We have also shown that they can improve the amino acid profile of soybean seed, increasing bound methionine and providing enough B12 in lettuce to meet the recommended daily allowance for that vitamin (Witzig and Holland 1998; Holland and Polacco 2006). Ongoing experiments in my lab are following a similar strategy to improve the folate content of several different crop species.

2.4

Outstanding Questions

A Mechanism I mentioned earlier that the mechanism whereby seed endophytes enhance germination is not clear. We know at least some of the compounds secreted by endophytes in culture that make a contribution to germination, but we have not accounted for the level of stimulation provided by the bacteria themselves. It is humbling to think that even if we did know which bacterial products were responsible for the effect, we might still not know the mechanism by which they work at a cellular level. So much of the biology surrounding germination is still a black box in plant physiology. Selectivity The phylloplane of plants is inhabited by a myriad of bacterial genera. As seeds are produced, is there some selective mechanism at work that determines which of those bacteria end up in the seed? The question has been approached regarding pathogenic microbes by Barret et al. (2015, 2016). Beneficial microbes that are seedtransmitted were considered in a recent review by Shahzad et al. (2018). The PPFM bacteria are slow-growing and only sluggishly motile. Note that although we have never failed to find them associated with seed, if they are not isolated on a selective medium, they likely will be overgrown by competitors and will go unnoticed. These characteristics suggest that they might be unlikely to colonize seeds. Our experience, however, tells us that they are reliably seed-transmitted. If some selection guarantees this, it remains unknown.

30

M. A. Holland

A Mystery Surrounding B12 Vitamin B12 is a molecule required in the metabolism of many organisms, but produced by only a few. As such, it has been recognized as a shared commodity that can influence the shape of microbial communities (Romine et al. 2017; Taylor and Sullivan 2008). Many of the micro- and macroalgal relatives of terrestrial plants are B12 auxotrophs (Croft et al. 2005; Helliwell et al. 2015). In my lab, we have isolated a B12-overproducing and B12-secreting PPFM mutant (Witzig and Holland 1998) and have demonstrated a marked stimulation in the growth and nutritional composition of microalgae when they are co-cultivated with these PPFM bacteria (Kelly 2015). We have isolated PPFMs from water inhabited by microalgae and from macroalgae using the leaf print technique (Fig. 2.1; Holland et al. 2000). The relationship between the algae and the PPFMs appears depending, at least in part, on the production of the vitamin by the bacterium. B12 appears again as a signaling molecule for growth and gametophore production in liverwort (Basile et al. 1985) and moss (see Pohjanen et al. 2014). So what is this “mystery” surrounding B12? Plants and fungi are presumed to live in a world without B12. Neither group is thought to require the vitamin. Yet it appears to be essential in establishing the relationship between Sinorhizobium meliloti and its host plant (Taga and Walker 2010). Lawrence et al. (2018) have shown that fluorescent analogs of cobalamin are taken up by the roots of Lepidium sativum (Brassicaceae) and are incorporated into the vacuoles of leaf. In a previously unpublished experiment from my lab looking at B12 effects on the germination of Brassica oleracea (collard), the B12-overproducing PPFMs alone stimulate germination and growth, but when supplemented with additional B12, the effect is orders of magnitude greater (Fig. 2.4). Other species of seed have given similar results. Does this work reveal an unsuspected role for B12 in plants? Is it possible that B12 is just a vehicle for the delivery of cobalt, an element that has been suggested to be an essential nutrient for some plants? Treating collard seed with B12 PPFMs supplemented with cobalt gives a result similar to that seen with the B12 PPFMs supplemented with B12 (Fig. 2.4). The question of why B12 should stimulate germination at all remains to be answered.

2.5

Conclusions

The important role of seed endophytes as coevolved partners in plant biology has only recently been recognized. Taking what we know of the PPFM bacteria as a model bacterial endophyte, we can assign to seed endophytes in general: effects on seed germination, plant growth and yield, disease resistance, and drought and stress tolerance. We have also imagined ways in which the PPFMs and other seed endophytes can be used for plant improvement (Holland 2016). It will be interesting to see what future research in this area brings.

2

Thinking About PPFM Bacteria as a Model of Seed Endophytes: Who Are They?. . .

31

References American Society for Microbiology (2014) Plants prepackage beneficial microbes in their seeds. Science Daily. www.sciencedaily.com/releases/2014/09/140929180055.htm Barret M, Briand M, Bonneau S, Preveaux A, Valiere S, Bouchez O, Hunault G, Simoneau P, Jacques M-A (2015) Emergence shapes the structure of the seed microbiota. Appl Environ Microbiol 81 (4):1257–1266 Barret M, Guimbaud JF, Darrasse A, Jaques MA (2016) Plant microbiota affects seed transmission of phytopathogenic microorganisms. Mol Plant Pathol 17(6):791–795. https://doi.org/10.1111/ mpp.12382 Barrow JR, Lucero ME, Reyes-Vera I, Havstad KM (2008) Do symbiotic microbes have a role in plant evolution, performance and response to stress? Commun Integr Biol 1(1):69–73 Basile DV, Slade LL, Corpe WA (1969) A association between a bacterium and a liverwort, Scapania nemorosa. Bull Torrey Bot Club 96(6):711–714 Basile DV, Basile MR, Li QY, Corpe WA (1985) Vitamin B12-stimulated growth and development of Jungermannia leiantha Grolle and Gymnocolea inflata (Huds.) Dum. (Hepaticae). Bryologist 88(2):77–81 Bokulich NA, Lewis ZT, Boundy-Mills K, Mills DA (2016) A new perspective on microbial landscapes within food production. Curr Opin Biotechnol 37:182–189. https://doi.org/10.1016/ j.copbio.2015.12.008 Briand CH, Holland MA (1999) Microbial symbionts and the evolution of fruit. In: Presented at the annual meeting of the American Society of Plant Physiologists, Baltimore, MD, 24–28 July 1999 Butler HSK (2001) Contribution of PPFM (Methylobacterium mesophilicum), a bacterial symbiont, to cytokinin content and biomass accumulation in soybean [Glycine max (L.) Merr.] seedlings. Masters Thesis. University of Maryland Eastern Shore, Princess Anne, MD Compant S, Sessitsch A, Mathieu F (2012) The 125th anniversary of the first postulation of the soil origin of endophytic bacteria – a tribute to MLV Galippe. Plant Soil. https://doi.org/10.1007/ s11104-012-1204-9 Corpe WA (1985) A method for detecting methylotrophic bacteria on solid surfaces. J Microbiol Methods 3:215–321 Corpe WA, Basile DV (1982) Methanol utilizing bacteria associated with green plants. Dev Ind Microbiol 23:483–493 Corpe WA, Rheem S (1989) Ecology of the methylotrophic bacteria living on leaf surfaces. FEMS Micobiol Ecol 62:243–250 Croft MT, Lawrence AD, Raux-Deery E, Warren MJ, Smith AG (2005) Algae acquire vitamin B12 through a symbiotic relationship with bacteria. Nature 438(7064):90–93 Cruz RS, Yanez-Ocampo G, Wong-Villareal A (2014) Effect of nodulating bacteria on the seed germination of Capsicum spp. Afr J Microbiol Res 8(7):659–663. https://doi.org/10.5897/ AJMR2013.6494 Delshadi S, Ebrahimi M, Shirmohammadi E (2017) Influence of plant-growth-promoting bacteria on germination, growth and nutrients’ uptake of Onobrychis sativa L. under drought stress. J Plant Interact 12(1):200–208. https://doi.org/10.1080/17429145.2017.1316527 Doroninqa NV, Ivanova EG, Trotsenko I (2002) New evidence for the ability of methylobacteria and methanotrophs to synthesize auxins. Microbiology 71:116–118 Dunleavy JM (1988) In vitro expression of the cellulose gene in Methylobacterium mesophilicum, a seed-transmitted bacterium ubiquitous in soybean. In: Presented at 2nd biennial conference on the molecular and cellular biology of the soybean, Ames, IA, 25–27 July 1988 Fernbach MA (1888) De l’absence des microbes dans les tissus vegetaux. Ann Inst Past 2(10):567 Frank AC, Guzmain JPS, Shay JE (2017) Transmission of bacterial endophytes. Microorganisms 5:70. https://doi.org/10.3390/microorganisms5040070 Freyermuth SK, Long RL, Mathur S, Holland MA, Holtsford TP, Stebbins NE, Morris RO, Polacco JC (1996) Metabolic aspects of plant interaction with commensal methylotrophs. In: Lidstrom M, Tabita R (eds) Microbial growth on C1 compounds. Kluwer Academic, pp 277–284

32

M. A. Holland

Gopal M, Gupta A (2016) Microbiome selection could spur next-generation plant breeding strategies. Front Microbiol 7:1971. https://doi.org/10.3389/micb.2016.01971 Grossman A (2017) Nutrient acquisition: the generation of bioactive vitamin B12 by microalgae. Curr Biol 26:R319–R337. https://doi.org/10.1016/j.cub.2016.02.047 Gunfel PE, Landesmann JB, Martinez-Ghersa MA, Ghersa CM (2007) Effects of Neotyphoduim endophyte infection on seeds viability and germination vigor in Lolium multiflorum under accelerated aging conditions. New Zealand Grassland Association: Endophyte symposium. https:// www.grassland.org.nz/publications/nzgrassland_publication_2363.pdf Hassani MA, Duran P, Hacquardo S (2018) Microbial interactions within the plant holobiont. Microbiome 6:58. https://doi.org/10.1186/s40168-018-0445-0 Helliwell KE, Wheeler GL, Leptos KC, Goldstein RE, Smith AG (2011) Insights into the evolution of vitamin B12 auxotrophy from sequenced algal genomes. Mol Biol Evol 28(10):2921–2933. https://doi.org/10.1093/molbev/msr124 Helliwell KE, Collins S, Kazamia E, Purton S, Wheeler GL, Smith A (2015) Fundamental shift in vitamin B12 eco-physiology of a model alga demonstrated by experimental evolution. ISME J 9:1446–1455. https://www.nature.com/articles/ismej2014230 Herrera SD, Grossi C, Zawoznik M, Groppa MD (2016) Wheat seeds harbor bacterial endophytes with potential as plant growth promoters and biocontrol agents of Fusarium graminearum. Microbiol Res 186–187:37–43 Holland MA (1997) Methylobacterium and plants. Rec Res Dev Plant Phys 1:207–213 Holland MA (2011) Nitrogen: give and take from phylloplane microbes. In: Polacco JC, Todd CD (eds) Ecological aspects of nitrogen metabolism in plants. Wiley-Blackwell, London, pp 217–230. ISBN 978-0-8138-1649-4 Holland MA (2016) Probiotics for plants? What the PPFMs told us and some ideas about how to use them. J Wash Acad Sci 102(1):31–42 Holland MA, Polacco JC (1992) Urease-null and hydrogenase-null phenotypes of a phylloplane bacterium reveal altered nickel metabolism in two soybean mutants. Plant Physiol 98:942–948 Holland MA, Polacco JC (1994) PPFMs and other covert contaminants: is there more to plant physiology than just plant? Annu Rev Plant Physiol Plant Mol Biol 45:197–209 Holland MA, Polacco JC (1996) Seeds, coated or impregnated with a pink pigmented facultative methylotroph, having improved germinability. U.S. Patent # 5,512,069 Holland MA, Polacco JC (2006) A method for altering the metabolism of a plant. U.S. Patent # 8,153,118 Holland MA, Davis R, Moffitt S, O’Laughlin K, Peach D, Sussan S, Wimbrow L, Tayman B (2000) Using “leaf prints” to investigate a common bacterium. Am Biol Teach 62(2):128–131 Holland MA, Long RLG, Polacco JC (2002) Methylobacterium spp.: Phylloplane bacteria involved in cross-talk with the plant host? In: Lindow SE, Hecht-Poinar EI, Elliot VJ (eds) Phyllosphere microbiology. APS Press, St. Paul, MN, pp 125–135 Jalilian J, Modarres-Sanavy SAM, Saberali SF, Sadat-Asilan K (2012) Effects of the combination of beneficial microbes and nitrogen on sunflower seed yields and seed quality traits under different irrigation regimes. Field Crop Res 127:26–34. https://doi.org/10.1016/j.fcr.2011.11.001 Jimenez-Gomez A, Celador-Lera L, Fradejas-Bayon M, Rivas R (2017) Plant probiotic bacteria enhance the quality of fruit and horticultural crops. AIMS Microbiol 3(3):483–501. https://doi. org/10.3934/miocrobiol.2017.3.483 Johnston-Monje D, Raizada M (2011) Conservation and diversity of seed associated endophytes in Zea across boundaries of evolution, ethnography and ecology. PLoS One 6(6):e20396 Joshi JM, Holland MA (1999) Method for treating plants. U.S. Patent # 5,961,687 Joshi JM, Holland MA (2001a) Method for treating plants. US Patent # 6,174,837 Joshi JM, Holland MA (2001b) Method for treating plants. U.S. Patent # 6,329,320 Kelly SM (2015) Altering growth rates and nutritional qualities of microalgal feedstocks with symbiotic bacteria. Masters Thesis. Salisbury University, Salisbury, MD

2

Thinking About PPFM Bacteria as a Model of Seed Endophytes: Who Are They?. . .

33

Khalaf EM, Raizada MN (2018) Bacterial seed endophytes of domesticated cucurbits antagonize fungal and oomycete pathogens including powdery mildew. Front Microbiol 9:42. https://doi. org/10.3389/micb.2018.00042 Koenig RL, Morris RO, Polacco JC (2002) tRNA is the source of low-level trans-zeatin production in Methylobacterium spp. J Bacteriol 184:1832–1842 Lawrence AD, Nemoto-Smith E, Deery E, Boshoff HI, Barry CE III, Warren MJ (2018) Construction of fluorescent analogs to follow the uptake and distribution of cobalamin (Vitamin B12) in bacteria, worms, and plants. Cell Chem Biol. https://doi.org/10.1016/j.chembiol.2018.04.012 Madhaiyan M, Poonguzhali S, Ryu J, Sa T (2006) Regulation of ethylene levels in canola (Brassica campestris) by 1-aminocyclopropane-1-carboxylate deaminase-containing Methylobacterium fujisawaense. Planta 224(2):268–278 Mitter B, Sessitsch A, Naveed M (2012) Method for producing plant seed containing endophytic micro-organisms. Patent Application EP267536A1 Mitter B, Pfaffenbichler N, Flavell R, Compant S, Antonielli L, Petric A, Berninger T, Naveed M, Sheibani-Tezerji R, vonMaltzahn G, Sessitsch A (2017) A new approach to modify plant microbiomes and traits by introducing beneficial bacterial at flowering into progeny seeds. Front Microbiol 8:11. https://doi.org/10.3389/fmicb.2017.00011 Morsy M (2015) Microbial symbionts: a potential bio-boom. J Investig Genom 2(1):00015 Mundt JO, Hinkle NF (1976) Bacteria within ovules and seeds. Appl Environ Microbiol 32 (5):694–698 Munsanje EM (1999) Potential of a cytokinin-excreting Methylobacterium as a biofertilizer in soybean production. PhD Dissertation. University of Maryland Eastern Shore, Princess Anne, MD Munsanje EM, Holland MA, Joshi JM (1998) The significance of PPFM foliar spray on soybean yield. In: Dadson RB, Noureldin RA (eds) Soybeans in Egypt: research, production, economics, nutrition and health. University of Maryland Press, Bethesda, MD, pp 69–78 O’Callaghan M (2016) Microbial inoculation of seed for improved crop performance: issues and opportunities. Appl Microbiol Biotechnol 100:5729–5746. https://doi.org/10.1007/s00253-0167590-9 Omer ZS, Tombolini R, Gerhardson B (2004) Plant colonization by pink-pigmented facultative methylotrophic bacteria (PPFMs). FEMS Microbiol Ecol 47:319–326 Pitzschke A (2016) Developmental peculiarities and seed-borne endophytes in quinoa: omnipresent, robust bacilli contribute to plant fitness. Front Micro 7:article 2. https://doi.org/10.3389/ fmicb.2016.00002 Pohjanen J, Koskimaki JJ, Pirttila AM (2014) Interactions of meristem-associated endophytic bacteria. In: Verma VJ, Gange AC (eds) Advances in endophytic research. Springer. https:// doi.org/10.1007/978-81-322-1575-2 Polacco JC, Holland MA (1993) A method for altering the metabolism of a plant. U.S. Patent # 5,268,171 Rahman M, As Sabir A, Mukta JA, Khan MMA, Mohi-Ud-Din M, Miah MG, Rahman M, Islam MT (2018) Plant probiotic bacteria Bacillus and Paraburkholderia improve growth, yield and content of antioxidants in strawberry fruit. Sci Rep 8:2504. https://doi.org/10.1038/s41598-01820235-1 Rodrigues Pereira AS, Houwen PWJ, Deurenberg-Vos HWJ, Pey EBF (1972) Cytokinins and the bacterial symbiosis os Ardisia species. Z Pflanzenphysiol 68:170–177 Romine MF, Rodionov DA, Maezato Y, Andersona LN, Nandhikonda P, Rodionova IA, Carred A, Li X, Xu C, Clauss TRW, Kim YM, Metz TO, Wright AT (2017) Elucidation of roles for vitamin B12 in regulation of folate, ubiquinone, and methionine metabolism. Proc Natl Acad Sci USA. doi:https://doi.org/10.1073/pnas.1612360114 Samova LA, Pechurkin NS, Sarangove AB, Pisman TI (2001) Effect of bacterial population density on germination wheat seeds and dynamics of simple artificial ecosystems. Adv Space Res 27 (9):1611–1615

34

M. A. Holland

Sanchez-Lopez AS, Pintelon I, Stevens V, Imperato V, Timmermans JP, Gonzalez-Chavez C, Carillo-Gonzalez R, Van Hamme J, Vangronsveld J, Thijs S (2018) Seed endophyte microbiome of Crotalaria pumila unpeeled: identification of plant-beneficial methylobacteria. Int J Mol Sci 19:291. https://doi.org/10.3390/jims19010291 Sato K, Kudo Y, Muramatsu K (2004) Incorporation of a high level of vitamin B12 into a vegetable, kaiware daikon (Japanese radish sprout), by the absorption from its seeds. Biochim Biophys Acta 1672:135–137 Shahzad R, Khan AL, Bilal S, Asaf S, Lee I (2018) What is there in seeds? Vertically transmitted endophytic resources for sustainable improvement in plant growth. Front Plant Sci 9:24. https:// doi.org/10.3389/fpls.2018.00024 Shuang S, Zhenfang G, Xiaolei G (2015) The effect of bacteria on seed germination in sorghum and rape under cadmium and petroleum conditions. Int J Biotechnol Wellness Indus 4:123–127 Siddikee A, Hamayun M, Han G-H, Sa T-m (2010) Optimization of gibberellic acid production by Methylobacterium oryzae CBMB20 Md. Korean J Soil Sci Fert 43(4):522–527 Sudre C, Akiba F (2015) Influence of effective microorganisms on seed germination and plantlet vigor of selected crops. https://www.researchgate.net/publication/265524659 Sy A, Giraud E, Jourand P, Garcia N, Willems A, De L Prin Y, Neyra M, Gillis M, Boivin M, Dreyfus B (2001) Methylotrophic Methylobacterium bacteria nodulate and fix nitrogen in symbiosis with legumes. J Bacteriol 183:214–220 Taga ME, Walker GC (2010) Sinorhizobium meliloti requires a cobalamin-dependent ribonucleotide reductase for symbiosis with its plant host. MPMI 23(12):1643–1654. https://doi.org/10. 1094/MPMI-07-10-0151 Taylor GT, Sullivan CW (2008) Vitamin B12 and cobalt cycling among diatoms and bacteria in Antarctic sea ice microbial communities. Limnol Oceanogr 53(5):1862–1877 Truyens S, Weyens N, Cuypers A, Vangronsveld J (2015) Bacterial seed endophytes: genera, vertical transmission and interaction with plants. Environ Microbiol Rep 7(1):40–50 Vaughan MJ, Mitchell T, Mc Spadden Gardener BB (2015) What’s inside the bean we brew? A new approach to mining the coffee microbiome. Appl Environ Microbiol 81(19):6518–6527. https://doi.org/10.1128/AEM.01933-15 Walitang DI, Kim K, Madhaiyan M, Kim YK, Kang Y, Sa T (2017) Characterizing endophytic competence and plant growth promotion of bacterial endophytes inhabiting the seed endosphere of rice. BMC Microbiol 17:209. https://doi.org/10.1186/s12866-1117-0 White JF Jr, Johnson H, Torres MS Irizarry I (2012) Nutritional endosymbiotic systems in plants: bacteria function like “quasi-organelles” to convert atmospheric nitrogen into plant nutrients. J Plant Pathol Microb 3:7. https://doi.org/10.4172/2157-7471.1000e104 Witzig SB, Holland MA (1998) A microbial symbiont used to alter the nutritional quality of plants. Presented at the annual meeting of the American Society of Plant Physiologists, Madison, WI, 27 June–2 July Wu CH, Bernard SM, Andersen GL, Chen W (2009) Developing microbe-plant interactions for applications in plant-growth promotion and disease control, production of useful compounds, remediation and carbon sequestration. Microb Biotechnol 2(4):428–440 Yarzabal LA, Monserrate L, Buela L, Chica E (2018) Antarctic Pseudomonas spp. promote wheat growth at low temperature. Polar Biol. https://doi.org/10.1007/s00300-018-2374-6 Yousaf A, Qadir A, Anjum T, Ahmad A (2015) Identification of microbial metabolites elevating vitamin contents in barley seeds. J Agric Food Chem 63:7301–7310 Yousaf A, Qadir A, Anjum T, Khan Dr RI, Naughton D, Yousaf A (2017) Evaluation of bacterial strains for the induction of plant biochemicals, nutritional contents and isozymes in barley. J Nutr Food Sci 7(5):1000623. https://doi.org/10.4172/2155-9600.1000623 Zhu YL, She XP, Wang JS, Lv HY (2017) Endophytic bacterial effects on seed germination and mobilization of reserves in Ammodendron biofolium. Pak J Bot 49(5):2029–2035

3

Seed Endophytes and Their Potential Applications Haiyan Li, Shobhika Parmar, Vijay K. Sharma, and James Francis White, Jr

Contents 3.1 3.2 3.3 3.4

Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Seed Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Assessing Seed Endophytic Communities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Roles of Seed-Borne Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1 Seed Endophytes and Plant Growth Enhancement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2 Seed Endophytes Mitigating Heavy Metal Toxicity/Stress . . . . . . . . . . . . . . . . . . . . . . . 3.5 Mechanism of the Growth Promotion and Heavy Metal Stress Tolerance . . . . . . . . . . . . . . . 3.5.1 Heavy Metal Resistance Genes Conferring Metal Resistance . . . . . . . . . . . . . . . . . . . . . 3.6 Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

36 37 40 41 42 43 44 46 47 48

Abstract

With growing interest in the role of endophyte to the host plant ecology, health, and productivity, this chapter discusses seed-inhabiting endophytes. These endophytes were less recognized when compared with those found in the other parts of the plant. However, they cannot be ignored as they are the first one colonizing young seedlings and further determining the fate of the plant. These endophytes often have potential to improve seed germination and seedling growth. Recent advances in seed endophytes have proved that they can confer stress tolerance to the host plants, especially the heavy metal resistance. Microbial dynamic equilibrium with plant systems is vital for the germination, growth, and reproductive phases of plant life cycle. The colonization and transmission of seed endophytes suggests that host plants select an endophytic community having beneficial traits that can be passed to successive generations. Seed endophytes can H. Li (*) · S. Parmar · V. K. Sharma Medical School of Kunming University of Science and Technology, Kunming, China J. F. White, Jr Department of Plant Biology, Rutgers University, New Brunswick, NJ, USA # Springer Nature Switzerland AG 2019 S. K. Verma, J. F. White, Jr (eds.), Seed Endophytes, https://doi.org/10.1007/978-3-030-10504-4_3

35

36

H. Li et al.

facilitate the improvement of seed quality and plant growth of agriculturally important crops via different biotechnological applications; they have prospects in endophyte-mediated phytoremediation applications. Keywords

Seed endophytes · Plant-microbe interaction · Plant growth promotion · Stress tolerance · Heavy metal toxicity

3.1

Background

Researchers believe that 400 million years ago when higher plants first came into existence on the earth, endophytic microbes also existed and may have facilitated plant evolution (Sun et al. 2012). The first endophytes to be described were outgrowths in wheat leaves called “exanthemata” (Unger 1833). Leveille (1846) documented these outgrowths as fungal structures in the leaves of wheat and called them “endophytic fungi” (Leveille 1846). However the term “endophytes” first of all was defined by de Bary in 1866 to symbolize all microbes that reside inside the living healthy plants. The earliest documentation of mutualistic symbiosis dates back to the Paleozoic era in the roots of the fossil tree Amyelon radicians which indicates that plant-fungal association was present (Bacon and Hill 1996). Krings et al. (2007) recorded three fungal endophytes in the thin sections of the early Devonian Rhynie chert plant Nothia aphylla that confirms the existence of endophyte 400 million years ago (Krings et al. 2007). Since then endophytic relationships may have evolved considerably. Several definitions of endophytes have been advanced by investigators as follows. Endophytes are microbes, generally bacteria or fungi, colonizing internal living tissues of plants without causing any disease symptoms (Petrini 1991; Hardoim et al. 2012; Lopez et al. 2012). Endophytes are any fungi isolated from internal symptomless plant tissues (Cabral et al. 1993). Endophytes are fungi that colonize the plant without causing any visible disease symptom at any specific moment (Schulz and Boyle 2005). Endophytes are the microbes that colonize living, internal tissues of plants without causing any immediate, overt negative effect (Bacon and White 2000). Endophyte was defined as a topographical term that includes bacteria, fungi, actinomycetes, and algae, which spend their whole or a period of life cycle either in symplast or apoplast region of healthy plant tissues without producing any disease or clinical symptoms (Kharwar et al. 2014). Ernst et al. (2003) defined endophyte establishment as a symbiotic relationship between plants and fungi. There are two types of endophyte mutualism: one is constitutive endophytic mutualism and another one is inducible endophytic mutualism. In constitutive endophytic mutualism, endophytes can flourish in infected plants, additionally vertically transmitted via seeds to next generation of the host. On the other hand, in inducible endophytic mutualism, the interior of the host tissues is severely colonized with localized infections which can evolve by numerous fungi (Carroll 1988). Endophytic microbes have attracted attention because they are

3

Seed Endophytes and Their Potential Applications

37

prospective resource of novel natural products for application in industry, medicine, and agriculture field (Strobel 2003; Smith et al. 2008; Selim et al. 2012). Endophyte-host plant interaction is a mutualistic association in which the endophyte takes nutrition and shelter in the host plant and in turn provides various benefits to host plants. It is predicted that plants which do not contain endophytes in their living tissues might be more susceptible to abiotic stress and pathogen actions (Smith et al. 2008; Timmusk et al. 2011). Endophytes can confer on host plant herbivore resistance (Bacon et al. 1977; Finch et al. 2016), invertebrate pest resistance (Ball et al. 1995; Wilkinson et al. 2000), increased drought tolerance (West 1994), disease resistance (Siegel and Latch 1991; Wiewióra et al. 2015; James and Mathew 2015), micronutrient fortification (Zhang et al. 2012a), resistance against salt stress (Naveed et al. 2014), and improved tolerance against heavy metal stress (Bonnet et al. 2000; Monnet et al. 2001; Ezzouhri et al. 2009). In addition, natural compounds secreted by endophytes were well documented by various workers; these compounds belong to various chemical groups like alkaloids, terpenoids, flavonoids, steroids, polyketides, etc. exhibiting a range of bioactivities such as antimicrobial activity, antimalarial properties, anticancer activity, insecticidal activity, and many more (Tan and Zou 2001; Strobel 2003; Strobel and Daisy 2003; Verma et al. 2009; Kharwar et al. 2011; Zhang et al. 2015). Approximately 300,000 plant species exist on earth and each may host more than one endophyte species, and few of the endophytes have been characterized (Smith et al. 2008). Endophytes have been observed in all green plants ranging from algae, bryophytes, pteridophytes, gymnosperms, and angiosperms, including underground root to all aerial parts of host (Kharwar et al. 2014). Endophytic fungi are ubiquitous and have also been isolated from host plants that are adapted to extreme environmental conditions such as water-stressed deserts, cold-stressed arctic, antarctic ocean, geothermal soils, highly diverse rain forests, dry deciduous and coastal forests, and mangrove swamps (Kharwar et al. 2014). Endophytes have been observed in many plant spheres such as rhizosphere, phyllosphere, caulosphere, laminosphere, dermosphere, carposphere, spermosphere, and anthosphere (Shahzad et al. 2018). Among all, seed endophytes are ecologically important as they are the microbial group that colonizes seeds and they are also the first microbial group that colonizes the new seedling. The present article explores the endophytic association of bacteria and fungi with the seed and its influence on germination, growth, and stress tolerance.

3.2

Seed Endophytes

In general, most endophytes originate from the epiphytic communities belonging to rhizosphere, phyllosphere, or the other plant parts, but some may be transmitted through the seed (Bacon and Hinton 2006). Seed endophytes are passed to successive plant generations through vertical transmission and thus are guaranteed to be present in the next generation of seedlings (Cope-Selby et al. 2017; Shade et al. 2017; Vujanovic and Germida 2017). Seed-borne endophytes as a concept were first

38

H. Li et al.

discussed by Baker and Smith (1966). However, initial reports of bacteria colonizing seeds date back to the 1970s (Mundt and Hinkle 1976), while symbiotic association of seed-borne fungi dates back to 1950 (Boursnell 1950). In general endophytes are present in all seeds, and if the seed germinates, all progeny seedlings inherit parent endophytes (Siegel et al. 1984). But sometimes incomplete systemic infections can also be observed within plants. In tall fescue and perennial ryegrasses, uninfected seeds were observed from the infected plants (Welty et al. 1994). Endophytic diversity in seeds is often lower than other plant parts (Ganley and Newcombe 2006; Vega et al. 2010). Seed endophytes are adapted for a symbiotic life cycle inside plants, vertically transmitted via seed at the cost of competitiveness and ability to survive in most environments outside the plant (Cope-Selby et al. 2017). “Vertical transmission” is the direct transfer of the endophyte from parent to offspring. This type of transmission should always favor mutualism against pathogenicity, while the endophyte remains dependent on host for survival and reproduction (Ewald 1987; Rudgers et al. 2009). It is still very difficult to differentiate at initial stages whether a seedinhabiting microbe is endophyte or pathogen. True seed endophytes will result in a healthy seedling, perhaps providing prenatal care to the plant. Seed endosymbiosis is a vital relationship that confers enhanced seed vigor, germination, and resilience and thus overall improved growth under stressful conditions (Vujanovic and Germida 2017). The concept of “competent endophyte” was introduced for microorganisms that successfully colonize the plant tissues and that have the capacity to stimulate plant physiology and be selectively favored, providing beneficial assistance to the plant-microbe association (Hardoim et al. 2008, 2012). Whether the selection of seed-borne endophytes is host dependent, mainly to increase the fitness of the next generation, or whether seeds are only vectors for dissemination and colonization of new environments is still a question of research (Hardoim et al. 2012). Some selective genera like Bacillus and Paenibacillus were common in cucurbit seeds, while rare in other plants, suggesting specific ecological roles (Khalaf and Raizada 2016). Truyens et al. (2013) studied culturable seed endophytes of Arabidopsis thaliana for several generations exposed to cadmium concentrations and without cadmium. Their results supported the hypothesis of selective transfer of certain endophytic species to the next generation for its vital role in subsequent germination and early seedling development (Truyens et al. 2013). Seed endophytes can play a role in seed preservation and preparation of the environment for germination, while during germination they may directly and indirectly promote plant growth and health (Nelson 2004; Truyens et al. 2015). How these endophytes interact with the host plant, genetic drivers of their niche adaption and functionality, is still not clear. Different isolates of the same species colonizing the same ecological niche can interact distinctively with the host plant. Comparative analysis of three strains of Pantoea ananatis isolated from maize seeds showed that although genomes of these strains were highly similar, there were genomic differences in genes encoding protein secretion systems and putative effectors (Sheibani-Tezerji et al. 2015).

3

Seed Endophytes and Their Potential Applications

39

Vertically transmitted endophyte, remains throughout life cycle of plant, colonize all plant parts Vertically transmitted endophyte, remains throughout life cycle of plant, show preferential colonization in some plant parts Vertically transmitted endophyte, remains throughout life cycle of plant, show preferential colonization, Exits to rhizospheric soil for its beneficial role like nutrient acquisition

Transmission of endophytes

Horizontally transferred endophyte, But selectively transferred to the next generation for its beneficial roles, colonize seed, may become vertically transmitted endophyte in subsequent generations if beneficial Horizontally transferred, via exposure to a nearby infected plant, do not colonize seed, may/may not shows preferential colonization in some tissue Horizontally transferred, from soil microbiota, do not colonize seed, may/may not shows preferential colonization in some tissue

Fig. 3.1 Transmission of endophytic microbiota to plants

There can be different modes of entry and establishment of the endophytes within seeds. Maude (1996) recognized that microbial entry to seeds are mainly via nonvascular or xylem tissues in the parental plant, by floral pathways through stigma of parental plant, and through some external pathway in which seeds are colonized from the external environment (Maude 1996). Truyens et al. (2015) discussed three main modes of endophyte transmission into seeds: (1) they can use the vascular tissue for transmission from the parent vegetative parts into the seed endosperm; (2) they can also be directly transmitted to the endosperm or embryo through gametes; and (3) they can be vertically transferred as in the case of reproductive meristems that finally give rise to ovules and seeds. Seed endophytes must possess efficient motility and means to enter and establish in the seeds (Shahzad et al. 2018). Endophytes often secrete cell wall-degrading enzymes to colonize and use nutrientrich intercellular spaces of host plants for migration, whereas endophytes that colonize at later stages must withstand high osmotic pressure of seeds often over months or years (Pitzschke 2018). Cope-Selby et al. (2017) proposed that transmission of bacterial endophytes to seeds involves adaptation of host and microbes. The mode of endophyte transmission (horizontal vs. vertical) is important and determines efficiency of transmission (Vandenkoornhuyse et al. 2015). Shade et al. (2017) suggested that mode of transmission is dependent on selection exerted by the host plant (Fig. 3.1). Some studies suggest that the rate of vertical transmission for many fungal endophytes is greater than 90% (Ngugi and Scherm 2006). Apparently vertical transmission is probably widespread and more interesting as it provides a chance to successive plant generation to utilize the beneficial attributes of selective endophytic community passed from the parent plant (Hodgson et al. 2014; Ferreira et al. 2008). Vertical transmission of seed-borne endophytes was reported in various

40

H. Li et al.

plant species. Freeman (1903) was first to describe the entire scenario of life history of Lolium temulentum and its endophyte. He observed that hyphae enter the embryo much before seed maturation, and after seed germination the endophyte establishes itself to the aboveground plant tissues later colonizing buds and inflorescences (Freeman 1903). The transmission of seed endophytic bacteria Pantoea agglomerans tagged with gfp to seedlings supported vertical transmission in Eucalyptus species (Ferreira et al. 2008). Hodgson et al. (2014) showed that vertical transmission of fungal endophytes was common in forbs and observed fungal growth with pollen tubes, suggesting this as the probable path of entry to the developing seeds. Viable fungal propagules were observed in Phragmites australis that later colonized developing seedlings suggesting vertical transmission (Ernst et al. 2003). Recently, Walitang et al. (2018) recorded similar populations of endophytic bacteria in rice seeds even after crossbreeding and repeated inbreeding of rice at different locations, indicating conservation of bacteria across generations and environments; further, they suggested that contributions of microbes from parent plants were decisive in shaping the endophytic community of successive generations of seeds. However, they did not rule out the possibility of acquiring similar populations of endophytes from the soil generation after generation. Plant genotype may also influence seed microbial community dynamics throughout germination. During radicle protrusion, certain bacterial and fungal taxa may decrease as a result of increase in relative abundance of microbiota possessing fast-growing abilities (Barret et al. 2014).

3.3

Assessing Seed Endophytic Communities

A seed not only carries genetic information for the next generation but additionally is a reservoir and vector of a microbial community (Nelson 2004, 2018). Seed-borne endophytes have been studied from a diverse range of plant species (Truyens et al. 2015; Shahzad et al. 2018). Shahzad et al. (2018) summarized endophytic microbes isolated and characterized from the seeds of some plants. Nevertheless, most studies were limited to culture-based techniques and some DNA fingerprinting methodologies. Evaluation of the seed endophytic communities is very important for its ecological and functional aspects. Factors that control the seed endophytic community are important for biotechnological applications (Truyens et al. 2016). Understanding the richness that the seed microbiome brings to the promotion of plant growth becomes an important stepping stone in our comprehension of plant development, as the microbiome resides on the interface between crop health and(or) productivity and the associated environment (Vujanovic and Germida 2017). Traditionally, surface sterilization of the seeds, followed by isolation and characterization of the microbiota, is the most widely used method to obtain endophytes (Ganley and Newcombe 2006; Ferreira et al. 2008; Donnarumma et al. 2011; Herrera et al. 2016). However, advancement in the molecular techniques has provided the facility and ease of culture-independent approaches (Ikeda et al. 2006; Qin et al. 2016; Cope-Selby et al. 2017; Zhang et al. 2018; Alibrandi et al.

3

Seed Endophytes and Their Potential Applications

41

2018; Sánchez-López et al. 2018). Next-generation sequencing (NGS) can allow identification of endophytic microbial community and offers detailed insights of taxonomy and phylogeny of the microbial community in evolutionary studies (Kaul et al. 2016). Endophytic colonization can be localized and visualized in seeds and developing seedlings employing light microscopy and suitable stain; detailed structures and stages can be obtained through TEM (Cope-Selby et al. 2017). Hameed et al. (2015) characterized seed-borne endophytes of rice through DGGE; the study further showed the transforming ability of endophytes into rhizobacteria in rice. Khalaf and Raizada (2016) identified 169 unique bacterial strains in the seeds of seven economically important cucurbits using culturable approach and 16S rRNA gene sequencing. Qin et al. (2016) employed 454 pyrosequencing to characterize seed fungal endophytes of Suaeda salsa, a coastal plant, exhibiting low species richness in seeds. Culturable endophytic bacteria in surface-sterilized alfalfa seeds were identified to 40 separate genera and distributed within 4 taxa through MALDITOF analysis (López et al. 2017). Studies that aimed to investigate the endophytic communities among a large number of genotypes require fast and reliable techniques such as high-throughput sequencing and metagenomics. Zhang et al. (2018) investigated endophytic bacterial communities in rice seeds through Illuminabased 16S rRNA gene sequencing, and the results suggested that various rice genotypes do not have significantly different endophytic bacterial communities in seeds. However, the abundance was different, particularly of dominant genera. Gagne-Bourgue et al. (2013) used species-specific primers and PCR to identify bacterial endophytes in different cultivars of field-grown switchgrass. Change in microbial community composition was observed in different Brassicaceae during germination and emergence by next-generation sequencing (Barret et al. 2014). Endophytic bacterial communities were evaluated in different Zea sp. through culturing, cloning, and DNA fingerprinting using terminal restriction fragment length polymorphism (TRFLP) of 16S rDNA; further gfp tagging with wide-host vector pDSK-GFPuv proved systemic presence of several seed endophytes through the plant (Johnston-Monje and Raizada 2011). Cope-Selby et al. (2017) used PhyloChip™ G3 Assay (Second Genome) to evaluate seed bacterial diversity of Miscanthus spp. This technology does not have limitations of clone library and contains 1.1 million 16S rDNA sequence probes representing 60,000 microbial taxa (Cope-Selby et al. 2017).

3.4

Roles of Seed-Borne Endophytes

Responses of seed endophytes during germination and their potential transfer to seedlings have been rarely studied (Nelson 2018). Previous studies suggest that the seed microbial community can be the source of the endophytic and rhizospheric communities (Kaga et al. 2009; Johnston-Monje and Raizada 2011; Hardoin et al. 2012). Endophytic bacteria that colonize the interior of the seed also infect the subsequent generation and become the dominant endophytic species in the mature plant of rice (Kaga et al. 2009). Dissemination of seed-originating endophytic bacteria

42

H. Li et al.

was reported in the shoot, root, rhizosphere, and surrounding soil using surfacesterilized seeds of rice and sterile soils (Hardoin et al. 2012). Like plant growthpromoting rhizobacteria (PGPR), various seed endophytes supply growth improving advantages to their host plants (Hardoim et al. 2008). In addition, some seed endophytes can improve host plant metal tolerance and assist host plants in acquiring inorganic nutrients from unusual soils (Puente et al. 2009; Mastretta et al. 2009).

3.4.1

Seed Endophytes and Plant Growth Enhancement

Stagonospora isolates of Phragmites australis seed increased biomass and stem lengths of hosts in axenic microcosm experiments (Ernst et al. 2003). Seedassociated bacterial endophytes from cucurbit seeds were reported to enhance nutrient acquisition and comparatively improved growth of the hosts (Khalaf and Raizada 2016). Seed endophytes reduced wheat susceptibility to heat and drought, and improved its agricultural traits, which suggested potential applications in agriculture (Hubbard et al. 2012, 2014). Bacterial seed endophytes of a wheat cultivar showed potential as plant growth promoters and biocontrol agents of Fusarium graminearum (Herrera et al. 2016). Bacterial seed endophytes of Oryza sativa also showed plant growth promotion chiefly through phytohormone production (Shahzad et al. 2016). In another study, bacterial seed endophytes of O. sativa were found to considerably enhance germination rate and rice seedling development; potential physiological traits observed were pectinase and cellulase activity as well as salt and osmotic tolerance (Walitang et al. 2017). Increased seedling development was recorded in another important food crop, Zea mays, by its own seed endophyte Pantoea ananatis (Sheibani-Tezerji et al. 2015). Some seed endophytes travel to rhizoplane and take part in weathering of rocks as well as mineral transformations (Puente et al. 2009). Symbiotic association of the cactus Pachycereus pringlei and endophytic bacteria was found responsible for seedling establishment and growth on barren rock (Puente et al. 2009). Seed germination and other plant growth-enhancing properties were also exhibited by Cladosporium cladosporioides, a seed-borne fungal endophyte of a coastal plant Suaeda salsa (Qin et al. 2016). Recently, seed endophytic bacteria of Anadenanthera colubrina were reported to enhance plant growth through increasing mineral nutrient availability and induced production of plant hormones (Alibrandi et al. 2018). Quinoa exhibits exceptional germination velocity, salt stress tolerance, and heavy metal tolerance (Pitzschke 2018). Seedborne bacteria of Quinoa were observed to actively contribute to host cell expansion and therefore support rapid germination of hosts; additionally induction of mitogenactivated protein kinase in hosts was attributed partly to plant development and stress performance (Pitzschke 2018). Seeds of Phragmites australis colonized with endophytic fungi showed increased seed germination and seedling growth (Shearin et al. 2018). Genomics-assisted breeding using beneficial microbiomes (enhanced microbial activities and microbiome characteristics) can be exploited in the future for crop improvement (Vujanovic and Germida 2017).

3

Seed Endophytes and Their Potential Applications

3.4.2

43

Seed Endophytes Mitigating Heavy Metal Toxicity/Stress

Heavy metal (HM) toxicity is of serious concern due to the persistent and bioaccumulative nature of HMs (Parmar and Singh 2015; Khan et al. 2017). Heavy metals remain in the environment for long periods as they cannot be destroyed completely through any physical, chemical, or biological means, only transformed from one form to another. Although natural activities like weathering of mineral deposits, floods, brush burning, and windblown dust predominate as the sources of HM contamination, however, rapid urbanization, industrialization, and modern agriculture practices have increased HM pollution to an alarming level. Anthropogenic sources include mining, combustion of fossil fuels, extensive use of phosphate fertilizers and some pesticides, wastewater irrigation, paints and dyes, cement and printing industries, leather tanning industries, and e-wastes such as batteries, circuit boards, etc. (Tiller 1992; Facchinelli et al. 2001; Al-Khashman and Shawabkeh 2006; Su et al. 2014; Topalidis et al. 2017). Soil HM pollution is a global issue; overall HM pollution detrimentally influences nearly 12% of the agriculture production worldwide (Moffat 1999). In China, approximately, 20 Mha of agriculture lands were polluted with HMs that account for about one-fifth of the total agriculture area in use (Zhang and Huang 2000). As mentioned HMs cannot be degraded, but some microorganisms can alter the bioavailability of metals and their potential toxicity by altering their chemical form through microbe-mediated oxidation or reduction (Roane et al. 2015). Microorganisms can also influence HM accumulation in plants (Soleimani et al. 2010; Zhang et al. 2012a, b). To cope with HM pollution, endophytes isolated from seeds can give support to plants (Truyens et al. 2014). Inoculation of a cadmiumtolerant seed endophyte of Nicotiana tabacum reduced metal toxicity and improved host plant productivity suggesting the potential application of seeds as a carrier of beneficial microbes (Mastretta et al. 2009). Endophyte-infected seeds of Achnatherum inebrians showed better germination rates and index as compared to non-infected plants in the high concentration (100, 200, and 300 μM) of cadmium (Zhang et al. 2010). Similarly, colonization by an endophytic Epichloë endophyte positively affected seed germination and seedling growth of Elymus dahuricus exposed to high Cd concentrations; endophyte infection also influenced antioxidative enzyme activities and amounts of malondialdehyde and proline (Zhang et al. 2012b). Fungal Epichloë endophytes were also found to improve cadmium tolerance and bioaccumulation in two grass species; further the endophyte-infected plants showed better germination potential (Soleimani et al. 2010). Some of these microbes can increase zinc and iron biofortification in wheat by upregulation of genes facilitating higher Fe and Zn translocation in roots and shoots (Singh et al. 2018). Interestingly, it seems that these seed endophytes have evolved according to the needs of host plants. Endophytic fungal isolate FXZ2 that was isolated from hyperaccumulator, Arabis alpina, growing on a Pb-Zncontaminated site in Southwest China has shown to have a comparatively better tolerance to Pb, Zn, and Cd (40 mmol/L, 60 mmol/L, and 220 mmol/L, respectively) suggesting their functional role in increasing host plant stress tolerance as well as

44

H. Li et al.

influencing their metal accumulation (Chu et al. 2017). Transgenerational exposure to the hyperconcentration of heavy metal can affect the endophytic population of seed in Arabidopsis thaliana and Agrostis capillaris; some endophytes showed promising metal tolerance and plant growth-promoting potential (Truyens et al. 2013, 2014). The seed endophytes of A. capillaris having phosphorus-solubilizing properties, Cd tolerance, and ACC deaminase-, IAA-, siderophore-, and acetoinproducing ability were suggested beneficial for the phytoextraction and phytostabilization applications (Truyens et al. 2014). Artificially produced seeds of Phragmites australis containing endophytic Herbaspirillum frisingense that secrets indole-3-acetic acid and cadmium-binding siderophores encouraged seedling growth and cadmium tolerance (Gao and Shi 2018). Efforts can be made to try and replicate this strategy with other beneficial endophytes and plants to achieve desired beneficial traits or resistance to a particular toxicity. Methylobacterium was a dominant seed endophyte of Crotalaria pumila growing on metal mine residues and was suggested to contribute in nutrient uptake, plant growth promotion, and stress tolerance to their hosts under metal pollution (Sánchez-López et al. 2018). In support of successful germination and early seedling growth, beneficial seed endophytes are adapted for next-generation transfer (Rudgers et al. 2009; Truyens et al. 2013, 2016; Gao and Shi 2018; Sánchez-López et al. 2018). Quinoa seed endophytes possess high metal tolerance capacities suggesting their future utilization in endophyte-assisted phytoremediation applications (Pitzschke 2016).

3.5

Mechanism of the Growth Promotion and Heavy Metal Stress Tolerance

Mechanisms of direct plant growth enhancement may involve nitrogen fixation, nutrient mobilization (like phosphorus and iron through production of organic acids and siderophores), phytohormone production (like auxins, indole-2-acetic acid, and cytokinins), and suppression of stress ethylene production by 1-aminocyclopropane1-carboxylate (ACC) deaminase. Mechanisms of indirect growth enhancement involve checking phytopathogen activities through competition for space and nutrients, antagonism, secretion of hydrolytic enzymes, inhibition of toxins, and induction of plant defense mechanisms (Fig. 3.2). In addition, seed endophytes can enhance seed vigor, vitality, energy of germination and hydrothermal time of germination, and tolerance to environmental stresses such as drought and heat (Vujanovic et al. 2000; Hubbard et al. 2012, 2014). Pantoea, a seed endophyte of wheat, was recorded as IAA and siderophore producer and phosphate solubilizer and survived on N-free medium. Paenibacillus, another isolate, exhibited greater biocontrol of F. graminearum, while combined consortium of Paenibacillus with Pantoea increased chlorophyll content in barley seedlings (Herrera et al. 2016). Seed-associated bacterial endophytes from cucurbit seeds were found to improve growth of the host; 33% of strains produced indole-3-acetic acid, a plant hormone that induces growth of gourds and nutrient-acquiring roots (Khalaf and Raizada 2016). Later on, a majority of these endophytes displayed antagonistic activities

3

Seed Endophytes and Their Potential Applications

Direct involvement

45

Indirect involvement

• Nitrogen fixation • • • •

• Antagnostic activity against phytopathogens • Increased competition for space and nutrients • Secretion of hydrolytic enzymes • Inhibition of toxins • induced plant defence mechanisms

Nutrient mobilization (Fe, Zn, P, etc) Production of organic acids Siderophores production Phytohormones production (auxins, IAA, GA, cytokinins etc)

• induced antioxidant enzyme activities • Suppression of stress ethylene production by ACC) deaminase.

Benefits conferred to host • Enhanced seed vigor and vitality

• Enhanced seedling and plant growth • Resistance to pathogens • Tolerance to environmental stresses such as drought, heat, heavy metals • Improved antioxidant defense system

Fig. 3.2 Benefits conferred by seed endophytes to the host plant and mechanism involved

against phytopathogens suggesting their disease-suppression potential that could be utilized in developing commercial biocontrol agents (Khalaf and Raizada 2018). Endophyte colonization in Achnatherum inebrians increased its tolerance to Cd by improving the antioxidant defense system, mainly affecting antioxidative enzyme activities involving superoxide dismutase, catalase, peroxidase, and ascorbate peroxidase and affecting amounts of malondialdehyde and proline (Zhang et al. 2010). Similar results of growth-promoting properties through induced bioactivities were observed in other plants such as Zea sp., Elymus dahuricus, Lolium perenne, and Anadenanthera colubrina (Johnston-Monje and Raizada 2011; Zhang et al. 2012b; Briggs et al. 2013; Alibrandi et al. 2018). Endophytic bacteria Bacillus amyloliquefaciens isolated from seeds of Oryza sativa were found to be a gibberellin producer (Shahzad et al. 2016). Some seed bacterial endophytes of O. sativa also showed promising plant growth-promoting activities including hormone modulation, nitrogen fixation, siderophore production, and phosphate solubilization (Walitang et al. 2017). Bacilio-Jiménez et al. (2001) reported that endophytes from seeds defend young roots from colonization of pathogenic Azospirillum brasilense in rice. Culturable endophytic bacteria of alfalfa seeds were evaluated for the biochemical activities related to plant growth promotion and biocontrol. The percentage of genera that were found positive for a biochemical activity were 60% for pectinases, 5% for siderophore, 50% for auxin production, 47% for cellulases, 42.5% for amylases, 40% for proteases, 25% for phosphate solubilization, and 12.5% for chitinase activity, while 5% showed inhibition of fungal growth (López et al. 2017). Endophytic fungi Shiraia sp. isolated from Moso bamboo seeds showed

46

H. Li et al.

high production of antimicrobial hypocrellin A suggesting its role in plant disease defense (Shen et al. 2014). Various bacterial seed endophytes were reported to have antifungal properties (Gagne-Bourgue et al. 2013). Some seed endophytes, for instance, Bacillus and Pseudomonas, showed antagonistic activity against F. oxysporum f. sp. lycopersici, the causal organism of tomato wilt (Sundaramoorthy and Balabaska 2013). Improved growth and control of oxidative stress have also been observed in other plant-endophyte associations (Mendarte-Alquisira et al. 2017). However, the significance of the seed endophytes is magnified due to their systemic presence at the time of germination. Moreover, many of the previous mentioned studies have only focused on endophytes from a single plant organ or tissue. Most researchers have neglected systemic endophytes characteristically present throughout the life cycle of the plant. Isolation of an endophyte having beneficial traits, from any plant part other than seed, does not imply that it cannot attribute benefits to other parts. For example, fungal treatments of soybean seeds with its own root endophytes increased plant growth under salinity stress conditions (Radhakrishnan et al. 2013). Seed endophytes of P. pringlei were able to release inorganic nutrients such as phosphorus from pulverized rock via the production of organic acids (Puente et al. 2009). Improved germination and plant growth enhancement through endophytic colonization of host plants exposed to high heavy metal concentrations were often related to altering antioxidative enzyme activities and amounts of malondialdehyde, proline, ACC deaminase, IAA, siderophores, organic acids, and acetoin in the host plant (Zhang et al. 2012b; Truyens et al. 2013, 2014; Gao and Shi 2018). Other functions associated with seed endophytes are C and N fixation, oxidative phosphorylation, and photosynthesis activity (Sánchez-López et al. 2018).

3.5.1

Heavy Metal Resistance Genes Conferring Metal Resistance

Some seed-inhabiting microbes can confer heavy metal resistance to hosts, while some cannot; therefore, it is important to understand the genomics of the determinant metal resistance genes from microbes. Nies (1999) presented detailed insights of metal resistance systems in microorganisms. Protein families important for heavy metal transport are ABC, P-typeb, A-typec, RND, HoxN, CHR, MIT, and CDF (Nies 1999). Gene expression of nik, cop, mer, chr, czc, ncc, Mdt, and znt genes corresponded to heavy metal resistance in bacteria (Abdelatey et al. 2011; Margaryan et al. 2013; Maynaud et al. 2013). Whole-genome sequencing and integration of metallomics with proteomics and transcriptomics can be used to predict toxic and defensive mechanisms triggered by metals in microbes (González-Fernández et al. 2009). Genome sequencing and qRT-PCR analysis demonstrated genes associated to Chr, Czc, and Mer family genes involved in heavy metal tolerance of strain LZ-C closely related to Delftia lacustris (Wu et al. 2016). Furthermore, three gene clusters including czcCBA1, cadA2R, and colRS were suggested to be involved in metal efflux systems, while colRS, ColR, and ColS were part of signal transduction system involved in tolerance to Cd in cadmium-

3

Seed Endophytes and Their Potential Applications

47

resistant bacterium Pseudomonas putida CD2 (Hu and Zhao 2006). Besides ColRS also had a role in regulation of multi-metal tolerance through genetic complementation (Hu and Zhao 2006). Novel gene OmFCR1 that codes proteins belonging to PLAC8 family was identified in fungus Oidiodendron maius; expression of this gene confers cadmium tolerance (Di Vietro et al. 2014). Metallothionein genes were associated with oxidative and heavy metal stress in fungi (Andrews 2000; Loebus et al. 2013; Hložková et al. 2016). Phytochelatin synthase (PCS)-encoding genes were also demonstrated to increase tolerance to metal stress in fungi (Shine et al. 2015). Shen et al. (2015) illustrated the first transcriptomic level insights into the glutathione S-transferase (GST) gene of a heavy metal-resistant fungus Exophiala pisciplila and a primary interpretation for its heavy metal resistance mechanism. Variable expression of these GSTs was observed under different heavy metal stress, while upregulation was related to the heavy metal tolerance of E. pisciplila (Shen et al. 2015). Genomic analysis of three metal-resistant plant growth-promoting bacteria indicated different genes in all (Xie et al. 2015). Metal transporters from P-type including ATPase, CDF, HupE/UreJ, and CHR family were observed in Mesorhizobium amorphae, CopA/CueO system in Sinorhizobium meliloti, and ZntA transporter, CzcD, in Agrobacterium tumefaciens involved in resistance and homeostasis against different metals (Xie et al. 2015). The presence of operons and gene clusters such as cop, cus, czc, nik, and asc systems in copper-resistant Pseudomonas putida was predicted for the resistance to heavy metals (Chong et al. 2016).

3.6

Future Prospects

The endophytic microbial community composition in seeds is still poorly understood. In this context, NGS can offer detailed insight and future direction in seed endophyte research. Further diversity, community composition, and their potential function in seed germination and seedling development may be predicted. Although the role of plant breeders is combating food crises, developing improved and resistant crop varieties cannot be ignored. But recent advances in plant sciences suggest that microbial dynamic equilibrium with plant systems is vital for the germination, growth, and reproductive phases of the plant life cycle. Further, an important aim should be to increase seed vigor, one of the foremost factors that decides the fate of further growth, development, and production, using beneficial seed endophytes. Prenatal microbiome plays a decisive role in the development and outcome of seeds and should be considered as an important factor for the productive value in agriculture and horticulture (Truyens et al. 2015; Vujanovic and Germida 2017). Better knowledge of the seed endophytic community and factors that affect the community may facilitate improvement of seed quality and growth of agriculturally important crops via biotechnological applications (Truyens et al. 2013). Wholegenome sequencing (WGS) facilitates generation of genomic data resources of endophytic microbes that can provide information related to genes involved in diversity, distribution, and lifestyle of this endophytic symbiosis with host plants (Chaudhry et al. 2017). WGS can be used in future studies to reveal the molecular

48

H. Li et al.

mechanism of the interaction between microbe and plant. There are studies that illustrated differential expression of various transcripts during germination (Job et al. 2005; Czarna et al. 2016). During germination mitochondrial protein abundance changes inside seeds that might come from differential transcript expression which encodes them (Job et al. 2005). In dry seeds, there are a huge number of long-lived transcripts that are believed to have a central role in early stages of germination (Czarna et al. 2016). But all these studies have not considered the role of seed microbiota during germination. As discussed, seed-inhabiting microbes are an important part of seeds that can influence the future of the offspring. Therefore, omic approaches can be employed to study seed germination under controlled endophyte colonization and endophyte-free plants, in order to acquire a more detailed understanding of the mechanisms involved during seed germination at proteomic, transcriptomic, and metabolomic levels. In concluding remarks, it can be said that seed endophytes may have competence for enhancing germination under heavy metal conditions. Integration of proteomic, transcriptomic, and metabolomic approaches can give detailed insights into the mechanisms involved in the endophyte-seed-seedling interaction. These beneficial microbes should be considered in future microbe-assisted breeding programs for crop improvement. These microbes may also have promising applications in endophyte-mediated phytoremediation applications.

References Abdelatey LM, Khalil WK, Ali TH et al (2011) Heavy metal resistance and gene expression analysis of metal resistance genes in gram-positive and gram-negative bacteria present in Egyptian soils. J Appl Sci Environ Sanitation 6(2):201–212 Alibrandi P, Cardinale M, Rahman MM et al (2018) The seed endosphere of Anadenanthera colubrina is inhabited by a complex microbiota, including Methylobacterium spp. and Staphylococcus spp. with potential plant-growth promoting activities. Plant Soil 422(1–2):81–99 Al-Khashman OA, Shawabkeh RA (2006) Metals distribution in soils around the cement factory in southern Jordan. Environ Pollut 140(3):387–394 Andrews GK (2000) Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem Pharmacol 59(1):95–104 Bacilio-Jiménez M, Aguilar-Flores S, del Valle MV et al (2001) Endophytic bacteria in rice seeds inhibit early colonization of roots by Azospirillum brasilense. Soil Biol Biochem 33(2):167–172 Bacon CW, Hill NS (1996) Symptomless grass endophytes: products of coevolutionary symbioses and their role in the ecological adaptations of grasses. In: Redlin SC, Carris LM (eds) Endophytic fungi in grasses and woody plants: systematics, ecology, and evolution. APS Press, St Paul, MN, pp 155–178 Bacon CW, Hinton DM (2006) Bacterial endophytes: the endophytic niche, its occupants and its utility. In: Gnanamanickam SS (ed) Plant-associated bacteria. Springer, Amsterdam, pp 15–194 Bacon CW, White JF (2000) Microbial endophytes. Marcel Dekker, New York Bacon CW, Porter JK, Robbins JD et al (1977) Epichloe typhina from toxic tall fescue grasses. Appl Environ Microbiol 34(5):576–581 Baker KF, Smith SH (1966) Dynamics of seed transmission of plant pathogens. Annu Rev Phytopathol 4(1):311–332 Ball OJ, Prestidge RA, Sprosen JM (1995) Interrelationships between Acremonium lolii, peramine, and lolitrem B in perennial ryegrass. Appl Environ Microbiol 61(4):1527–1533

3

Seed Endophytes and Their Potential Applications

49

Barret M, Briand M, Bonneau S et al (2014) Emergence shapes the structure of the seed-microbiota. Appl Environ Microbiol 81(4):1257–1266 Bonnet M, Camares O, Veisseire P (2000) Effects of zinc and influence of Acremonium lolii on growth parameters, chlorophyll a fluorescence and antioxidant enzyme activities of ryegrass (Lolium perenne L. cv Apollo). J Exp Bot 51(346):945–953 Boursnell JG (1950) The symbiotic seed-borne fungus in the Cistaceae: I. Distribution and function of the fungus in the seeding and in the tissues of the mature plant. Ann Bot 14(54):217–243 Briggs L, Crush J, Ouyang L et al (2013) Neotyphodium endophyte strain and superoxide dismutase activity in perennial ryegrass plants under water deficit. Acta Physiol Plant 35(5):1513–1520 Cabral D, Stone JK, Carroll GC (1993) The internal mycobiota of Juncus spp.: microscopic and cultural observations of infection patterns. Mycol Res 97(3):367–376 Carroll G (1988) Fungal endophytes in stems and leaves: from latent pathogen to mutualistic symbiont. Ecology 69(1):2–9 Chaudhry V, Sharma S, Bansa K et al (2017) Glimpse into the genomes of rice endophytic bacteria: diversity and distribution of firmicutes. Front Microbiol 7:2115 Chong TM, Yin WF, Chen JW et al (2016) Comprehensive genomic and phenotypic metal resistance profile of Pseudomonas putida strain S13.1.2 isolated from a vineyard soil. AMB Exp 6(1):95 Chu L, Li W, Li XY et al (2017) Diversity and heavy metal resistance of endophytic fungi from seeds of hyperaccumulators. Jiangsu J Agric Sci 1:008 Cope-Selby N, Cookson A, Squance M et al (2017) Endophytic bacteria in Miscanthus seed: implications for germination, vertical inheritance of endophytes, plant evolution and breeding. GCB Bioenergy 9(1):57–77 Czarna M, Kolodziejczak M, Janska H (2016) Mitochondrial proteome studies in seeds during germination. Proteomes 4(2):19 De Bary A (1866) Morphologic und physiologie der plize, Flechten, und Myxomyceten. In: Hofmeister’s hand book of physiological botany, vol 2. Leipzig Di Vietro L, Daghino S, Abbà S et al (2014) Gene expression and role in cadmium tolerance of two PLAC8-containing proteins identified in the ericoid mycorrhizal fungus Oidiodendron maius. Fungal Biol 118(8):695–703 Donnarumma F, Capuana M, Vettori C et al (2011) Isolation and characterisation of bacterial colonies from seeds and in vitro cultures of Fraxinus spp. from Italian sites. Plant Biol 13 (1):169–176 Ernst M, Mendgen KW, Wirsel SG (2003) Endophytic fungal mutualists: seed-borne Stagonospora spp. enhance reed biomass production in axenic microcosms. Mol Plant Microbe Interact 16 (7):580–587 Ewald PW (1987) Transmission modes and evolution of the parasitism-mutualism continuum. Ann N Y Acad Sci 503(1):295–306 Ezzouhri L, Castro E, Moya M et al (2009) Heavy metal tolerance of filamentous fungi isolated from polluted sites in Tangier, Morocco. Afr J Microbiol Res 3(2):35–48 Facchinelli A, Sacchi E, Mallen L (2001) Multivariate statistical and GIS-based approach to identify heavy metal sources in soils. Environ Pollut 114(3):313–324 Ferreira A, Quecine MC, Lacava PT et al (2008) Diversity of endophytic bacteria from Eucalyptus species seeds and colonization of seedlings by Pantoea agglomerans. FEMS Microbiol Lett 287 (1):8–14 Finch SC, Pennell CGL, Kerby JWF et al (2016) Mice find endophyte-infected seed of tall fescue unpalatable–implications for the aviation industry. Grass Forage Sci 71(4):659–666 Freeman EM (1903) The seed-fungus of Lolium temulentum, L, the Darnel. Proc R Soc Lond 71 (467–476):27–30 Gagne-Bourgue F, Aliferis KA, Seguin P et al (2013) Isolation and characterization of indigenous endophytic bacteria associated with leaves of switchgrass (Panicum virgatum L.) cultivars. J Appl Microbiol 114(3):836–853

50

H. Li et al.

Ganley RJ, Newcombe G (2006) Fungal endophytes in seeds and needles of Pinus monticola. Mycol Res 110(3):318–327 Gao T, Shi X (2018) Preparation of a synthetic seed for the common reed harboring an endophytic bacterium promoting seedling growth under cadmium stress. Environ Sci Pollut Res 25 (9):8871–8879 González-Fernández M, García-Barrera T, Arias-Borrego A et al (2009) Metallomics integrated with proteomics in deciphering metal-related environmental issues. Biochimie 91 (10):1311–1317 Hameed A, Yeh MW, Hsieh YT et al (2015) Diversity and functional characterization of bacterial endophytes dwelling in various rice (Oryza sativa L.) tissues, and their seed-borne dissemination into rhizosphere under gnotobiotic P-stress. Plant Soil 394(1–2):177–197 Hardoim PR, Van Overbeek LS, Van Elsas JD (2008) Properties of bacterial endophytes and their proposed role in plant growth. Trends Microbiol 16(10):463–471 Hardoim PR, Hardoim CC, Van Overbeek LS et al (2012) Dynamics of seed-borne rice endophytes on early plant growth stages. PLoS One 7(2):e30438. https://doi.org/10.1371/journal.pone.0030438 Herrera SD, Grossi C, Zawoznik M et al (2016) Wheat seeds harbour bacterial endophytes with potential as plant growth promoters and biocontrol agents of Fusarium graminearum. Microbiol Res 186:37–43 Hložková K, Matěnová M, Žáčková P et al (2016) Characterization of three distinct metallothionein genes of the Ag-hyperaccumulating ectomycorrhizal fungus Amanita strobiliformis. Fungal Biol 120(3):358–369 Hodgson S, Cates C, Hodgson J et al (2014) Vertical transmission of fungal endophytes is widespread in forbs. Ecol Evol 4(8):1199–1208 Hu N, Zhao B (2006) Key genes involved in heavy-metal resistance in Pseudomonas putida CD2. FEMS Microbiol Lett 267(1):17–22 Hubbard M, Germida J, Vujanovic V (2012) Fungal endophytes improve wheat seed germination under heat and drought stress. Botany 90(2):137–149 Hubbard M, Germida JJ, Vujanovic V (2014) Fungal endophytes enhance wheat heat and drought tolerance in terms of grain yield and second-generation seed viability. J Appl Microbiol 116 (1):109–122 Ikeda S, Fuji SI, Sato T et al (2006) Community analysis of seed-associated microbes in forage crops using culture-independent methods. Microb Environ 21(2):112–121 James D, Mathew S (2015) Antagonistic activity of endophytic microorganisms against bacterial wilt disease of tomato. Int J Curr Adv Res 4:399–404 Job C, Rajjou L, Lovigny Y et al (2005) Patterns of protein oxidation in Arabidopsis seeds and during germination. Plant Physiol 138(2):790–802 Johnston-Monje D, Raizada MN (2011) Conservation and diversity of seed associated endophytes in Zea across boundaries of evolution, ethnography and ecology. PLoS One 6(6):e20396. https://doi.org/10.1371/journal.pone.0020396 Kaga H, Mano H, Tanaka F et al (2009) Rice seeds as sources of endophytic bacteria. Microb Environ 24(2):154–162 Kaul S, Sharma T, Dhar MK (2016) “Omics” tools for better understanding the plant–endophyte interactions. Front Plant Sci 7(955). https://doi.org/10.3389/fpls.2016.00955 Khalaf EM, Raizada MN (2016) Taxonomic and functional diversity of cultured seed associated microbes of the cucurbit family. BMC Microbiol 16(1):131 Khalaf EM, Raizada MN (2018) Bacterial seed endophytes of domesticated cucurbits antagonize fungal and oomycete pathogens including powdery mildew. Front Microbiol 9:42 Khan Z, Rehman A, Nisar MA et al (2017) Molecular basis of Cdþ 2 stress response in Candida tropicalis. Appl Microbiol Biotechnol 101(20):7715–7728 Kharwar RN, Mishra A, Gond SK et al (2011) Anticancer compounds derived from fungal endophytes: their importance and future challenges. Nat Prod Rep 28(7):1208–1228 Kharwar RN, Mishra A, Sharma VK et al (2014) Diversity and biopotential of endophytic fungal flora isolated from eight medicinal plants of Uttar Pradesh, India. In: Kharwar R, Upadhyay R, Dubey N, Raghuwanshi R (eds) Microbial diversity and biotechnology in food security. Springer, New Delhi, pp 23–39

3

Seed Endophytes and Their Potential Applications

51

Krings M, Taylor TN, Hass H et al (2007) Fungal endophytes in a 400-million-yr-old land plant: infection pathways, spatial distribution, and host responses. New Phytol 174(3):648–657 Leveille JH (1846) Considérations mycologiques, suivies d’une nouvelle classification des champignons. Imprimerie de I Martinet, Paris Loebus J, Leitenmaier B, Meissner D et al (2013) The major function of a metallothionein from the aquatic fungus Heliscus lugdunensis is cadmium detoxification. J Inorg Biochem 127:253–260 Lopez BR, Tinoco-Ojanguren C, Bacilio M et al (2012) Endophytic bacteria of the rock-dwelling cactus Mammillaria fraileana affect plant growth and mobilization of elements from rocks. Environ Exp Bot 81:26–36 López JL, Alvarez F, Príncipe A et al (2017) Isolation, taxonomic analysis, and phenotypic characterization of bacterial endophytes present in alfalfa (Medicago sativa) seeds. J Biotechnol 267:55–62 Margaryan AA, Panosyan HH, Birkeland NK et al (2013) Heavy metal accumulation and the expression of the copA and nikA genes in Bacillus subtilis AG4 isolated from the Sotk Gold Mine in Armenia. Biol J Armenia 65(3):51–57 Mastretta C, Taghavi S, Van Der Lelie D et al (2009) Endophytic bacteria from seeds of Nicotiana tabacum can reduce cadmium phytotoxicity. Int J Phytoremed 11(3):251–267 Maude RB (1996) Seed-borne diseases and their control: principles and practice. CAB International, Wallingford Maynaud G, Brunel B, Mornico D et al (2013) Genome-wide transcriptional responses of two metal-tolerant symbiotic Mesorhizobium isolates to zinc and cadmium exposure. BMC Genom 14(1):292 Mendarte-Alquisira C, Gutiérrez-Rojas M, González-Márquez H et al (2017) Improved growth and control of oxidative stress in plants of Festuca arundinacea exposed to hydrocarbons by the endophytic fungus Lewia sp. Plant Soil 411(1–2):347–358 Moffat AS (1999) Engineering plants to cope with metals. Science 285:369–370 Monnet F, Vaillant N, Hitmi A et al (2001) Endophytic Neotyphodium lolii induced tolerance to Zn stress in Lolium perenne. Physiol Plantarum 113(4):557–563 Mundt JO, Hinkle NF (1976) Bacteria within ovules and seeds. Appl Environ Microbiol 32 (5):694–698 Naveed M, Mitter B, Reichenauer TG et al (2014) Increased drought stress resilience of maize through endophytic colonization by Burkholderia phytofirmans PsJN and Enterobacter sp. FD17. Environ Exp Bot 97:30–39 Nelson EB (2004) Microbial dynamics and interactions in the spermosphere. Annu Rev Phytopathol 42:271–309 Nelson EB (2018) The seed microbiome: origins, interactions, and impacts. Plant Soil 422 (1–2):7–34 Ngugi HK, Scherm H (2006) Biology of flower-infecting fungi. Annu Rev Phytopathol 44:261–282 Nies DH (1999) Microbial heavy-metal resistance. Appl Microbiol Biotechnol 51(6):730–750 Parmar S, Singh V (2015) Phytoremediation approaches for heavy metal pollution: a review. J Plant Sci Res 2(2):135 Petrini O (1991) Fungal endophytes of tree leaves. In: Andrews J, Hirano S (eds) Microbial ecology of leaves. Springer, New York, pp 179–197 Pitzschke A (2016) Developmental peculiarities and seed-borne endophytes in quinoa: omnipresent, robust bacilli contribute to plant fitness. Front Microbiol 7:2 Pitzschke A (2018) Molecular dynamics in germinating, endophyte-colonized quinoa seeds. Plant Soil 422(1–2):135–154 Puente ME, Li CY, Bashan Y (2009) Endophytic bacteria in cacti seeds can improve the development of cactus seedlings. Environ Exp Bot 66(3):402–408 Qin Y, Pan X, Yuan Z (2016) Seed endophytic microbiota in a coastal plant and phytobeneficial properties of the fungus Cladosporium cladosporioides. Fungal Ecol 24:53–60 Radhakrishnan R, Khan AL, Lee IJ (2013) Endophytic fungal pre-treatments of seeds alleviates salinity stress effects in soybean plants. J Microbiol 51(6):850–857

52

H. Li et al.

Roane TM, Pepper IL, Gentry TJ (2015) Microorganisms and metal pollutants. In: Pepper IL, Gerba CP, Gentry TJ (eds) Environmental Microbiology. Academic Press, Amsterdam, pp 415–439 Rudgers JA, Afkhami ME, Rúa MA et al (2009) A fungus among us: broad patterns of endophyte distribution in the grasses. Ecology 90(6):1531–1539 Sánchez-López AS, Thijs S, Beckers B et al (2018) Community structure and diversity of endophytic bacteria in seeds of three consecutive generations of Crotalaria pumila growing on metal mine residues. Plant Soil 422(1–2):51–66 Schulz B, Boyle C (2005) The endophytic continuum. Mycol Res 109(6):661–686 Selim KA, El-Beih AA, AbdEl-Rahman TM et al (2012) Biology of endophytic fungi. Curr Res Environ Appl Mycol 2(1):31–82 Shade A, Jacques MA, Barret M (2017) Ecological patterns of seed microbiome diversity, transmission, and assembly. Curr Opin Microbiol 37:15–22 Shahzad R, Waqas M, Khan AL et al (2016) Seed-borne endophytic Bacillus amyloliquefaciens RWL-1 produces gibberellins and regulates endogenous phytohormones of Oryza sativa. Plant Physiol Biochem 106:236–243 Shahzad R, Khan AL, Lee IJ (2018) What is there in seeds? Vertically transmitted endophytic resources for sustainable improvement in plant growth. Front Plant Sci 9:24 Shearin ZR, Filipek M, Desai R et al (2018) Fungal endophytes from seeds of invasive, non-native Phragmites australis and their potential role in germination and seedling growth. Plant Soil 422 (1–2):183–194 Sheibani-Tezerji R, Naveed M, Jehl MA et al (2015) The genomes of closely related Pantoea ananatis maize seed endophytes having different effects on the host plant differ in secretion system genes and mobile genetic elements. Front Microbiol 6:440 Shen XY, Cheng YL, Cai CJ et al (2014) Diversity and antimicrobial activity of culturable endophytic fungi isolated from Moso bamboo seeds. PLoS One 9(4):e95838. https://doi.org/ 10.1371/journal.pone.0095838 Shen M, Zhao DK, Qiao Q et al (2015) Identification of glutathione S-transferase (GST) genes from a dark septate endophytic fungus (Exophiala pisciphila) and their expression patterns under varied metals stress. PLoS One 10(4):e0123418. https://doi.org/10.1371/journal.pone.0123418 Shine AM, Shakya VP, Idnurm A (2015) Phytochelatin synthase is required for tolerating metal toxicity in a basidiomycete yeast and is a conserved factor involved in metal homeostasis in fungi. Fungal Biol Biotechnol 2(1):3 Siegel MR, Latch GC (1991) Expression of antifungal activity in agar culture by isolates of grass endophytes. Mycologia 83:529–537 Siegel MR, Johnson MC, Varney DR et al (1984) A fungal endophyte in tall fescue: incidence and dissemination. Phytopathology 74(8):932–937 Singh D, Geat N, Rajawat MVS et al (2018) Deciphering the mechanisms of endophyte-mediated biofortification of Fe and Zn in wheat. J Plant Growth Regul 37(1):174–182 Smith SA, Tank DC, Boulanger LA et al (2008) Bioactive endophytes warrant intensified exploration and conservation. PLoS One 3(8):e3052. https://doi.org/10.1371/journal.pone.0003052 Soleimani M, Hajabbasi MA, Afyuni M et al (2010) Effect of endophytic fungi on cadmium tolerance and bioaccumulation by Festuca arundinacea and Festuca pratensis. Int J Phytoremed 12(6):535–549 Strobel GA (2003) Endophytes as sources of bioactive products. Microbes Infect 5(6):535–544 Strobel G, Daisy B (2003) Bioprospecting for microbial endophytes and their natural products. Microbiol Mol Biol Rev 67(4):491–502 Su C, Jiang L, Zhang W (2014) A review on heavy metal contamination in the soil worldwide: situation, impact and remediation techniques. Environ Skeptics Critics 3(2):24–38 Sun Y, Wang Q, Lu X et al (2012) Endophytic fungal community in stems and leaves of plants from desert areas in China. Mycol Prog 11(3):781–790 Sundaramoorthy S, Balabaskar P (2013) Evaluation of combined efficacy of Pseudomonas fluorescens and Bacillus subtilis in managing tomato wilt caused by Fusarium oxysporum f. sp. lycopersici (Fol). Plant Pathol J 12(4):154–161

3

Seed Endophytes and Their Potential Applications

53

Tan RX, Zou WX (2001) Endophytes: a rich source of functional metabolites. Nat Prod Rep 18 (4):448–459 Tiller KG (1992) Urban soil contamination in Australia. Soil Res 30(6):937–957 Timmusk S, Paalme V, Pavlicek T et al (2011) Bacterial distribution in the rhizosphere of wild barley under contrasting microclimates. PLoS One 6(3):e17968. https://doi.org/10.1371/jour nal.pone.0017968 Topalidis V, Harris A, Hardaway CJ et al (2017) Investigation of selected metals in soil samples exposed to agricultural and automobile activities in Macedonia, Greece using inductively coupled plasma-optical emission spectrometry. Microchem J 130:213–220 Truyens S, Weyens N, Cuypers A et al (2013) Changes in the population of seed bacteria of transgenerationally Cd-exposed Arabidopsis thaliana. Plant Biol 15(6):971–981 Truyens S, Jambon I, Croes S et al (2014) The effect of long-term Cd and Ni exposure on seed endophytes of Agrostis capillaris and their potential application in phytoremediation of metalcontaminated soils. Int J Phytoremed 16(7–8):643–659 Truyens S, Weyens N, Cuypers A et al (2015) Bacterial seed endophytes: genera, vertical transmission and interaction with plants. Environ Microbiol Rep 7(1):40–50 Truyens S, Beckers B, Thijs S et al (2016) Cadmium-induced and trans-generational changes in the cultivable and total seed endophytic community of Arabidopsis thaliana. Plant Biol 18 (3):376–381 Unger F (1833) Die Exantheme der Pflanzen und einige mit diesen verwandte Krankheiten der Gewächse. Carl Gerold, Wien, p 421 Vandenkoornhuyse P, Quaiser A, Duhamel M et al (2015) The importance of the microbiome of the plant holobiont. New Phytol 206(4):1196–1206 Vega FE, Simpkins A, Aime MC et al (2010) Fungal endophyte diversity in coffee plants from Colombia, Hawai’i, Mexico and Puerto Rico. Fungal Ecol 3(3):122–138 Verma VC, Kharwar RN, Strobel GA (2009) Chemical and functional diversity of natural products from plant associated endophytic fungi. Nat Prod Commun 4(11):1511–1532 Vujanovic V, Germida JJ (2017) Seed endosymbiosis: a vital relationship in providing prenatal care to plants. Can J Plant Sci 97(6):972–981 Vujanovic V, St-Arnaud M, Barabé D et al (2000) Viability testing of orchid seed and the promotion of colouration and germination. Ann Bot 86(1):79–86 Walitang DI, Kim K, Madhaiyan M et al (2017) Characterizing endophytic competence and plant growth promotion of bacterial endophytes inhabiting the seed endosphere of Rice. BMC Microbiol 17(1):209 Walitang DI, Kim CG, Jeon S et al (2018) Conservation and transmission of seed bacterial endophytes across generations following crossbreeding and repeated inbreeding of rice at different geographic locations. Microbiol Open. https://doi.org/10.1002/mbo3.662 Welty RE, Craig AM, Azevedo MD (1994) Variability of ergovaline in seeds and straw and endophyte infection in seeds among endophyte-infected genotypes of tall fescue. Plant Dis 78 (9):845–849 West CP (1994) Physiology and drought tolerance of endophyte-infected grasses. In: Bacon CW, White JF Jr (eds) Biotechnology of endophytic fungi of grasses. CRC, Boca Raton, FL, pp 87–99 Wiewióra B, Żurek G, Żurek M (2015) Endophyte-mediated disease resistance in wild populations of perennial ryegrass (Lolium perenne). Fungal Ecol 15:1–8 Wilkinson HH, Siegel MR, Blankenship JD et al (2000) Contribution of fungal loline alkaloids to protection from aphids in a grass-endophyte mutualism. Mol Plant Microbe Interact 13 (10):1027–1033 Wu W, Huang H, Ling Z et al (2016) Genome sequencing reveals mechanisms for heavy metal resistance and polycyclic aromatic hydrocarbon degradation in Delftia lacustris strain LZ-C. Ecotoxicology 25(1):234–247

54

H. Li et al.

Xie P, Hao X, Herzberg M et al (2015) Genomic analyses of metal resistance genes in three plant growth promoting bacteria of legume plants in Northwest mine tailings, China. J Environ Sci 27:179–187 Zhang J, Huang W (2000) Advances on physiological and ecological effects of cadmium on plants. Acta Ecol Sin 20(3):514–523 Zhang X, Fan X, Li C et al (2010) Effects of cadmium stress on seed germination, seedling growth and antioxidative enzymes in Achnatherum inebrians plants infected with a Neotyphodium endophyte. Plant Growth Regul 60(2):91–97 Zhang X, Lin L, Chen M et al (2012a) A nonpathogenic Fusarium oxysporum strain enhances phytoextraction of heavy metals by the hyperaccumulator Sedum alfredii Hance. J Hazard Mater 229:361–370 Zhang X, Li C, Nan Z (2012b) Effects of cadmium stress on seed germination and seedling growth of Elymus dahuricus infected with the Neotyphodium endophyte. Sci China Life Sci 55 (9):793–799 Zhang X, Wei W, Tan R (2015) Symbionts, a promising source of bioactive natural products. Sci China Chem 58(7):1097–1109 Zhang J, Zhang C, Yang J et al (2018) Insights into endophytic bacterial community structures of seeds among various Oryza sativa L. rice genotypes. J Plant Growth Regul. https://doi.org/10. 1007/s00344-018-9812-0

4

Exploring Endophytic Communities of Plants: Methods for Assessing Diversity, Effects on Host Development and Potential Biotechnological Applications Satish K. Verma, Ravindra N. Kharwar, Surendra K. Gond, Kathryn L. Kingsley, and James Francis White, Jr

Contents 4.1

4.2

4.3

4.4

4.5

4.6 4.7 4.8

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1 What Are Endophytes? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2 Challenges in the Study of Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isolation of Endophytic Microbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1 Media for Isolation of Fungal Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2 Media for Isolation of Bacterial Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identification of Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1 Molecular Tools to Identify Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2 Markers and Primers for Endophyte Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Techniques to Evaluate Endophyte Distribution in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.1 Hood and Shew Staining Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.2 Fluorescent Probes for Localization of Bacterial and Fungal Endophytes . . . . . . 4.4.3 ROS Staining to Study Bacterial Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endophyte Modulation of Seedling Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.1 Examining Modulation of Seedling Development Where Endophytes Are Not Culturable . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Application of Butyric Acid to Regulate Bacterial Entry into Plant Root Cells . . . . . . . . Use of Surrogate Hosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analysis of Endophyte Diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8.1 Non-culture Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8.2 Metagenomics and Pyrosequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8.3 Microarray: Gene Chips to Study the Expression and Mechanisms of Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

56 56 57 57 59 59 59 61 62 63 63 64 65 68 68 69 69 69 70 71 72

S. K. Verma (*) · R. N. Kharwar Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi, UP, India S. K. Gond Department of Botany, MMV, Banaras Hindu University, Varanasi, UP, India K. L. Kingsley · J. F. White, Jr (*) Department of Plant Biology, Rutgers University, New Brunswick, NJ, USA # Springer Nature Switzerland AG 2019 S. K. Verma, J. F. White, Jr (eds.), Seed Endophytes, https://doi.org/10.1007/978-3-030-10504-4_4

55

56

S. K. Verma et al.

4.9 Techniques for Bioactive Metabolite Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 4.10 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Abstract

Endophytic microbes colonize plants growing in diverse habitats and play important roles in modulating development and improving fitness of host plants. Endophytes may be major components of undiscovered microbial diversity. Further, endophytes may have applications in growth promotion of crop plants and protectors of plants from biotic and abiotic stresses. Endophytes have been a source of bioactive molecules of pharmaceutical importance. Major focus areas in the investigation of endophytes include (1) assessment of endophyte diversity, (2) determining the roles played by endophytes in modulation of host plant development and (3) assessing the biotechnological potentials of endophytes. The study of endophytes is particularly challenging because endophytic microbes often go unobserved in plants, many endophytes cannot be isolated, and plants free of endophytes sometimes cannot be obtained, making it difficult to conduct experiments. In this chapter we discuss some of the methodologies that are being used to overcome challenges to the study of endophytic microbes. Keywords

Bioactive molecules · Diversity · Endophytes · Reactive oxygen staining · Plantmicrobe interactions

4.1

Introduction

4.1.1

What Are Endophytes?

Endophytic microbes are those microbes that at some time in their life cycles colonize internal plant tissues without causing apparent harm to the host (Petrini 1991). Endophytic microbes provide hosts with protection against an array of biotic and abiotic stresses (Bacon and White 2000; Omacini et al. 2001; Redman et al. 2002). Endophytic microbes are ubiquitous and have been reported from all plants investigated (Petrini et al. 1982; Carroll 1988). They have been reported in algae (Hawksworth 1987), lichens (Petrini et al. 1990; Li et al. 2007), mosses (Schulz et al. 1993), conifers (Carroll and Carroll 1978) and angiosperms (Hyde et al. 1997; Verma et al. 2007, 2014; Gond et al. 2011), including parasitic plants (Suryanarayanan et al. 2000). Many investigations from the last few decades suggest that endophytes are promising sources of bioactive natural products (Kusari and Spiteller 2011; Verma et al. 2009a; Strobel and Daisy 2003; Stierle et al. 1993; Xu et al. 2014). However, commercialization of natural products from endophytes is not yet widespread. Endophytic microbes include fungi (Mishra et al. 2012; Verma et al. 2014) and bacteria (Gond et al. 2015a; Kumar et al. 2016). Ecophysiological roles of most endophytes

4

Exploring Endophytic Communities of Plants: Methods for Assessing. . .

57

Fig. 4.1 Foci of endophyte research

are not well understood but are assumed to vary according to microbe, plants and environmental conditions (Carroll 1995; Blodgett et al. 2007; Rodriguez et al. 2009).

4.1.2

Challenges in the Study of Endophytes

Studying endophytic microbes is often challenging to investigators because often microbes go unobserved in plants, many cannot be isolated, and it is difficult to conduct experiments where endophytes cannot be cultured or plants free of endophytes cannot be obtained. Further, methods used to study endophytes are still imperfect, and additional methodologies and techniques are needed to better understand endophytes and their potential applications (Gamboa et al. 2002; Rodriguez et al. 2009). In general, research on endophytic microbes has three major foci, including to assess diversity, explain plant-microbe interactions and assess biotechnological potentials (Fig. 4.1). The present review discusses the approaches, techniques and their limitations that may be employed to confront the challenges to the study of endophytes.

4.2

Isolation of Endophytic Microbes

Isolation of endophytic microorganisms is an important step in exploring the phylogeny, diversity, interactions with plants and applications as bio-inoculants for improvement of fitness of agricultural plants and for examining sources of biologically active molecules of industrial or medicinal importance (Hallmann et al. 2006; Strobel and Daisy 2003; Verma et al. 2009a; Kharwar et al. 2011b). Isolation procedures should be good enough to recover multiple endophytes and at the same

58

S. K. Verma et al.

Table 4.1 List of the most commonly used surface sterilization agents with their concentrations and sterilization times S. no. 1.

2. 3. 4.

5.

6. 7. 8.

Sterilization agents, dilution and duration Ethanol 96%, 30 s to 1 min; NaOCl 2.5–10% available Cl, 1–10 min; ethanol 70–96%, 10–30 s; rinse sterile water (this is most commonly used) Formaldehyde 37–40%, 1–5 min; NaOCl, 10% available Cl, 5 min Ethanol 75–96%, 30 s, rinse with sterile water Ethanol 70%, 1 min; H2O2 15–35%, 15 min; ethanol 70–99%, 1 min; sterile water Ethanol 96%, 1 min; peracetic acid 0.35%, 3–5 min; ethanol 96%, 30 s Ethanol 75–96%, 30 s, rinse with sterile water HgCl2 0.001–0.1%, 1–5 min; ethanol 70%, 1 min; sterile water, 1 min NaOCl 3–8% available Cl, 20–40 min; rinse three times with sterile distilled water

References Petrini and Müller (1979), Schulz et al. (1993), Espinosa-Garcia and Langenheim (1990), Mishra et al. (2012), Verma et al. (2014) Schulz et al. (1993) Fisher et al. (1994) Bissegger and Sieber (1994), Hata and Futai (1995) Fisher and Petrini (1990)

Fisher et al. (1986), Petrini and Fisher (1988) C. Booth (1971), Cabral (1985) Gond et al. (2015b), Verma et al. (2017)

time thorough enough to eliminate saprophytes and casual associates of plants (Hallmann et al. 2006). The procedure for isolation of endophytes is more or less the same for all kinds of endophytic microbes, including fungi and bacteria. Isolation involves surface sterilization, plating tissue pieces or crushed plant tissues on different media and then purification of microbes that grow from tissues (Verma et al. 2009b; Kharwar et al. 2011a; Mishra et al. 2012). Since endophytes are present in many organs, they can be isolated from different parts of the plant, including leaves, stem twigs, bark, roots, fruits and seeds (Bacon and White 1994; Ghimire and Hyde 2004). In general, it is suggested that the plant parts collected should be healthy and young in order to minimize unwanted opportunistic plant pathogens and saprobic species (Ghimire and Hyde 2004; Kharwar et al. 2011a; Verma et al. 2007). Endophytes are sometimes slow growing, and often faster-growing saprobes make it difficult to isolate endophytes (Bacon and White 1994). Plant parts should be kept in a refrigerator and processed in the shortest time possible after collection, if possible within 24 h. The samples for investigation should be cut into small pieces to facilitate sterilization and isolation processes. Most common surface sterilization techniques comprise dipping plant tissues in 75–90% ethanol for 1–2 min, then in 4% aqueous sodium hypochlorite for 5–10 min and finally in 90% ethanol for 30 s followed by rinsing with deionized sterile water at least three times to remove sterilizing agents (Petrini et al. 1992; Bills 1996; Verma et al. 2014; Stone et al. 2004; Kumar et al. 2016) (Table 4.1). Effectiveness of surface sterilization may be checked by making imprint of sterilized

4

Exploring Endophytic Communities of Plants: Methods for Assessing. . .

59

tissue sample (Schulz et al. 1998). Since different tissues of plants differ in their nature, thickness, roughness and permeability, therefore steps and concentrations of sterilizing agents may vary from plant to plant and tissue to tissue. Some work is thus necessary to optimize the surface sterilization procedure before isolation of endophytes (Hyde and Soytong 2008). Preferred isolation media differ depending on the kind of endophytes (fungi, bacteria and actinomycetes) to be isolated.

4.2.1

Media for Isolation of Fungal Endophytes

Bills and Polishook (1992) tested several media for endophytic fungal isolation and observed that malt-yeast extract media (1% malt extract, 0.2% yeast extract) gave the maximum species richness. Verma et al. (2011) used four media PDA (potato dextrose agar), MYA (malt-yeast extract agar), MCA (mycological agar: 1% papaic digest of soybean meal, 1% dextrose, 1.5% agar) and nutrient agar (0.5% peptone, 0.3% beef extract, 0.5% NaCl, 1.5% agar) for the isolation of endophytic fungi from the root and fruits of Azadirachta indica and suggested PDA and MCA as better isolation media for fungal endophytes than the others. Several researchers have used diverse isolation media to analyse endophytic fungal diversity but have also suggested using WA (water agar) for isolation to reduce contamination (Stone et al. 2004). Colony-limiting agents and antibiotics are recommended for primary isolations of endophytic fungi (Table 4.2).

4.2.2

Media for Isolation of Bacterial Endophytes

Procedures for the isolation of bacterial endophytes were described in detail by Hallmann et al. (2006). Crushed tissue samples in sterile water may be spread onto solid agar media of various types. Common media used for the isolation of bacterial endophytes are LBA (Luria-Bertani agar), TSA (tryptic soy agar; 10%), YESA (yeast extract sucrose agar) and nutrient agar. The use of antifungals like cycloheximide may also be used in media for the isolation bacterial endophytes (Table 4.2). Starch casein agar (SCA) with slight modification in ingredients is most commonly used for actinomycete isolation (Kutser and Williams 1964; Kumar et al. 2016). Media should be amended with antifungal agents like cycloheximide or nalidixic acid to restrict fungal growth (Verma et al. 2009b) (Table 4.2).

4.3

Identification of Endophytes

After isolation and purification of endophytes, the next important steps are to identify microbes and to assess species diversity (Sun and Guo 2012). Endophytes may be identified through culture-dependent methods or directly from the tissues using culture-independent methods (Sun and Guo 2012). The most common method to identify fungal endophytes is the cultivation-dependent approach which is

60

S. K. Verma et al.

Table 4.2 Some of the common media and antimicrobial agents used for the isolation of endophytes Name of S. no. media Endophytic fungi 1. Potato dextrose agar (PDA) 2.

Malt extract agar

3.

CzapekDox agar (CzDA)

Endophytic bacteria 1. LuriaBertani agar (LBA) 2.

3.

Tryptic soy agar (TSA, 10%)

Composition

Antimicrobials

References

Potato, 200.0 g; dextrose, 20.0 g; agar, 20.0 g; distilled water, 1 l

Streptomycin and/or ciprofloxacin (100–150 μg ml
1) (antibacterial) Streptomycin and/or ciprofloxacin (100–150 μg ml
1) (antibacterial)

Mishra et al. (2012), Verma et al. (2014) Mishra et al. (2012), Verma et al. (2014)

Streptomycin and/or ciprofloxacin (100–150 μg ml
1) (antibacterial)

Mishra et al. (2012), Verma et al. (2014)

Cycloheximide or nalidixic acid (100–150 μg ml
1) (antifungal) Cycloheximide or nalidixic acid (100–150 μg ml
1) (antifungal) Cycloheximide or nalidixic acid (100–150 μg ml
1) (antifungal)

Gond et al. (2015b), White et al. (2015) Gond et al. (2015b) White et al. (2015) Gond et al. (2015b), White et al. (2015)

Cycloheximide or nalidixic acid (100–150 μg ml
1) (antifungal)

Kumar et al. (2016), Kutser and Williams (1964)

Malt extract, 30.0 g; K2HPO4, 1.0 g; NH4Cl, 1.0 g; citric acid 1N, 15.0 ml; agar, 20.0 g; distilled water, 1l MgSO47H2O, 0.5 g; KCl, 0.5 g; NaNO3, 2.0 g; K2HPO4, 1.0 g; FeSO4, 0.01 g; sucrose, 30.0 g; agar, 15.0 g; distilled water, 1 l LBA, 15 g; distilled water, 1 l

Tryptic soy agar, 4 g; agar, 14 g; distilled water, 1 l

Yeast Yeast extract, 10 g; sucrose, extract 10 g; agar, 15 g; distilled sucrose agar water, 1 l (YESA) Endophytic actinomycetes 1. Starch Starch, 10 g; casein, 0.3 g; casein KNO3, 2 g; NaCl, 2 g; K2HPO4, 2 g; MgSO47H2O, potassium 0.05 g; CaCO3, 0.02 g; nitrate agar FeSO47H2O, 0.01 g; agar, (SCA) 18 g; distilled water, 1 l

frequently employed in assessing endophyte species diversity (e.g. Petrini and Fisher 1988; Fisher et al. 1986; Guo et al. 1998, 2003; Taylor et al. 1999; Sun et al. 2008; Kharwar et al. 2011a; Mishra et al. 2012; Verma et al. 2014), in the bioprospection of endophytes for bioactive metabolite production (e.g. Wang et al. 2006; Li et al. 2008; Kharwar et al. 2011a; Xu et al. 2014) and as mediators of host defence against biotic and abiotic stresses (e.g. Omacini et al. 2001; Redman et al. 2002, 2011; Dai et al. 2008; Gao et al. 2010; Gond et al. 2015a). This method involves isolation and purification of endophytic microbes from healthy tissues onto culture media (as previously described) followed by their identification. Endophytic fungi can

4

Exploring Endophytic Communities of Plants: Methods for Assessing. . .

61

sometimes be identified by their structural features in culture, i.e. growth patterns, colony colour, texture and fruiting structures, including spores, conidia, conidiophores, sporangiophores or sexual spores. This requires taxonomical expertise and references linking structural features of fungi to their identities. Some of the most routinely used standard taxonomic manuals for fungi are Illustrated Genera of Imperfect Fungi by Barnett and Hunter (1998), The Genera of Fungi Sporulating in Pure Culture by Von Arx (1978), More Dematiaceous Hyphomycetes by Ellis (1976) and The Fungi: An Advanced Treatise by Ainsworth et al. (1973). A most frequently encountered problem in fungal endophyte studies is the presence of mycelia sterilia (fungi which do not produce sexual or asexual spores), making their identification difficult (Ghimire and Hyde 2004; Sun and Guo 2012). Many times taxonomic manuals fail to provide enough information on spore-producing fungi to identify them to species level. In a study of fungal endophytes of Mahua plants (M. longifolia) from India, investigators were able to identify 28 species by morphological characters from 40 different morphotypes (Verma et al. 2014), in Adenocalymma alliaceum 14 species out of 17 morphotypes (Kharwar et al. 2011a) and in Tinospora cordifolia 24 out of 29 morphotypes (Mishra et al. 2012), and in Nyctanthes arbor-tristis 17 out of 19 morphotypes were identified by morphological characters (Gond et al. 2011). Due to the absence of differentiating morphological features, bacterial species generally cannot be distinguished using morphology alone.

4.3.1

Molecular Tools to Identify Endophytes

The morphological method of identification of endophytes may be useful for many endophytes (Kumar and Hyde 2004; Verma et al. 2014). However, a substantial number of isolates are reported as Mycelia sterilia due to failure to sporulate in culture. The number of Mycelia sterilia ranges significantly among studies of endophytes; in a study of endophytes in palm (Trachycarpus fortunei), 11% were reported as Mycelia sterilia, while in a study of endophytes of Quercus ilex in Switzerland, 54% were reported as Mycelia sterilia (Fisher et al. 1994; Frohlich et al. 2000; Ghimire and Hyde 2004). Some workers tried to improve sporulation in Mycelia sterilia in culture by changing the nutrient composition of the medium without much success (Guo et al. 1998; Frohlich et al. 2000; Kumaresan and Suryanarayanan 2002; Sun and Guo 2012). Even some of the spore-producing fungi are difficult to identify to correct taxonomic units because of the lack of literature (Verma et al. 2014). Further, some taxonomists believe that morphological characters are not reliable taxonomic features or sufficient to distinguish some taxa (Sun and Guo 2012). Molecular techniques therefore could be the best alternative to identify unidentified endophytic microbes. Molecular approaches have been used to determine the taxonomic status of fungi isolated from many habitats (White et al. 1990; Ma et al. 1997; Zhang et al. 1997; Ranghoo et al. 1999). Molecular identification requires isolation of total genomic DNA of all the recovered isolates from the host plant, PCR amplification of conserved sequences of DNA (rDNA) using specific or universal primers, sequencing of PCR products, BLAST options in

62

S. K. Verma et al.

Fig. 4.2 Schematic representation of fungal rRNA gene structure with position of different primers used for its amplifications

GenBank or other DNA databanks, retrieval of related sequences from the databank and construction of phylogenetic trees using molecular software (Saitou and Nei 1987). Genotype-based identification of microbes is believed to be more reliable because some portions of nucleic acid sequences are highly stable and conserved in a wide range of organisms (Lindahl et al. 2013). Certain locations of ribosomal DNA (rDNA) sequences have been shown to have the highest accuracy for identification of bacteria and fungi up to species level and further may be used in phylogenetic studies (Sugita and Nishikawa 2003).

4.3.2

Markers and Primers for Endophyte Identification

The fungal nuclear rRNA operon (rDNA) in multiple copies in fungal genomes is a continuous sequence made up of the 18S, ITS1, 5.8S, ITS2 and 28s subunit regions (Iwen et al. 2002). The gene for the smaller subunit is 18S (SSU:18S), and large subunit is 28S (LSU:28S) separated by the internal transcribed spacer (ITS1 and ITS2) regions. ITS1 and ITS2 are highly variable spacers and intercalated by another 5.8S gene (Fig. 4.2). Each rRNA gene is separated by two intergeneric spacers (IGS1 and IGS2) with intercalated another 5S gene. For good marker highly conserved regions of rDNA and its less variation across related taxa are needed, but a useful marker should also be able to differentiate among closely related taxa (Lindahl et al. 2013). Because of high interspecies variation and low intraspecies variation, nuclear rDNA genes have been exploited as an ideal marker for identification of fungi (Lindahl et al. 2013). Since the length of the amplified fragment is critical for the phylogenetic analysis, therefore primers should also amplify enough length which could require for correct analysis. ITS1, ITS2 and ITS4 are frequently used primers

4

Exploring Endophytic Communities of Plants: Methods for Assessing. . .

63

in fungal endophyte identification (Mishra et al. 2012; Verma et al. 2014). ITS1 (F) and ITS4 (R) primers amplify partial sequences of 18S, complete ITS1, ITS2 and 5.8S and partial sequences of 28S rRNA genes. Some authors argue to use NL1 and NL2 primers to amplify D1 and D2 region of 28S rRNA because it gives more resolution than ITS regions (Khot et al. 2009). Likewise many bacterial endophytes may be identified using the 16S rDNA gene since this gene is highly conserved and gives good resolution of species in most bacterial groups. Researchers have used a variety of primers to amplify 16S rDNA genes, amplifying around 1000–1500 base pairs of rDNA, for example, 8F, UnF, 11F, 27F, 6R, UnR, 907R, 1492R and 1525R (Barghouthi 2011; Gond et al. 2015a). Although 16S rDNA gene sequences allow bacterial identification that is usually more robust, reproducible and accurate than that obtained by phenotypic testing, however, due to limited variability of the 16S rRNA gene in some groups and limited representation in some databases, it is often difficult to identify species in some bacterial groups (Clarridge 2004).

4.4

Techniques to Evaluate Endophyte Distribution in Plants

Schulz et al. (1998) introduced a tissue blotting technique to check surface sterilization for the isolation of endophytes from the interior of healthy plant tissues. This technique may also be used to determine whether the hypothesized endophytes are also superficial on plant tissues. Endophytism can be further evaluated by doing microdissection and histological visualization of plant tissues through light, confocal or electron microscopy (Stone et al. 2004; Lucero et al. 2011; Sun and Guo 2012). Histological investigation of tissue is a method to observe all types of endophytes, including bacteria and fungi, within living host tissues. This enables us to see endophyte growth patterns, e.g. intra- or intercellular nature, and endophyte density in tissue and identify the part of tissue in which the endophyte is more concentrated. Most endophytes within plant tissues cannot be identified to any taxonomic category because they do not produce any identifying characters in vivo. For microscopic investigations of endophytes, the tissues must be stained with fungal specific or stain combination and mounted. Stone et al. (2004) have described a detailed protocol for clearing tissues for direct visualization of endophytic fungi in host tissues. The staining procedures must be optimized according to plant tissue and endophytic microbes (Hood and Shew 1996), described as follows.

4.4.1

Hood and Shew Staining Protocol

Leaf tissues are cut into small pieces (0.5 cm2) in size and washed thoroughly with running tap water; thereafter, tissues are surface sterilized followed by triple rinse in deionized water. Then leaf segments are treated with 2.5% KOH and heated at 40–60  C to remove chlorophylls. This process is repeated several days until the leaf becomes straw coloured. The KOH solution is changed daily 2–3 times for 5–10 days. The cleared leaf tissues are rinsed in distilled water three times and bleached

64

S. K. Verma et al.

Fig. 4.3 Histological visualization of endophytic fungal hyphae and spores in healthy leaf tissues of M. longifolia (a, b, c, d) and in tall fescue leaf (e)

3 min in 4% NaOCl and finally rinsed in distilled water twice. For staining KOH-aniline blue, solutions are prepared at least 2–3 h prior to use as 0.05% aniline blue dye in 0.067 M K2HPO4 at pH 9.0. Leaf sections are mounted on glass slides in small drops of stain solution and visualized under a compound light microscope (Fig. 4.3).

4.4.2

Fluorescent Probes for Localization of Bacterial and Fungal Endophytes

Fluorescent probing to determine internal endophyte distribution (intra- or intercellular) and density of endophytic microbes in different tissues of the plant has now become a popular method in the study of endophyte-plant interactions. There are two categories of fluorescent probes: one is green fluorescent protein (GFP) that autofluoresces, and the other is chemically linked fluorescent compounds that bind to proteins and nucleic acids or react with enzymes (Thirugnanasambandam et al.

4

Exploring Endophytic Communities of Plants: Methods for Assessing. . .

65

2011; Card et al. 2013). Tagging bacteria with GFP using compatible vector has now become popular to track the endophytic bacteria location inside plant tissues. However tagging with GFP is most crucial step in that. Mousa et al. (2016) have successfully tagged the endophyte Enterobacter sp. with GFP using vector (pDSKGFPuv) and visualized endophytic bacteria inside root tissues of maize, wheat and millets using confocal scanning microscopy. Several fluorescein-based compounds or dyes, including fluorescein diacetate (FDA), carboxyfluorescein diacetate (CFDA), chloromethylfluorescein diacetate (CMFDA), SYTO9 (S9), propidium iodide (PI), diamidino-2-phenylindole (DAPI), etc., have been used to observe in vivo tissue colonization by endophytic bacteria and fungi. FDA, CFDA and CMFDA fluorescence are activated by a cellular esterase enzyme which is widely found in plants and microbes. Card et al. (2013) evaluated FDA, CFDA, CMFDA and calcofluor white fluorescent compounds to visualize Epichloë coenophiala in tissues of tall fescue grass (Pooideae) and suggested that CMFDA is more a suitable stain in comparison to others; it produced better contrast between host tissues and endophytes. SYTO9, SYTO13, propidium iodide and diamidino-2-phenylindole are nucleic acid-staining dyes (Thomas and Reddy 2013). SYTO9 and SYTO13 have been shown very useful to stain and visualize living bacterial endophytes in different plant tissues (White et al. 2012; Thomas and Reddy 2013). Thomas and Reddy (2013) have compared SYTO9, PI and DAPI for the staining of endophytic bacteria in banana shoot tips and found that SYTO9 stained living bacterial cells; however propidium iodide (PI) and diamidino-2-phenylindole (DAPI) stained dead bacteria or bacteria within damaged plant tissues. White and his group also have used SYTO9 and SYTO13 to locate the bacterial endophyte in several plant tissues including vanilla, cotton, turf grasses, Amaranthus, etc. (Fig. 4.4). SYTO9 has excitation/emission band at 485/498 nm for DNA and 486/501 nm for RNA. For the staining of endophytic bacteria in vitro, surface-sterilized plant tissues were cut into fine sections and then stained with SYTO9 or SYTO13 (10–20 μM) in water for 5–10 min and observed using a fluorescent microscope. Endophytic bacteria around root cells and root hairs of seedlings can be visualized directly by staining without sectioning (White et al. 2012).

4.4.3

ROS Staining to Study Bacterial Endophytes

Reactive oxygen species (ROS) including hydrogen peroxide (H2O2), superoxide (•O
2) and hydroxyl radical (•OH) are recognized as important signalling molecules in various metabolic and developmental processes in plants. H2O2 is the most stable ROS in tissue, and H2O2 levels are found to be increased under biotic and abiotic stresses (Choudhury et al. 2013). It has been found that invasion of plant cells by many bacterial endophytes is associated with secretion of hydrogen peroxide in cells in the vicinity of intracellular bacteria (White et al. 2014a). White et al. (2014a) reported that 3,30 -diaminobenzidine tetrachloride (DAB), which has been used to stain cellular production of hydrogen peroxide in many plants tissues and organs (Daudi and O’Brien 2012), is a good staining agent for bacterial endophytes. DAB

66

S. K. Verma et al.

Fig. 4.4 SYTO@13 fluorescence staining of endophytic bacteria, (a) and (b) in root cells and root hairs of fescue grass, (c) in Amaranthus leaf, (d) intracellular localization of bacteria in leaf cells of vanilla orchid; red arrows indicate presence of bacteria (White et al. 2014b)

with counterstain aniline blue/lactophenol was found suitable to visualize endophytic (intra- and intercellular) bacteria as well as epiphytic colonization of bacteria in several vascular plant species through light microscopy (White et al. 2014a, 2015). ROS staining steps include (1) washing the roots properly with sterilized water, (2) putting the root of seedlings into DAB solution (2.5 mM) overnight, (3) washing with deionized water, (4) counterstaining with aniline blue and mounting on slide and (5) observing under a light microscope. In this staining procedure,

4

Exploring Endophytic Communities of Plants: Methods for Assessing. . .

67

Fig. 4.5 ROS staining by DAB-aniline stains to visualize endophytic bacteria: clusters of endophytic bacteria in the root hairs (a) in root parenchyma cells (b) of Cynodon sp. and in the root hairs (c) root parenchyma cells (d) of rice seedlings

bacteria on the surface are generally stained with aniline blue, and endophytic bacteria stained with DAB are dark brown in colour (Fig. 4.5). This staining method is selective to stain bacteria with their localization inside and outside the cell. DAB-aniline blue stain combination also permits the visualization of deformation and lysis of bacteria in plant root cells. Several different shapes and swollen endophytic bacteria (L-forms) have been observed in root cells of many plants (White et al. 2014a, 2015). White and his group presently are working with this staining procedure to visualize the endophytic bacterial community of many vascular plants including Cynodon sp., Phragmites sp., Daucus carota, Oryza sativa, Triticum aestivum, etc. with satisfactory results (Fig. 4.5). However this staining procedure is not effective to visualize endophytes in shoot systems due to failure of the stain to penetrate the waxy cuticle and pigmentation.

68

4.5

S. K. Verma et al.

Endophyte Modulation of Seedling Development

One of the more exciting discoveries regarding endophytes is the extent to which they play roles in modulation of host development. In experiments using grasses, it has been shown that root hair development is a function of the presence of bacteria that become intracellular in root cells and trigger hair formation, and they exit hairs at the hair tip where the cell wall is thinnest (White et al. 2015, 2017). The rate of seedling development in some plants is being found to largely depend on activities of microbial endophytes. Without intracellular bacterial endophytes, seedlings do not produce root hairs, and seedling growth is repressed. To assess endophyte modulation of development, we have conducted experiments on seedlings. Typically we used seedlings where we surface-sterilized seeds and germinated them on water agarose media. In these experiments we used plants: Bermuda grass (Cynodon dactylon) and annual bluegrass (Poa annua). These seeds could be surface-sterilized for approximately 40 min with continuous agitation in 4% sodium hypochlorite solution to remove all surface microbes (White et al. 2015, 2017). Seeds were then plated onto 0.7% agarose media in Petri dishes without nutrient additives. Bacterial aqueous suspensions were inoculated onto seeds and plates incubated at room temperature for 6–7 days. Seedlings were assessed for gravitropic affects, root hair development and root length extension. Without endophytic bacteria most of the seedling roots fail to show gravitropic growth; instead they remain on the surface of agarose without vertical growth. For the few roots that penetrate, the agarose root hairs do not form, and root length is often reduced. Bacteria may be visualized within seedling root cells by flooding plates bearing seedlings with DAB and incubating overnight at laboratory ambient temperature (White et al. 2014a). In all probability a similar in vitro assay system could be developed for testing effects of endophytes on seedling development in other species of plants.

4.5.1

Examining Modulation of Seedling Development Where Endophytes Are Not Culturable

In some cases endophytes are not isolatable, and it may be difficult to remove endophytes from seedlings if endophytes are vectored within seeds. One instance where we encountered this situation is with a bacterial endophyte in tomato (Solanum lycospersicum). Through microscopic examination of DAB-stained (White et al. 2014a) seedlings derived from surface-sterilized seeds, we saw evidence of an intracellular bacterial endophyte that we could not isolate. Through the use of a combination of rigorous surface disinfection using 4% sodium hypochlorite for 40 min to remove all surface bacteria, followed by an overnight soak in a 100–200 mg/l solution of streptomycin sulphate to reduce or remove internal bacteria, we were able to suppress development of the bacterial endophyte in seedlings germinated on agarose (Verma et al. 2017). Suppression of the bacterial endophyte in tomato seedlings resulted in suppression of root hairs on tomato roots. We also found similar effect in rice seedling experiments (Verma et al. 2017).

4

Exploring Endophytic Communities of Plants: Methods for Assessing. . .

4.6

69

Application of Butyric Acid to Regulate Bacterial Entry into Plant Root Cells

In plant roots bacteria enter root cells at the root meristem tips where cell walls are thin. As root cells differentiate, bacteria in the epidermal cells stimulate root hair development and exit through the hair tip. We have actively been looking for ways to control the entry of bacteria into root meristem cells. Recently we have identified butyric acid as a regulatory molecule for plant intracellular invasion by bacteria. By incorporation of butyric acid (0–1 mM concentration) into agarose in which grass seedlings (Poa annua) bearing a Pseudomonas endophyte, we were able to regulate the entry of bacteria into cells of the grass. In agarose that did not contain butyric, we saw the highest level of bacterial entry into plant cells (with the longest root hair development); at the 1 mM concentration of butyric acid, bacteria did not enter into plant cells (and root hairs did not form); and intermediate level of butyric acid (0.5 mM) showed some entry of bacteria into meristematic cells (and short root hairs formed). Butyric acid may be useful to regulate intracellular invasion of meristematic cells and evaluate endophyte effects on growth and development of many plant species.

4.7

Use of Surrogate Hosts

In some cases experiments cannot be conducted using the host of the endophyte due to inability to obtain sufficient plant material, failure of seeds to germinate or slowness of plant growth under laboratory conditions. In several studies we have had success in using surrogate hosts to conduct experiments to determine the likely effects of endophytes on its host plant. For assessing the effects of endophytes from grass hosts, we use turfgrass species Poa annua (cool-season grass) and Cynodon dactylon (warm-season grass) to conduct in vitro experiments. Seeds of these species are readily purchased, and many of their seed-transmitted microbes may be removed by vigorous surface disinfection (40 min in 4% sodium hypochlorite). These species also readily germinate in agarose so that observations may be made on developing seedlings. Because some endophytes may be adapted to a particular host species, in selection of surrogate plants, it is probably advisable to select surrogate test plants that are taxonomically close to the original host of the endophyte.

4.8

Analysis of Endophyte Diversity

Diversity of isolated endophytic microbes on different culture media can be studied through nonmolecular methods. There are several ways to assess the diversity of isolated microbes, i.e. colonization frequency, isolation frequency, alpha diversity indices (Simpson’s, Shannon-Wiener diversity indices), species richness-evenness, etc. (Hata and Futai 1995; Mishra et al. 2012; Verma et al. 2007, 2014). The colonization frequency (%CF) of endophytic fungi may be calculated manually

70

S. K. Verma et al.

using the formula given by Hata and Futai (1995). %CF ¼ Ncol / Nt  100, where Ncol ¼ number of segments colonized by each fungus and Nt ¼ total number of segments studied. Diversity indices: Simpson’s and Shannon-Wiener diversity indices and species richness may be calculated manually or by several programs available including PAST, BioDiversity Pro and Origin software (Verma et al. 2014; Orduna et al. 2011) based on the formula of Simpson’s diversity ¼ 1
∑( pi)2 and Shannon-Wiener diversity ¼
∑ s ( pi log pi), where pi ¼ proportion of frequency of the ith species in a sample. Species evenness can be calculated as evenness (E) ¼ H/log(S), where H ¼ Shannon-Wiener diversity and S ¼ species richness (i.e. total number of species). Statistical verification can be done by ANOVA by SPSS 16.0 or a newer version of software to assess significant differences among mean diversity indices of samples collected from different locations and tissues (to check the temporal and spatial variation in diversity). Kharwar and his co-workers used the same approach to analyse the diversity of endophytic fungi of more than ten medicinal plants of Northern India. To further determine the interrelationship and tissue and site specificity of endophytic microbial populations among different tissue samples and sites, ecological associations among endophytes with the different tissues and different localities of the host, principal component analysis (PCA) may be performed (Orduña et al. 2011; Verma et al. 2014).

4.8.1

Non-culture Methods

There is a difference between the number of microbes isolated into pure culture and numbers present as endophytes in plants (as few as 1–5% of the microbes may be isolated from the natural habitats) (Stewart 2012; Staley and Konopka 1985). It is likely that numerous microbes are never isolated and a majority of endophytic diversity, like other microbial habitats, are missed by cultivation-based methods (Hugenholtz et al. 1998). This is because many microbes do not grow under the conditions currently being employed to isolate them. In the past few years, molecular approaches have been employed to elucidate diversity of microbes within host tissues (Guo et al. 2001). Guo et al. (2001) amplified the 18S rRNA gene from the total DNA extracted from frond tissues of Livistona chinensis followed by cloning, sequencing and phylogenetic analysis to elucidate fungal endophyte diversity. They recovered some novel fungal endophytes that had not previously been isolated by culture methods. Technology has advanced the study of molecular diversity and phylogeny of endophytes (Fig. 4.6) (De Hoog et al. 2005; Green et al. 2004; Duong et al. 2006). Denaturing gradient gel electrophoresis (DGGE) has been employed successfully in studying direct endophyte diversity in leaves of Magnolia liliifera (Duong et al. 2006).

4

Exploring Endophytic Communities of Plants: Methods for Assessing. . .

71

Diversity Analysis Plant Tissue Sampling (Roots, Leaf, Stem etc) Surface sterilization Direct Molecular Approach (Cultivation Independent) METAGENOMICS

Traditional Approach (Cultivation Dependent) Tissue Segments/Ground (0.5 cm2)

Total DNA Isolation from Tissues (Leaf, Roots, Stems)

Plot on Different Media (PDA, MEA, LBA etc.)

Biotechnological Applications

Purification of Isolates Morphological Identification

Cloning Library, RFLP/DGGE (Molecular Fingerprinting)

Unidentified (Mycelia Sterilia)

Isolation of DNA, ITS/18S/16S rDNA PCR

PCR Amplification of ITS/18S/16S rDNA

PCR Product Sequencing, Analysis Sequencing and BLAST (Molecular Identification)

Calculation of Diversity Indices, Phylogenetic Analysis

Fig. 4.6 Schematic way for the steps of analysis of diversity and phylogeny of endophytes

4.8.2

Metagenomics and Pyrosequencing

Metagenomics (environmental genomics) involves the study of all genetic material recovered directly from any environmental samples, i.e. water, soil, plant tissue and animal tissue samples. This technique offers a powerful tool for surveying microbes and has been employed in the study of the plethora of endophytes in several studies (Guo et al. 2001; Duong et al. 2006; Marco 2011). Metagenomics is not restricted to phylogenetic description based on the 16S or 18S rRNA genes but provides much important information about functional genes and their diversity composition in communities. Pyrosequencing has been applied to the study of the composition of endophytic community diversity in several host plants (Romero et al. 2014; Manter et al. 2010). High-throughput sequencing technologies (pyrosequencing) or next-generation sequencing (NSG) does not require a cloning step, and greater yields of sequence data can be obtained (Mardis 2008; Segata et al. 2013). Advancement in nextgeneration sequencing has minimized the cost of analysis of complex metagenomic sequences. It has revolutionized the metagenomic study. Shotgun metagenomics and pyrosequencing provide information about organism’s diversity and their theoretical metabolic functional role in host plant (Kaul et al. 2014; Tian et al. 2015). This could be very important in the study of functional endophytism. Recently metagenomics

72

S. K. Verma et al.

and pyrosequencing technologies have been applied successfully to study the functional metabolic role of endophytes in several host plants along with their structural diversity (e.g. Sessitsch et al. 2012; Tian et al. 2015). Sessitsch et al. (2012) reported functional roles of the endophytic community of rice roots, and their data suggested that endophytes are involved in promoting plant growth and enhancing stress tolerance against biotic and abiotic stresses. They also found that endophytes might play important roles in the entire nitrogen cycle including nitrogen fixation, denitrification and nitrification. Tian et al. (2015) investigated and compared functional genomics of healthy and nematode-infected tomatoes root-endophyte microbiomes. They showed that the bacterial community was involved in nematode pathogenesis. Using 454 pyrosequencing, Jumpponen and Jones (2009) analysed fungal communities in the phyllosphere of Quercus macrocarpa and compared fungal diversity and distribution among trees located in different locations. Using 454 pyrosequencing techniques, Toju et al. (2013) also described the association of both endophytic fungi and mycorrhizal fungi in roots of different plant species. Metagenomics and NGS technologies have been employed extensively in biological science in the past few decades; however its application in revealing endophyte functional diversity and their composition in plant is limited to few studies. Some limitations of the techniques include datasets that are very complex and comprehensive requiring novel tools, statistical software, extensive storage, visualization and analysis with existing datasets (Thomas et al. 2012). Further, there is insufficient annotation of submitted sequences in public databases, and this may be the one major limitation of metagenomic approaches.

4.8.3

Microarray: Gene Chips to Study the Expression and Mechanisms of Interaction

DNA microarray (commonly known as DNA chip or gene chip) along with real-time PCR (RT-qPCR) analysis is one of the fastest-growing new molecular techniques. RT-PCR is now very common to study the gene expression in host plant tissue, and it has been now widely applied in enabling endophyte role in modulation of gene expression in host plant (Gond et al. 2015a). However it is limited to few gene studies at a time. DNA microarray has the advantage of studying whole-gene expression altogether in the organism, and it has been used for the study of differential gene expression in biological systems. This important technology may be used to understand the molecular mechanisms of plant-endophyte interactions (Dinkins et al. 2010; Kaul et al. 2014). A diagrammatic representation of DNA microarray is shown in Fig. 4.7. Since very little information about the molecular mechanisms of the plantendophyte interactions are known, therefore microarray study followed by RT-PCR may put forth more light on that aspect. Some studies have sought to analyse differential gene expression in endophyte-free and endophyte-containing plants using DNA chip techniques (Johnson et al. 2003; Felitti et al. 2004, 2006; Dinkins et al. 2010). Commercial gene chips are available for many crop and medicinal plants. There is an opportunity for researchers to analyse and differentiate the relative

4

Exploring Endophytic Communities of Plants: Methods for Assessing. . .

73

Plant-Microbe Interactions Transcriptomics of Plant – Endophyte Interface Q-PCR/RT-PCR of Specific Gene

Isolation of Total m-RNA of Interface

DNA Microarray/ Genechip

Isolation of Total m-RNA of Interface C-DNA and tag with Fluorescent

Q-PCR (Data Analysis)

Hybridization with Gene chip

Gene chip Fluorescent Reader (Data Analysis)

Comparison with Control for Gene Expression (Up-regulated/ Down-regulated/No-change)

Fig. 4.7 Schematic representation of steps to study gene expression during plant-endophyte interaction

expression of a large number of genes simultaneously for at least those plants for whom microarray platforms are available or with their close relatives (Ciannamea et al. 2006; Sawbridge et al. 2003; Dinkins et al. 2010). Microarray analysis of transcriptomes of Arabidopsis-Pseudomonas fluorescens (endophyte) interactions showed the up-regulation of many genes involved in critical metabolism, signal transduction and stress management. Significantly putative auxin- and nodulinrelated genes were up-regulated, and ethylene-responsive genes were downregulated (Wang et al. 2005). Dinkins et al. (2010), with some success, studied the differential gene expression in tall fescue with their obligate endophyte Epichloë coenophiala using the Affymetrix Wheat Genome Array GeneChip® and Barley1 Genome Array GeneChip®. Irizarry and White have also applied Affymetrix cotton GeneChip to study the role Bacillus sp. (endophyte) in cotton seedling differential gene expression. They found that endophytes up-regulated some key genes related with nitrogen metabolism and auxin synthesis (not published). They have also observed the phenotypic effect of the same bacteria on the development of better root architecture than control (Irizarry and White 2017).

74

S. K. Verma et al.

Phenotypic effects of endophytes on hosts include growth promotion, stress tolerance, enhanced ability to acquire mineral from the soils and improved nitrogen utilization (Malinowski and Belesky 2000). Microarray analysis could be used to understand the molecular mechanisms of beneficial impacts of endophytes on plants. A major limitation of microarray analysis is the lack of availability of gene chip platforms for specific plants and compatible reference datasets for statistical analysis.

4.9

Techniques for Bioactive Metabolite Analysis

Many endophytic microbes, particularly fungi, synthesize bioactive metabolites that may be candidates for treating many current and newly emerging diseases in humans, plants and animals (Strobel and Daisy 2003; Kusari and Spiteller 2011, 2012). Many endophytes have been found to produce metabolites similar to those produced by host plants. Some examples are paclitaxel (Taxol), podophyllotoxin, deoxypodophyllotoxin, camptothecin, hypericin, pipericin and emodin (Chithra et al. 2014; Gond et al. 2014a, b; Shweta et al. 2010; Tan and Zou 2001) and azadirachtin (Kusari et al. 2012). There is potential to discover novel and useful molecules from endophytes by manipulating culture conditions. Cultural conditions include media types, source of nitrogen, temperature, etc. (Kusari et al. 2012). A procedure for isolating bioactive molecules is shown in Fig. 4.8. Steps in metabolite isolation include (1) culture of endophytic isolates in broth media (different types of broth media may be tested); (2) extraction of secondary metabolites from the medium at different time intervals using different organic solvents (e.g. hexane, ethyl acetate, chloroform, etc.) followed by drying of the crude extract (Duarte et al. 2012); (3) screening (e.g. antimicrobial, anticancer, antioxidant, antimalarial, antiviral test, etc.) of bioactivity of the crude extract; (4) purification of pure bioactive compounds from the active crude extract by (4a) thin-layer chromatography (TLC), (4b) column chromatography, (4c) highperformance liquid chromatography (HPLC) and (4d) gas chromatography; and (5) identification of structure of pure compound by (5a) nuclear magnetic resonance (NMR), (5b) LCMS, (5c) GCMS and (5d) X-ray crystallography (Fig. 4.8). Several forms of nuclear magnetic resonance (1D and 2D NMR) spectroscopy are frequently applied techniques for structural elucidation of bioactive compounds isolated from endophytes (Strobel et al. 1999, 2002; Kusari et al. 2009). Several forms of 2D proton NMR like correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY) and nuclear Overhauser effect spectroscopy (NOESY) help in the determination of the correct conformation of molecules (Duarte et al. 2012). LCMS and HPLC and other mass-based techniques are generally used for the identification of already known compounds (Stierle et al. 1993; Liu et al. 2007; Kusari et al. 2008). GCMS analysis is frequently successful for the isolation and identification of oily or volatile compounds (Gond et al. 2014a, b). In some cases pure compounds may crystallize, and structures may be elucidated by crystallography (Kharwar et al. 2009; Strobel et al. 2002). Purification and identification steps are critical and may require optimization and the expertise of an organic analytical

4

Exploring Endophytic Communities of Plants: Methods for Assessing. . .

75

Biotechnological Applications Purification of Endophyte Isolates Cultivation in Different Broth Media

Extraction of Secondary Metabolites (Organic solvents; Ethyl acetate, Chloroform, Hexane etc.) Culture Purification of Compounds from Extract

Separate Pure Band (Check Bioactivity)

V

V IV

Column Chromatography (Solvent combinations)

IV

Thin Layer Chromatography (Solvent combinations)

Extracts

II

HPLC (Purity check ; >99%)

0

I

I

II

III

III

FTIR LCMS (for Volatiles GCMS)

If Crystallized X-Ray Crystallography

NMR

Combine Data Identify Pure / Novel Compounds

Fig. 4.8 Schematic showing steps in extraction and identification of bioactive molecules from endophytic microbes

chemist for deduction of correct structures. A large number of bioactive compounds have been reported from endophytic fungi in the past few decades using the above methods. Many good reviews are available on endophytic fungal metabolites, their sources and molecular structures (Tan and Zou 2001; Schulz et al. 2002; Strobel and Daisy 2003; Gunatilaka 2006; Kusari and Spiteller 2011, 2012; Kharwar et al. 2011b; Xu et al. 2014; Gond et al. 2014a, b).

76

4.10

S. K. Verma et al.

Conclusions

Endophytic microbes are now recognized as important components of microbial communities of plants. However, we still do not understand the extent to which endophytes and hosts have come to be interdependent. In the extreme case of interdependency, we ask the question: Can plants survive or seedlings develop without involvement of endophytic microbes? This question has not been answered, but it is increasingly looking relevant. From a less extreme perspective, endophytes may be probiotics of plants. Here too, it is important to work out the mechanisms involved in plant-endophyte interactions at the molecular level. New technologies including transcriptomics (RT-qPCR), DNA microarray analysis, high-throughput sequencing (NGS) and protein-based array analysis may help to develop an understanding of the interaction mechanisms and functional roles. Conducting relevant experiments is the most important approach to determine the extent of the interdependency of endophytes and hosts. Once the mechanisms of interactions and functional roles are understood, we may understand why plants maintain endophytic microbial communities. Developing a better understanding of endophytes and their effects on hosts and host ecology will also enable us to find applications for endophytes in agriculture and medicine. Acknowledgements The authors are thankful to the Department of Plant Biology, Rutgers University, NJ, for providing the facilities. SKV acknowledges UGC, India, for providing a Raman Post Doctoral fellowship No. F 5-11/2016(IC) for the year (2016–2017) to work in the USA. The SKV and RNK are also grateful to the Head and Coordinator CAS and DST-FIST and PURSE of Botany, BHU, Varanasi, for providing the facilities and leave to pursue endophyte research. SKV acknowledges the support from UGC (Project – UGC-BSR startup-M14-26). The authors are also grateful for the support from the John E. and Christina C. Craighead Foundation, USDA-NIFA Multistate Project W3147 and the New Jersey Agricultural Experiment Station.

References Ainsworth GC, Sparrow FK, Sussman AS (1973) The fungi: an advanced treatise, vol 4A. Academic Press, New York Bacon CW, White JF (1994) Stain, media and procedure for analyzing endophytes. In: Bacon CW, White JF (eds) Biotechnology of endophytic fungi of grasses. CRC Press, Boca Raton, FL, pp 47–56 Bacon CW, White JF (2000) Microbial endophytes. Dekker, New York Barghouthi SA (2011) A universal method for the identification of bacteria based on general PCR primers. Indian J Microbiol 51(4):430–444 Barnett HL, Hunter BB (1998) Illustrated genera of imperfect fungi, 4th edn. The American Phytopathological Society, St. Paul, MN Bills GF (1996) Isolation and analysis of endophytic fungal communities from woody plants. In: Redlin SC, Carris LM (eds) Endophytic fungi in grasses and woody plants. APS Press, St. Paul, MN, pp 31–65 Bills GF, Polishook JD (1992) Recovery of endophytic fungi from Chamaecyparis thyoides. Sydowia 44:1–12 Bissegger M, Sieber TN (1994) Assemblages of endophytic fungi in coppice shoots of Castanea sativa. Mycologia 86:648–655

4

Exploring Endophytic Communities of Plants: Methods for Assessing. . .

77

Blodgett JT, Swart WJ, Lnow SVd M, Weeks WJ (2007) Soil amendments and water influence the incidence of endophytic fungi in Amaranthus hybrids in South Africa. Appl Soil Ecol 35:311–318 Booth C (1971) The genus Fusarium. Commonwealth Mycological Institute, Kew Cabral D (1985) Phyllosphere of Eucalyptus viminalis: dynamics of fungal populations. Trans Br Mycol Soc 85:501–511 Card SD, Tapper BA, Loyd-West C, Wright KM (2013) Assessment of fluorescein-based fluorescent dyes for tracing Neotyphodium endophytes in planta. Mycologia 105(1):221–229 Caroll GC (1995) Forest endophytes: patterns and process. Can J Bot 73:1316–1324 Carroll GC (1988) Fungal endophytes in stems and leaves: from latent pathogen to mutualistic symbionts. Ecology 69:2–9 Carroll GC, Carroll FE (1978) Studies on the incidence of coniferous needle endophytes in the Pacific Northwest. Can J Bot 56:3034–3043 Chithra S, Jasim B, Sachidanandan P, Jyothis M, Radhakrishnan EK (2014) Piperine production by endophytic fungus Colletotrichum gloeosporioides isolated from Piper nigrum. Phytomedicine 21:534–540 Choudhury S, Panda P, Sahoo L, Panda SK (2013) Reactive oxygen species signaling in plants under abiotic stress. Plant Signal Behav 8:e23681. https://doi.org/10.4161/psb.2368 Ciannamea S, Busscher-Lange J, de Folter S, Angenent GC, Immink RGH (2006) Characterization of the vernalization response in Lolium perenne by a cDNA microarray approach. Plant Cell Physiol 47:481–492 Clarridge JE (2004) Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clin Microbiol Rev 17:840–862 Dai CC, Yu BY, Li X (2008) Screening of endophytic fungi that promote the growth of Euphorbia pekinensis. Afr J Biotechnol 7:3505–3509 Daudi A, O’Brien JA (2012) Detection of hydrogen peroxide by dab staining in arabidopsis leaves. Bio-protocol 2(18). http://www.bio-protocol.org/e263 De Hoog GS, Göttlich E, Platas G, Genilloud O, Leotta G, van Brummelen J (2005) Evolution, taxonomy and ecology of the genus Thelebolus in Antarctica. Stud Mycol 51:33–76 Dinkins RD, Barnes A, Waters W (2010) Microarray analysis of endophyte-infected and endophyte-free tall fescue. J Plant Physiol 167:1197–1203 Duarte K, Rocha-Santos TAP, Freitas AC, Duarte AC (2012) Analytical techniques for discovery of bioactive compounds from marine fungi. Trends Analytic Chem 34(1):97–110 Duong LM, Jeewon R, Lumyong S, Hyde KD (2006) DGGE coupled with ribosomal DNA gene phylogenies reveal uncharacterized fungal phylotypes. Fungal Divers 23:121–138 Ellis MB (1976) More dematiaceous hyphomycetes. Commonwealth Mycological Institute, Kew Espinosa-Garcia FJ, Langenheim JH (1990) The endophytic fungal community in leaves of coastal redwood population diversity and spatial patterns. New Phytologist 116:89–97 Feletti SA, Shields K, Ramsperger M, Tian P, Webster T, Ong EK, Sawbridge T, Spagenberg G (2004) Gene discovery and microarray based transcriptome analysis in grass endophytes. In: Hopkins A et al (eds) Proceedings of the 3rd international symposium, molecular breeding of forage and turf. Kluwer Academic, Dordrecht, pp 145–153 Felitti S, Shields K, Ramsperger M, Tian P, Sawbridge T, Webster T et al (2006) Transcriptome analysis of Neotyphodium and Epichloe grass endophytes. Fungal Genet Biol 43:465–475 Fisher PJ, Petrini O (1990) A comparative study of fungal endophytes in xylem and bark of Alnus species in England and Switzerland. Mycol Res 94:313–319 Fisher PJ, Anson AE, Petrini O (1986) Fungal endophytes in Ulex europaeus and Ulex galli. Trans Br Mycol Soc 86:153–156 Fisher PJ, Pertini O, Petrini LE, Sutton BC (1994) Fungal endophytes from leaves and twigs of Quercus ilex L. from England, Majorca and Switzerland. New Phytol 127:133–137 Fröhlich J, Hyde KD, Petrini O (2000) Endophytic fungi associated with palms. Mycol Res 104:1202–1212 Gamboa MA, Laureano S, Bayman P (2002) Measuring diversity of endophytic fungi in leaf fragments: Does size matter? Mycopathologia 156:41–45

78

S. K. Verma et al.

Gao F, Dai C, Liu X (2010) Mechanisms of fungal endophytes in plant protection against pathogens. Afr J Biotechnol Res 4(13):1346–1351 Ghimire SR, Hyde KD (2004) Fungal endophytes. In: Varma A, Abbott L, Werner D, Hampp R (eds) Plant surface microbiology. Springer, Berlin Gond SK, Mishra A, Sharma VK, Verma SK, Kumar J, Kharwar RN, Kumar A (2011) Diversity and antimicrobial activity of endophytic fungi isolated from Nyctanthes arbor-tristis, a wellknown medicinal plant of India. Mycoscience 53:113–121 Gond SK, Mishra A, Sharma VK, Verma SK, Kharwar RN (2014a) Isolation and characterization of antibacterial naphthalene derivative from Phoma herbarum, an endophytic fungus of Aegle marmelos. Curr Sci 105:167–169 Gond SK, Kharwar RN, White JF Jr (2014b) Will fungi be the new source of the blockbuster drug taxol? Fung Biol Rev 28:77–84 Gond SK, Torres MS, Bergen MS, Helse Z, White JF Jr (2015a) Induction of salt tolerance and up-regulation of aquaporin genes in tropical corn by rhizobacterium Pantoea agglomerans. Lett Appl Microbiol 60:392–399 Gond SK, Bergen MS, Torres MS, White JF (2015b) Endophytic Bacillus spp. produce antifungal lipopeptides and induce host defence gene expression in maize. Microbiol Res 172:79–87 Green SJ, Freeman S, Hadar Y, Minz D (2004) Molecular tools for isolate and community studies of perinomycete fungi. Mycologia 96:439–451 Gunatilaka AAL (2006) Natural products from plant-associated microorganisms: distribution, structural diversity, bioactivity and implications of their occurrence. J Nat Prod 69:509–526 Guo LD, Hyde KD, Liew ECY (1998) A method to promote sporulation in palm endophytic fungi. Fungal Divers 1:109–113 Guo LD, Hyde KD, Liew ECY (2001) Detection and taxonomic placement of endophytic fungi within frond tissues of Livistona chinensis based on rDNA sequences. Mol Phylogenet Evol 20:1–13 Guo LD, Huang GR, Wang Y, He WH, Zheng WH, Hyde KD (2003) Molecular identification of white morphotype strains of endophytic endophytic fungi from Pinus tabulaeformis. Mycol Res 107(6):680–688 Hallmann J, Berg G, Schulz B (2006) Isolation procedures for endophytic microorganisms. In: Schulz BJE, Boyle CJC, Sieber TN (eds) Microbial root endophytes, vol 9. Soil biology. pp 299–319 Hata F, Futai K (1995) Endophytic fungi associated with healthy pine needles and needles infested by pine needle gall midge Thecodiplosis japonensis. Can J Bot 73:384–390 Hawksworth DL (1987) Observations on three algicolus microfungi. Notes R Bot Gard, Edinb 44:549–560 Hood ME, Shew HD (1996) Applications of KOH-aniline blue fluorescence in the study of plantfungal interactions. Phytopathology 86:704–708 Hugenholtz P, Goebel BM, Pace NR (1998) Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J Bacteriol 180(18):4765–4774 Hyde KD, Soytong K (2008) The fungal endophyte dilemma. Fungal Divers 33:163–173 Hyde KD, Frohlich J, Taylor JE (1997) In: Hyde KD (ed) Diversity of Ascomycetes on palms in the tropics. Biodiversity of tropical microfungi. Hong Kong University Press, Hong Kong, pp 141–156 Irizarry I, White JF (2017) Application of bacteria from non-cultivated plants to promote growth, alter root architecture and alleviate salt stress of cotton. J Appl Microbiol. https://doi.org/10. 1111/jam.13414 Iwen PC, Hinrichs SH, Rupp ME (2002) Utilization of the internal transcribed spacer regions as molecular targets to detect and identify human fungal pathogens. Med Mycol 40:87–109 Johnson LJ, Johnson RD, Schardl CL, Panaccione DG (2003) Identification of differentially expressed genes in the mutualistic association of tall fescue with Neotyphodium coenophialum. Physiol Mol Plant Pathol 63:305–317 Jumpponen A, Jones KL (2009) Massively parallel 454 sequencing indicate hyperdiverse fungal communities in temperate Quercus macrocarpa phyllosphere. New Phytol 18:438–448

4

Exploring Endophytic Communities of Plants: Methods for Assessing. . .

79

Kaul S, Sharma T, Dhar MK (2014) Omics tools for better understanding the plant endophyte interactions. Front Plant Sci. https://doi.org/10.3389/fpls.2016.00955 Kharwar RN, Verma VC, Kumar A, Gond SK, Harper JK, Hess WM, Ma C, Ren Y, Strobel GA (2009) Javanicin, an antibacterial naphthaquinone from an endophytic fungus of Neem, Chloridium sp. Curr Microbiol 58:233–238 Kharwar RN, Verma SK, Mishra A, Gond SK, Sharma VK, Afreen T, Kumar A (2011a) Assessment of diversity, distribution and antibacterial activity of endophytic fungi isolated from a medicinal plant Adenocalyma alliaceum Miers. Symbiosis 55:39–46 Kharwar RN, Mishra A, Gond SK, Stierle A, Stierle D (2011b) Anticancer compounds derived from fungal endophytes: their importance and future challenges. Nat Prod Rep 28:1208–1228 Khot PD, Ko DL, Fredricks DN (2009) Sequencing and analysis of fungal rRNA operons for development of broad-range fungal PCR assays. Appl Environ Microbiol 75:1559–1565 Kumar DSS, Hyde KD (2004) Biodiversity and tissue-recurrence of endophytic fungi in Tripterygium wilfordii. Fungal Divers 17:69–90 Kumar J, Sharma VK, Singh DK, Mishra A, Gond SK, Verma SK, Kumar A, Kharwar RN (2016) Epigenetic activation of antibacterial property of an endophytic Streptomyces coelicolor Strain AZRA 37 and identification of the induced protein using MALDI TOF MS/MS. PLoS One 11 (2):e0147876. https://doi.org/10.1371/journal.pone.0147876 Kumaresan V, Suryanarayanan TS (2002) Endophyte assemblage in young, mature and senescent leaves of Rhizophora apiculata: evidence for the role of endophytes in mangrove litter degradation. Fungal Divers 9:81–91 Kusari S, Spiteller M (2011) Are we ready for industrial production of bioactive plant secondary metabolites utilizing endophytes? Nat Prod Rep 28:1203–1207 Kusari S, Spiteller M (2012) Camptothecin: recent advances in plant endophyte research. In: Patro LR (ed) Natural resources conservation and management. Manglam Publications, New Delhi, pp 1–32 Kusari S, Lamshoft M, Zuhlke S, Spiteller M (2008) An endophytic fungus from Hypericum perforatum that produces hypericin. J Nat Prod 71:159–162 Kusari S, Zuhlke S, Spiteller M (2009) An endophytic fungus from Camptotheca acuminata that produces camptothecin and analogues. J Nat Prod 72:2–7 Kusari S, Hertweck C, Spiteller M (2012) Chemical ecology of endophytic fungi: origins of secondary metabolites. Chem Biol 19(7):792–798 Kutser E, Williams ST (1964) Selection of media for the isolation of Streptomyces. Nature 202:928–929 Li WC, Zhou J, Guo SY, Guo LD (2007) Endophytic fungi associated with lichens in Baihua mountain of Beijing, China. Fungal Divers 25:69–80 Li E, Tian R, Liu S, Chen X, Guo L, Che Y (2008) Pestalotheols A-D, bioactive metabolites from the plant endophytic fungus Pestalotiopsis theae. J Nat Prod 71(4):664–668 Lindahl BD, Nilsson RH, Tedersoo L et al (2013) Fungal community analysis by high-throughput sequencing of amplified markers – a user’s guide. New Phytol 199:288–299 Liu X, Dong M, Chen X, Jiang M, Lv X, Yan G (2007) Antioxidant activity and phenolics of an endophytic Xylaria sp. from Ginkgo biloba. Food Chem 105:548–554 Lucero ME, Unc A, Cooke P, Dowd S, Sun SL (2011) Endophyte microbiome diversity in micropropagated Atriplex canescens and Atriplex torreyi var. griffithsii. PLoS One 6:e17693 Ma LJ, Catramis CM, Rogers SO, Starmer WT (1997) Isolation and characterization fungi entrapped in glacial ice. Inoculum 48:23–24 Malinowski DP, Belesky DP (2000) Adaptations of endophyte-infected cool-season grasses to environmental stresses: mechanisms of drought and mineral stress tolerance. Crop Sci 40:923–940 Manter Daniel K, Delgado JA, Holm DJ, Stong RA (2010) Pyrosequencing reveals a highly diverse and cultivar-specific bacterial endophyte community in potato roots. Microbial Ecol 60 (1):157–166 Marco D (2011) Metagenomics: current innovations and future trends. Caister Academic Press. ISBN 978-1-904455-87-5

80

S. K. Verma et al.

Mardis ER (2008) The impact of next-generation sequencing technology on genetics. Trends Genet 24(3):133–141 Mishra A, Gond SK, Kumar A, Sharma VK, Verma SK, Kharwar RN, Sieber TN (2012) Season and tissue type affect fungal endophyte communities of the Indian medicinal plant Tinospora cordifolia more strongly than geographic location. Microb Ecol 64:3288–3398 Mousa WK, Shearer C, Limay-Rios V, Ettinger CL, Eisen JA, Raizada MN (2016) Root-hair endophyte stacking in finger millet creates a physicochemical barrier to trap the fungal pathogen Fusarium graminearum. Nat Microbiol 16167. https://doi.org/10.1038/NMICROBIOL.2016.167 Omacini M, Chaneton EJ, Ghersa CM, Müller CB (2001) Symbiotic fungal endophytes control insect host–parasite interaction webs. Nature 409:78–81 Orduña FNR, Sanchez RAS, Bustamante ZRF, Rodriguez JNG, Cotera LBF (2011) Diversity of endophytic fungi of Taxus globosa (Mexican yew). Fungal Divers 47:65–74 Petrini O (1991) Fungal endophytes of tree leaves. In: Adrews J, Hirano S (eds) Microbial ecology of leaves. Springer, Berlin, pp 179–197 Petrini O, Fisher PJ (1988) A comparative study of fungal endophytes in xylem and whole stems of Pinus sylvestris and Fagus sylvatica. Trans Br Mycol Soc 91:233–238 Petrini O, Muller E (1979) Pilzliche Endophyten, am Beispiel von Juniperus communis L. Sydowia 32:224–251 Petrini O, Stone JK, Carroll FE (1982) Endophytic fungi in evergreen shrubs in western Oregon: a preliminary study. Can J Bot 60:789–796 Petrini O, Hake U, Dreyfuss MM (1990) An analysis of fungal communities isolated from fruticose lichens. Mycologia 82:444–451 Petrini O, Sieber TN, Toti L, Viret O (1992) Ecology, metabolite production and substrate utilization in endophytic fungi. Nat Toxin 1:185–196 Ranghoo VM, Hyde KD, Liew ECY, Spatafora JW (1999) Family placement of Ascotaiwanian and Ascolacicola based on DNA sequences from the large subunit rRNA gene. Fungal Divers 2:159–168 Redman RS, Sheehan KB, Stout RG, Rodriguez RJ, Henson JM (2002) Thermotolerance generated by plant/fungal symbiosis. Science 298:1581 Redman RS, Kim YO, Woodward CJDA, Greer C, Espino L et al (2011) Increased fitness of rice plants to abiotic stress via habitat adapted symbiosis: a strategy for mitigating impacts of climate change. PLoS One 6(7):e14823. https://doi.org/10.1371/journal.pone.0014823 Rodriguez RJ, White JF, Arnold JAE, Redman RS (2009) Fungal endophytes: diversity and functional roles. New Phytol 182:314–330 Romero FM, Marina M, Pieckenstain FI (2014) The community of tomato (Solanum lycopersicum L.) leaf endophytic bacteria, analyzed by 16s-ribosomal RNA gene pyrosequencing. FEMS Microbiol Lett 351:187–194 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425 Sawbridge T, Ong E-K, Binnion C, Emmerling M et al (2003) Generation and analysis of expressed sequence tags in perennial ryegrass (Lolium perenne L.). Plant Sci 165:1089–1100 Schulz B, Wanke U, Draeger S (1993) Endophytes from herbaceous and shrubs: effectiveness of surface sterilization methods. Mycol Res 97:1447–1450 Schulz B, Guske S, Dammann U, Boyle C (1998) Endophyte–host interaction II. Defining symbiosis of the endophyte–host interaction. Symbiosis 25:213–227 Schulz B, Boyle C, Draeger S, Mmert AKR, Krohn K (2002) Endophytic fungi: a source of novel biologically active secondary metabolites. Mycol Res 106(9):996–1004 Segata N, Boernigen D, Tickle TL, Morgan XC, Garrett WS, Huttenhower C (2013) Computational meta'omics for microbial community studies. Mol Syst Biol 9(666):666. https://doi.org/10. 1038/msb.2013.22 Sessitsch P, Hardoim J, Doring A, Weilharter A, Krause T, Woyke B et al (2012) Functional characteristics of an endophyte community colonizing rice roots as revealed by metagenomic analysis. Mol Plant Microbe Interact 25:28–36 Shweta S, Zuehlke S, Ramesha BT, Priti V, Kumar PM, Ravikanth G, Spiteller M et al (2010) Endophytic fungal strains of Fusarium solani, from Apodytes dimidiata E. Mey. ex Arn

4

Exploring Endophytic Communities of Plants: Methods for Assessing. . .

81

(Icacinaceae) produce camptothecin, 10-hydroxycamptothecin and 9-methoxycamptothecin. Phytochemistry 71:117–122 Staley JT, Konopka A (1985) Measurement of in situ activities of non photosynthetic microorganisms in aquatic and terrestrial habitats. Annu Rev Microbiol 39:321–346 Stewart EJ (2012) Growing unculturable bacteria. J Bacteriol 194:4151–4416 Stierle A, Strobel GA, Stierle D (1993) Taxol and taxane production by Taxomyces andreanae an endophytic fungus of Pacific yew. Science 260:214–216 Stone JK, Polishook JD, White JF (2004) Endophytic fungi. In: Mueller G, Bills GF, Foster MS (eds) Biodiversity of fungi: inventory and monitoring methods. Elsevier, Burlington, MA, pp 241–270 Strobel GA, Daisy B (2003) Bioprospecting for microbial endophytes and their natural products. Microbiol Mol Biol Res 67:491–502 Strobel GA, Miller RV, Miller C, Condron M, Teplow DB, Hess WM (1999) Cryptocandin, a potent antimycotic from the endophytic fungus Cryptosporiopsis cf. quercina. Microbiology 145:1919–1926 Strobel G, Ford E, Worapong J, Harper JK, Arif AM, Grant DM, Fung PCW, Chau RMW (2002) Isopestacin, an isobenzofuranone from Pestalotiopsis microspora, possessing antifungal and antioxidant activities. Phytochemistry 60:179–183 Sugita T, Nishikawa A (2003) Fungal identification method based on DNA sequence analyisis: reassessment of the method of Pharmaceutical society of Japan and the Japanese Pharmacopoeia. J Health Sci 49(6):531–533 Sun X, Guo L-D (2012) Endophytic fungal diversity: review of traditional and molecular techniques. Mycology 3(1):65–76 Sun JQ, Guo LD, Zang W, Ping WX, Chi DF (2008) Diversity and ecological distribution of endophytic fungi associated with medicinal plants. Sci China Ser C 51:751–759 Suryanarayanan TS, Senthilarasu G, Muruganandam V (2000) Endophytic fungi from Cuscuta reflexa and its host plants. Fungal Divers 4:117–123 Tan RX, Zou WX (2001) Endophytes: a rich source of functional metabolites. Nat Prod Rep 18:448–459 Taylor JE, Hyde KD, Jones EBG (1999) Endophytic fungi associated with the temperate palm, Trachycarpus fortunei, within and outside its natural geographic range. New Phytol 142:335–346 Thirugnanasambandam A, Wright KM, Atkins SD, Whisson SC, Newton AC (2011) Infection of Rrs1 barley by an incompatible race of the fungus Rhynchosporium secalis expressing the green fluorescent protein. Plant Pathol 60:513–521 Thomas P, Reddy KM (2013) Microscopic elucidation of abundant endophytic bacteria colonizing the cell wall–plasma membrane peri-space in the shoot-tip tissue of banana. AoB PLANTS 5: plt011. https://doi.org/10.1093/aobpla/plt011 Thomas T, Gilbert J, Meyer F (2012) Metagenomics – a guide from sampling to data analysis. Microb Inform Exp 2:3. http://www.microbialinformaticsj.com/content/2/1/3 Tian BY, Cao Y, Zhang K-Q (2015) Metagenomic insights into communities, functions of endophytes, and their associates with infection by root-knot nematode, Meloidogyne incognita, in tomato roots. Sci Rep 5:17087 Toju H, Yamamoto S, Sato H, Tanabe AS, Gilbert GS, Kadowaki K (2013) Community composition of root-associated fungi in a Quercus-dominated temperate forest: “codominance” of mycorrhizal and root-endophytic fungi. Ecol Evol 3:1281–1293. https://doi.org/10.1002/ece3.546 Verma VC, Gond SK, Kumar A, Kharwar RN, Strobel GA (2007) Endophytic mycoflora from leaf, bark, and stem of Azadirachta indica A Juss. from Varanasi, India. Microb Ecol 54:119–125 Verma VC, Kharwar RN, Strobel GA (2009a) Chemical and functional diversity of natural products from plant associated endophytic fungi. Nat Prod Commun 4(11):1511–1532 Verma VC, Gond SK, Kumar A, Mishra A, Kharwar RN, Gange A (2009b) Endophytic actinomycetes from Azadirachta indica A. Juss.: isolation, diversity, and anti-microbial activity. Microbial Ecol 57:749–756

82

S. K. Verma et al.

Verma VC, Gond SK, Kumar A, Kharwar RN, Boulanger LA, Strobel GA (2011) Endophytic fungal flora from roots and fruits of an Indian neem plant Azadirachta indica A. Juss., and impact of culture media on their isolation. Indian J Microbiol 51(4):469–476 Verma SK, Gond SK, Mishra A, Sharma VK, Kumar J, Singh DK, Kumar A, Goutam J, Kharwar RN (2014) Impact of environmental variables on the isolation, diversity and antibacterial activity of endophytic fungal communities from Madhuca indica Gmel. at different locations in India. Ann Microbiol 64(2):721–734 Verma SK, Kingsley K, Irizarry I, Bergen M, Kharwar RN, White JF (2017) Seed vectored endophytic bacteria modulate development of rice seedlings. J Appl Microbiol 22:1680–1691 Von Arx JA (1978) The genera of fungi sporulating in pure culture. Gantner AR, Verlag KG, Vaduz Wang Y, Ohara Y, Nakayashiki H, Tosa Y, Mayama S (2005) Microarray analysis of the gene expression profile induced by the endophytic plant growth promoting rhizobacteria bacteria, Pseudomonas fluorescens FPT9601-T5 in Arabidopsis. Mol Plant Microbe Interact 18:385–396 Wang FW, Ye YH, Chen JR, Wang XT, Zhu HL, Song YC, Tan RX (2006) Neoplaether, a new cytotoxic and antifungal endophyte metabolite from Neoplaconema napellum IFB-E016. FEMS Microbiol Lett 261:218–223 White JF Jr, Crawford H, Torres MS, Mattera R, Irizarry I, Bergen M (2012) A proposed mechanism for nitrogen acquisition by grass seedlings through oxidation of symbiotic bacteria. Symbiosis 57:161–117 White JF Jr, Torres MS, Somu MP, Johnson H, Irizarry I, Chen Q, Zhang N, Walsh E, Tadych M, Bergen M (2014a) Hydrogen peroxide staining to visualize bacterial infections of seedling root cells. Microscop Res Techniq 77:566–573 White JF, Torres MS, Sullivan RF, Jabbour RE, Chen Q, Tadych M et al (2014b) Occurrence of Bacillus amyloliquefaciens as a systemic endophyte of vanilla orchids. Microsc Res Tech 77 (11):874–885. https://doi.org/10.1002/jemt.22410 White TF, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky FS, White TT (eds) PCR protocol: a guide to methods and applications. Academic Press, San Diego, pp 315–322 White JF, Chen Q, Torres MS, Mattera R, Irizarry I, Tadych M, Bergen M (2015) Collaboration between grass seedlings and rhizobacteria to scavenge organic nitrogen in soils. AoB PLANTS 7:plu093. https://doi.org/10.1093/aobpla/plu093 White JF, Kingsley K, Kowalski KP, Irizarry I, Micci A, Soares M, Bergen MS (2017) Disease protection and allelopathic interactions of seed-transmitted endophytic pseudomonads of invasive reed grass (Phragmites australis). Plant Soil. https://doi.org/10.1007/s11104-016-3169-6 Xu J, Yang X, Lin Q (2014) Chemistry and biology of Pestalotiopsis-derived natural products. Fungal Divers 66:37–68 Zhang W, Wildel JF, Clark LG (1997) Bamboozled again! Inadvertent isolation of fungal rDNA sequences from bamboos (Poaceae: Bambusoideae). Mol Phylogenet Evol 8:205–217

5

Understanding the Indigenous Seed Microbiota to Design Bacterial Seed Treatments Birgit Wassermann, Eveline Adam, Tomislav Cernava, and Gabriele Berg

Contents 5.1 The Impact of Domestication on Plants and Seeds: Diversification and Diversity Loss . . . 5.2 The Plant and Seed Microbiota and Their Main Drivers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1 Plants Harbor Distinct Habitat-Specific and Species-Specific Microbial Signatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2 The Seed Microbiota and Its Specific Microbial Signatures and Drivers . . . . . . . . . 5.3 Microbial Diversity and Health Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.1 The Interconnected Microbiome Highlights the One Health Concept . . . . . . . . . . . . 5.3.2 The Role of Soil and Seed Microbiomes to Maintain Microbial Diversity . . . . . . . 5.4 Biotechnological Solutions for Sustainable Agriculture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

84 85 85 86 90 90 93 94 96 96

Abstract

Within millennia of domestication, crops and their seeds underwent traceably different adaptive trends, allowing rapid speciation and divergence that lead to phenotypic and genotypic distinction to their wild ancestors. Promoted by these dynamic processes, also the microbiotas have secretly coevolved with the host plants. Recent studies revealed an unexpected microbial diversity and abundance within seeds with bacterial endophytes as symbiotic components. Soil type, climate, geography and plant genotype were identified as main drivers of the seed microbiota. In addition, domestication and intensive agricultural management changed the seed microbiota. This resulted in a loss of diversity, which has consequences for one health-related issues. In order to restore microbial diversity, bacterial seed treatments can be designed. They can be reconstructed based on the rich diversity of seeds of wild ancestors or other native plants. The resulting seed B. Wassermann · E. Adam · T. Cernava · G. Berg (*) Institute of Environmental Biotechnology, Graz University of Technology, Graz, Austria e-mail: [email protected]; [email protected]; [email protected]; [email protected] # Springer Nature Switzerland AG 2019 S. K. Verma, J. F. White, Jr (eds.), Seed Endophytes, https://doi.org/10.1007/978-3-030-10504-4_5

83

84

B. Wassermann et al.

biologicals can be harnessed for sustainable agricultural approaches by improving stress tolerance and resilience of modern crops. Keywords

Seed microbiome · Holobiont · Domestication · One health · Seed biologicals

5.1

The Impact of Domestication on Plants and Seeds: Diversification and Diversity Loss

The domestication of plants and animals was a precondition for the expansion of civilization and the transformation of worldwide demography (Diamond 2002). It gave rise to the recent onset of rapid evolution and accompanied immense diversification and a global spread of domesticated plants. These dynamics inspired Charles Darwin in the process of formulating the thesis on the origin of species through natural selection (Darwin 1859). The cultivation of crop plants started 13,000 years ago, and today’s divergence of domesticated plants to their wild ancestors emerged as a consequence of selecting wild plants that were gathered and cultivated by hunter–gatherers in early domestication periods (Darwin 1859). In contrast to the early periods of domestication, which resulted in a diversification of plant genotypes, today’s agriculture and human lifestyle push domestication processes to a distinct outcome: a global landscape highly dominated by modern crops, accompanied by the homogenization of plant genotypes (Purugganan and Fuller 2009). In addition, nutritional demand of the growing world population is constantly increasing, and due to constraints in time and space, agriculture is focusing on extensive breeding and cultivation of specific crop cultivars with desired genotypic and phenotypic characteristics (Gruber 2017). Today, 90% of world’s energy demand is accomplished by only 15 crops, and two-thirds of the world’s calorie intake depends on rice, maize, and wheat (Food and Agriculture Organization of the United Nations). Specialized breeding and crop selection engender the loss of heirloom breeds; a sheer amount of 70% of wild relatives of modern crops are in risk of getting lost (Castañeda-Álvarez et al. 2016). Due to recent developments, the genetic diversity of plants is in urgent need of protection as an untold numbers of plant genotypes are going extinct. Altogether, domestication and especially intense agriculture causes long-lasting anthropogenic environmental impacts as it replaces natural vegetation, and thereby decreases diversity, and alters biogeochemical cycles. Therefore, a new humandominated geological epoch, the Anthropocene was defined (Lewis and Maslin 2015). There are many examples for significant anthropogenic signatures, which are related to agriculture. The conversion of atmospheric nitrogen to ammonia by the Haber–Bosch process for fertilizer production has altered the global nitrogen cycle so fundamentally that the nearest suggested geological comparison refers to events about 2.5 billion years ago (Canfield et al. 2010). A likewise global effect was induced by the land use conversion for agriculture. Large-scale conversions resulted in species extinctions some 100–1000 times higher than background rates and

5

Understanding the Indigenous Seed Microbiota to Design Bacterial Seed Treatments

85

probably constitute the beginning of the sixth mass extinction in Earth’s history. Crops, domesticated animals, and pathogens are efficiently exchanged around the world; this leads to a global homogenization of Earth’s biota. Seeds transmit the footprint of domestication (Berg and Raaijmakers 2018), and especially their altered morphology over time is therefore frequently studied by archeobotanic and genomic research. Cultivation pressure on plant seeds started about 8000 years ago and was primarily applied on seeds of the plant family Poaceae. Targeted traits were the improvement of germination, with increased soil disturbance and sowing depth, as well as facilitated harvesting (Harlan 1973). Those traits were accompanied by the two major alterations on seed phenotypes, namely, increased grain size and the selection of non-shattered cultivars (Baskin and Baskin 1998). The latter is considered as the most characteristic trait for plant domestication as it predicates successful seed-dispersal on human activity (Purugganan and Fuller 2009). Between then and now, extended multistage processes altered the genotype and phenotype of crop seeds. However, present seed treatments are considered to be among the most severe trends since the early stages of plant domestication.

5.2

The Plant and Seed Microbiota and Their Main Drivers

5.2.1

Plants Harbor Distinct Habitat-Specific and Species-Specific Microbial Signatures

Plants and their associated microbes have been interacting with each other for a long time, forming assemblages of species that are referred to as holobionts (Vandenkoornhuyse et al. 2015). The plant-associated microbiota has the ability to contribute multiple aspects to the functioning of the plant holobiont, such as (1) seed germination and growth support, (2) nutrient supply, (3) resistance against biotic stress factors (pathogen defense), (4) resistance against abiotic factors, and (5) production of bioactive metabolites (Berg 2016). Plants harbor distinct habitatspecific microbial signatures, which are mainly shaped by abiotic factors. The phyllosphere comprises all aboveground organs, which are exposed to the air and permanently changing abiotic factors such as ultraviolet (UV) radiation, temperature, and water, and a general low nutrient availability (Remus-Emsermann and Schlechter 2018). The phyllosphere can be further subdivided into the caulosphere (stems), phylloplane (leaves), anthosphere (flowers), and carposphere (fruits). Endophytic communities represent an intimate core of the plant microbiota, and distinct connections of the different plant microhabitats and development stages are of special importance for health issues (Hardoim et al. 2015). A reservoir for the plant’s endophytes is the rhizosphere, which represents the below-ground interface with the highly diverse soil microbiota (Berg et al. 2005). Due to this importance for the holobiont, the factors that shape the plant microbiome have been studied for a long time. After a longer debate, it is accepted that the plant genotype and the soil quality are the crucial factors influencing the composition of the rhizosphere microbiota (Berg and Smalla 2009). Both have an impact, but the

86

B. Wassermann et al.

extent depends on many factors (plant’s morphology and secondary metabolism and soil type) and is triggered by plant root exudates and signaling (Badri and Vivanco 2009; Doornbos et al. 2012). The spermosphere is the zone surrounding seeds where interactions between the soil, microbial communities, and germinating seeds take place (Schiltz et al. 2015). This microenvironment links the above and below-ground microbiome of plants. Plant domestication processes have impacted the plant microbiota assembly and its functions via habitat expansion and via changes in crop management practices, root exudation, root architecture, and plant litter quality (Pérez-Jaramillo et al. 2016). The authors proposed a “back to the roots” framework that comprises the exploration of the microbiome of indigenous plants and their native habitats for the identification of plant and microbial traits, with the ultimate goal to reinstate beneficial associations that may have been undermined during plant domestication.

5.2.2

The Seed Microbiota and Its Specific Microbial Signatures and Drivers

For a long time, it was assumed that the emerging seedling is colonized by microorganisms from its surrounding environment, with soil being the main source, controlled by the plant through different strategies, such as the specific profile of root exudates and its immune system (Truyens et al. 2014; Sánchez-Cañizares et al. 2017; Shade et al. 2017). Therefore, the study of the seed’s microbiota was often neglected in the past or focused only on the occurrence of pathogens. Moreover, the relevant literature is largely based on culture-dependent investigations (Nelson 2018). The seed itself was the last “wasteland” in the landscape of plant microbiology. In the last decade, seeds have been discovered as source for the transmission of a plant-specific core microbiota; an overview of selected studies and their main findings is shown in Table 5.1. Noteworthy, all of these studies revealed an unexpectedly high diversity and abundance of the seed-associated microbiota (Berg and Raaijmakers 2018). In some of these studies, up to 20,000 microbial species and up to two billions of bacterial cells were detected in one seed (Adam et al. 2018; Johnston-Monje et al. 2016; Shade et al. 2017). In general, the seed microbiota consists of bacteria, archaea, and fungi. The presented studies also focus on the main drivers of the seed microbiota. Microbial compositions of seeds are described to vary between different geographical sites (Klaedtke et al. 2016), soil types, and soil-associated microbiomes (Hardoim et al. 2012; Liu et al. 2013). In addition, microbial inoculants including pathogens and beneficials were shown to shape the seed’s microbiota (Mormile 2016; Rezki et al. 2016). However, plant genotype specificity of the seed microbiome has been described frequently (Barret et al. 2015; Adam et al. 2018; Rybakova et al. 2017; Wassermann et al., unpublished data) and it was shown that seed endophytes can even be highly conserved across generations of a plant species (Johnston-Monje and Raizada 2011; Links et al. 2014). Besides the horizontal transfer of microbiota from diverse environmental sources, thus, vertical transfer of microbiota to the next generation via

5

Understanding the Indigenous Seed Microbiota to Design Bacterial Seed Treatments

87

Table 5.1 Overview of the current studies on seed microbiomes with their main findings Plant species/cultivar Maize (Zea mays)

Rice (Oryza sativa)

Brassica and Triticum species

Bean (Phaseolus vulgaris) Radish (Raphanus sativus)

Maize (Z. mays)

Tall fescue (Schedonorus arundinaceus)

Sueda salsa

Quinoa

Cucurbitaceae species

Pepper (Capsicum annuum), soybean (Glycine max), T. aestivum

Main findings Domesticated maize and its wild ancestor share a significant core microbiota within seeds with potential plant growth-promoting activities. The seed microbiota is conserved across boundaries of evolution, ethnography and ecology Seeds of two consecutive rice generations were shown to share 45% of bacterial endophytes. Soil type is a major driver of the relative abundance of seed-borne strains A conserved epiphytic core microbiota on seeds of geographically and ecologically distinct crops of the same species was identified. It included bacterial strains with antagonistic potential toward a fungal plant pathogen The seed microbiome is affected by the host’s terroir The bacterial seed microbiome was not changed by application of phytopathogenic bacteria, while application of a fungal pathogen changed the seed mycobiome, without affecting bacterial assemblages Plants grown in sterile and non-sterile soils shared the same dominant rhizosphere microbiota, suggesting seeds to be the primary inoculum Infection by Epichloë coenophiala promoted fitness of the host plant by influencing the microbiome composition of seeds The dominant seed endophyte Cladosporium cladosporioides improved host germination rate Peculiarities of quinoa regarding stress resistance and germination ability is in part explainable by seed endophyte activity, particularly by seed-borne Bacillus species Seeds of 21 cucurbit varieties shared a cultivable core microbiota consisting of Bacillus species, potentially promoting host plants A potential biocontrol agent was introduced into seeds of various crop species. The accompanied modification of seed microbiota enhanced plant growth of treated seeds compared to control seeds in field trials

References JohnstonMonje and Raizada (2011)

Hardoim et al. (2012)

Links et al. (2014)

Klaedtke et al. (2016) Rezki et al. (2016)

JohnstonMonje et al. (2016) Mormile (2016)

Qin et al. (2016)

Pitzschke (2016)

Khalaf and Raizada (2016)

Mitter et al. (2017)

(continued)

88

B. Wassermann et al.

Table 5.1 (continued) Plant species/cultivar Malvaceae species

Oilseed rape (B. napus)

Soybean (Glycine max)

Rice (O. sativa)

Radish (R. sativus)

Muskmelon (Cucumis melo) Browntop millet (Brachiaria sp.) Cucurbitaceae species

Bean (P. vulgaris)

Bean (P. vulgaris), radish (R. sativus)

Barley (Hordeum vulgare)

Cucumber (Cucumis sativus)

Main findings Natural cotton seeds harbored plant beneficial bacteria that promoted growth and alleviated salt stress when they were applied on cultivated plants under abiotic stress conditions High genotype-specific bacterial diversity in seeds entailed colonization resistance toward potential pathogens and applied biologicals Seed microbial diversity was higher compared to sprout microbial diversity and taxonomy suggested sprouts to contain beneficial bacteria transmitted from seeds Regardless of physiological salinity tolerance of the host, seeds of different plants shared a similar microbiota with stress tolerance alleviation and plant growth-promoting activities The composition of seed microbiota was related to host community membership. Ecological drift and dispersal drives bacterial and fungal seed endophytes Groups of seed endophytes are specialized to specific niches within seeds Indigenous seed endophytes promoted seedling development and protected seedlings from fungal pathogens Cultivable seed endophytes possessed significant disease suppression potential against five major fungal and oomycete pathogens, by secretion of bioactive VOCs and extracellular ribonucleases Plant beneficial Azospirillum brasilense was vertically transmitted from the mother plant, forming significant intercellular population in seeds Changing nutrient availability was followed by a selection of microbiota with functional traits linked to copiotrophy by different plant species Barley seed endophytes showed high rhizosphere competence and plant growthpromoting effects. They induced resistance against a Blumeria pathogen in a greenhouse assay Microbes, recruited by germinating seeds, modified seed exudates to reduce encystment and germination of phytopathogenic Pythium species

References Irizarry and White (2017)

Rybakova et al. (2017)

Huang et al. (2017)

Walitang et al. (2017)

Rezki et al. (2018)

Glassner et al. (2018) Verma and White (2018) Khalaf and Raizada (2018)

Malinich and Bauer (2018)

Gloria et al. (2018)

Rahman et al. (2018)

Jack and Nelson (2018)

(continued)

5

Understanding the Indigenous Seed Microbiota to Design Bacterial Seed Treatments

89

Table 5.1 (continued) Plant species/cultivar Crotalaria pumila

Phragmites australis

Salvia miltiorrhiza

Ground-ivy (Glechoma hederacea)

Pumpkin (Cucurbita pepo)

Main findings A bacterial microbiota was shared across three consecutive seed generations. It included a high abundance of bacteria that supported the host growing in metal mine residues Seed microbiota improved seed germination and plant growth of P. australis and protected the host from damping off disease, while mortality of competitor plants was increased The seed core microbiome supports plant health and showed indications to supplement secondary metabolic capabilities of the host plant A similar pool of bacteria and fungi were vertically transmitted from the mother plant to the offshoots in clonal plants. A significant effect of the distance between mother and daughter plants was found Seed microbiomes have stronger genotype specificity but lower diversity compared to the rhizosphere

References Sánchez-López et al. (2018)

White et al. (2018)

Chen et al. (2018)

Vannier et al. (2018)

Adam et al. (2018)

seeds plays a key role in adjusting the seed microbiome (Truyens et al. 2014). The mother plant is suggested to be responsible for the recruitment (Nelson 2018), and the plant genotype to be the main driver of a specialized seed microbiota. Hence, threats of plant extinction, driven by the implications of recent trends in human culture, affect the whole genomic entirety of the holobiont. Incidentally, the plant microbiota influences evolution of plants, as well as their phenotypic and epigenetic plasticity (Van der Heijden et al. 2016); thereby biodiversity-loss forges ahead. A clear and drastic impact of domestication on seed microbiota was identified (Pérez-Jaramillo et al. 2017). Together with the centralized production and intensive treatment on seeds, plant genotype-specific seed microbiota is most probably homogenized and reduced, taking their functional and metabolic secrets with them (Fig. 5.1). The indigenous seed microbiota is characterized by a high diversity and abundance of bacteria, archaea, and fungi. The seed microbiome consists of up to 20,000 microbial species and up to two billions of bacterial cells in one seed. The composition of the microbiota is influenced by the soil type and its microbial population, by climate and geography, as well as by biotic factors such as pathogens and pests. However, the plant genotype is the main driver; therefore, crop domestication has a crucial impact on the seed microbiota.

90

B. Wassermann et al.

Fig. 5.1 Factors influencing the composition of the seed microbiota. Negative effects of crop domestication are highlighted together with a potential countermeasure

When the impact of domestication on crop seed microbiomes is studied, the seed microbiomes of plants from natural ecosystems are especially of interest, as solely undisturbed environments are appropriate to explain indigenous plant–microbeinteractions. Seeds of plants from natural ecosystems have to feature high adaptations in dispersal, persistence, and germinative ability under diverse environmental conditions (Fenner and Thompson 2005). Seeds and seedlings are exposed to a range of hazards like drought, resource limitation, herbivores, and eukaryotic or prokaryotic pathogens (Bever et al. 2015). The seed microbiome, considered as the primary inoculum for plants (Barret et al. 2015), might have a major impact on the plant’s possibilities to combat this plethora of biotic and abiotic stressors. Different visualization techniques can be applied to verify the colonization of seeds by distinct microorganisms. Scanning electron microscopy (SEM) and confocal laser scanning microscopy in combination with fluorescent in situ hybridization (FISH-CLSM) were used to visualize native micro niches of bacteria and fungi colonizing the seed surfaces (Fig. 5.2) and internal seed tissues (Fig. 5.3) of natural plants from the east alpine region of Austria. Studies that target seed microbiomes of plants from natural ecosystems are, however, still rare.

5.3

Microbial Diversity and Health Issues

5.3.1

The Interconnected Microbiome Highlights the One Health Concept

The microbiota of soil and plants plays a crucial role in plant and ecosystem health (Berg et al. 2017; Laforest-Lapointe et al. 2017). Recently, the importance of the plant microbiota for human health was evidenced (David et al. 2014). The plant-

5

Understanding the Indigenous Seed Microbiota to Design Bacterial Seed Treatments

91

Fig. 5.2 Scanning electron micrographs visualizing the seed surface of natural plants (Gentiana asclepiadea, Gentianella germanica and Parnassia palustris) from the east alpine region of Austria. All seeds, sampled exclusively from healthy plants of healthy populations, were densely colonized by various bacteria and fungi, as indicated by white and yellow arrows, respectively

92

B. Wassermann et al.

Fig. 5.3 Confocal laser scanning micrographs of endophytic colonization patterns of P. palustris and G. asclepiadea seeds, by bacteria (indicated by white arrows) and fungi (yellow arrows). Bacteria were stained by fluorescent in situ hybridization and fungal structures were visualized by calcofluor white staining. Plants were gathered from the east alpine region of Austria

associated microbial diversity can be transferred to the gut microbiome, because fruits and vegetables are the major components of a healthy diet. However, loss of microbial diversity in the gut is associated with acute outbreaks as well as with chronic disease, e.g., allergies, obesity, and mental diseases (Turnbaugh et al. 2006). Increasing chronic diseases in children can be explained by the missing microbe theory published by Blaser (2014). In 2017, this was further developed into the theory of disappearing microbiota and the epidemics of chronic diseases, which postulates that losses of particular bacterial species of our ancestral microbiota have altered the context in which immunological, metabolic and cognitive development occur in early life, resulting in increased disease susceptibility (Blaser 2017). Already in 2012, Hanski et al. showed that microbial biodiversity, human

5

Understanding the Indigenous Seed Microbiota to Design Bacterial Seed Treatments

93

microbiota, and allergy are interrelated. Structural and especially functional microbial diversity is already established as a key factor in preventing human diseases (Jakobsson et al. 2014) and is suggested as biomarker for plant health as well (Berg et al. 2017). Moreover, overlapping compositions and interconnected microbiomes of human, animal, and plant in connection with health should be considered and used to expand the version of one health that includes environmental health and its relation to human cultures and habits (Flandroy et al. 2018). Berg and Raaijmakers (2018) postulated the “domestication syndrome” for plants and humans. It was found that changes in relative abundances of gut microbiota, more precisely between Bacteroidetes and Firmicutes, contribute remarkably on the pathophysiology of obesity in humans (Turnbaugh et al. 2006). For plants, the domestication footprint is expressed in a shift from Bacteroidetes to Proteobacteria (Germida and Siciliano 2001; Adam et al. 2018; Pérez-Jaramillo et al. 2017).

5.3.2

The Role of Soil and Seed Microbiomes to Maintain Microbial Diversity

Soil acts as a microbial seed bank. A protective and supportive impact of a diverse soil microbiome on plant health and resilience has been frequently reported (Raaijmakers and Mazzola 2016). Bender and Heijden (2015), for example, observed that soil microbial diversity is directly correlated with increased nutrient uptake efficiency of crop plants and subsequent increase of crop yields. A loss of microbial symbionts reduces the capability of plants and seeds to deal with pathogen attacks, adverse environmental conditions, and impacts of a changing climate (Truyens et al. 2014; Nelson 2018). Moreover, reduced soil biodiversity is assumed to facilitate the proliferation of plant pathogens (Mendes et al. 2013; Raaijmakers and Mazzola 2016). Besides the direct impact on plant health and performance, a reduced dynamic reservoir of soil biodiversity is increasingly recognized to have profound impacts on human and ecosystem health (Wall et al. 2015). Low microbial soil diversity is described to support the accumulation of soil-borne human pathogens (Berg et al. 2005) that are hereinafter likely to contaminate staples, drinking water (Oliver and Gregory 2015), and even the air we breathe (Garrison et al. 2003). In fact, the reduction of soil microbial diversity might result in a decreased capacity of soil food webs to perform substantial functions for the whole ecosystem (Wall et al. 2015), with tremendous impact on health conditions of the human population. As a consequence, ever-stronger human interventions and pesticides are required (Oerke 2006; Gruber 2017). In between the microbiome connection, seeds also play a crucial role. Here, plants store their own beneficial inoculum to maintain plant health over generations.

94

5.4

B. Wassermann et al.

Biotechnological Solutions for Sustainable Agriculture

Seed germination and seedling development are among the most vulnerable stages in a plant’s life cycle (Leck et al. 2008), and the importance of the associated microbiome for seed and plant health is high. Seed-associated microbiomes contribute significantly to improve seed vigor and promote germination (Glick et al. 1998; Darrasse et al. 2010); several plant families, e.g., Orchidaceae or Sphagnaceae, depend on beneficial microorganisms during germination. Today, various modern cultivars need chemical protection for their establishment in soil. Many of those seed treatments are controversially discussed, e.g., copper seed treatments in organic agriculture. Neonicotinoids represent another one as they are the most widely used class of insecticides in the world for seed treatments. Due to their impact on bees, a ban by the European Union came into force in 2018. Modern cultivars, banned pesticides, and missing microbial diversity require novel solutions in plant biotechnology. Several solutions are already suggested in literature, and other ones are already commercialized. Berg and Raaijmakers (2018) proposed a so-called “back to the future” approach: unraveling the seed microbiomes of wild relatives and ancient heirloom breeds of crop cultivars to save beneficial seed microbiomes for agriculture. Harnessing seed microbiomes of wild relatives of crop plants from natural ecosystems potentially enables a matching symbiosis between the plant and its specific seed microbiota. Conservational patterns of seed microbiota across boundaries of evolution were discovered by Johnston-Monje and Raizada (2011), comparing the seed microbiomes of modern Zea cultivars and their wild ancestors. Another solution was suggested by Zachow et al. (2013); the authors developed a direct selection strategy to obtain cultivable microorganisms from promising bio resources (alpine mosses, lichens, and primrose) using the bait plants and seeds. In a recent study, seeds of the Styrian oil pumpkin (Cucurbita pepo subsp. pepo var. Styriaca) were treated with fluorescent protein-tagged, beneficial Serratia plymuthica strains. These seeds naturally lack the lignification of the outer seed coat and thus provide less protection against microbial intrusion. It was found that the bacterial strains colonize outer and inner seed compartments after seed priming (Fig. 5.4a, b). Due to the localization of beneficial bacteria in the inner seed compartments, this seed treatment leads to an early protection of the cotyledons and the rhizoplane of the emerging seedlings (Fig. 5.4c). Moreover, the strains were highly abundant on roots (Fig. 5.4d) and the first true leaves of young plants. This treatment is a promising alternative for conventional seed treatments with chemical fungicides containing, for example, fludioxonil. In addition, Mitter et al. (2017) demonstrated the feasibility and promising utility of using seed microbiota for sustainable crop cultivation. The authors succeeded to insert a potential biological control agent into seeds. During field trials, those treated seeds showed faster plant development compared to control seeds.

5

Understanding the Indigenous Seed Microbiota to Design Bacterial Seed Treatments

95

Fig. 5.4 Confocal laser scanning micrographs (seeds and roots) and whole-plant visualization of the Styrian oil pumpkin (blue signal). Seeds were primed with fluorescent protein-tagged Serratia plymuthica strains (green signal), and the colonization was visualized 2 days after the treatment: (a) vascular bundle in the chlorenchyma layer of the outer seed coat and (b) root tip of the roothypocotyl-embryo; (c) S. plymuthica colonization of a pumpkin seedling 9 days after seed priming, visualized with a Bio-Rad ChemiDocTM XRS System; (d) CLSM of a densely colonized root of a seed-primed pumpkin plant, 16 days after inoculation

Current knowledge on the indigenous seed microbiota allows to draw some conclusions for several applied aspects and biotechnology. 1. The structure of the seed microbiota can be used as novel biomarker in breeding strategies. Moreover, joint breeding strategies of the plant and the indigenous plant-associated microbiota are promising. (continued)

96

B. Wassermann et al.

2. Breeding strategies can be successfully combined with biocontrol strategies. Biocontrol and stress-protecting agents can be designed and applied as seed treatments. 3. Currently global seed production and management focus on uniform, pathogen-free and clean seeds. Learning from the seed studies would suggest a local production of indigenous cultivars. This could reduce the amount of required pesticides, because the plants are better adapted to the certain environment. 4. Seed cleanings and assessments can be evaluated using microbial diversity as criterion. 5. Conservation strategies for seeds to preserve genetic diversity, which already exists, should include conservation strategies for seed microbes as well.

5.5

Conclusion

As a process of evolution and species diversification, domestication created a rich genetic diversity of early ancestors of modern crops. The coevolution of plants and microorganisms resulted also in genotype-dependent seed microbiomes, which need to be better understood. Since multi-omics technologies allow us deeper insights into the functioning of the holobiont, we should intensively focus on the following issues: How does native seed microbiota perform under stressful conditions? How stable is the seed microbiome? How does horizontally transmitted seed microbiota overcome the plant’s defense strategies to become endophytes? Bacterial seed treatments can be designed, which allow a better functioning of the crop holobiont to cope with pathogen pressure and even climate change.

References Adam E, Bernhart M, Müller H, Winkler J, Berg G (2018) The Cucurbita pepo seed microbiome: genotype-specific composition and implications for breeding. Plant Soil 422(1–2):35–49 Badri DV, Vivanco JM (2009) Regulation and function of root exudates. Plant Cell Environ 32 (6):666–681 Barret M, Briand M, Bonneau S, Préveaux A, Valière S, Bouchez O, Jacques MA (2015) Emergence shapes the structure of the seed microbiota. Appl Environ Microbiol 81(4):1257–1266 Baskin CC, Baskin JM (1998) Seeds: ecology, biogeography, and, evolution of dormancy and germination. Elsevier, San Diego Bender SF, Heijden MG (2015) Soil biota enhance agricultural sustainability by improving crop yield, nutrient uptake and reducing nitrogen leaching losses. J Appl Ecol 52(1):228–239 Berg G (2016) Analysing the plant microbiome for control of pathogens. In: Recent trends in PGPR research for sustainable crop productivity, p 45 Berg G, Raaijmakers JM (2018) Saving seed microbiomes. ISME J 12:1167–1170 Berg G, Smalla K (2009) Plant species and soil type cooperatively shape the structure and function of microbial communities in the rhizosphere. FEMS Microbiol Ecol 68(1):1–13

5

Understanding the Indigenous Seed Microbiota to Design Bacterial Seed Treatments

97

Berg G, Eberl L, Hartmann A (2005) The rhizosphere as a reservoir for opportunistic human pathogenic bacteria. Environ Microbiol 7(11):1673–1685 Berg G, Köberl M, Rybakova D, Müller H, Grosch R, Smalla K (2017) Plant microbial diversity is suggested as the key to future biocontrol and health trends. FEMS Microbiol Ecol 93(5) Bever JD, Mangan SA, Alexander HM (2015) Maintenance of plant species diversity by pathogens. Annu Rev Ecol Evol Syst 46:305–325 Blaser MJ (2014) Missing microbes: how the overuse of antibiotics is fueling our modern plagues. Macmillan, London Blaser MJ (2017) The theory of disappearing microbiota and the epidemics of chronic diseases. Nat Rev Immunol 17(8):461 Canfield DE, Glazer AN, Falkowski PG (2010) The evolution and future of Earth’s nitrogen cycle. Science 330(6001):192–196 Castañeda-Álvarez NP, Khoury CK, Achicanoy HA, Bernau V, Dempewolf H, Eastwood RJ, Guarino L, Harker RH, Jarvis A, Maxted N, Müller JV (2016) Global conservation priorities for crop wild relatives. Nat Plants 2(4):16022 Chen H, Wu H, Yan B, Zhao H, Liu F, Zhang H, Liang Z (2018) Core microbiome of medicinal plant Salvia miltiorrhiza seed: a rich reservoir of beneficial microbes for secondary metabolism? Int J Mol Sci 19(3):672 Darrasse A, Darsonval A, Boureau T, Brisset MN, Durand K, Jacques MA (2010) Transmission of plant-pathogenic bacteria by nonhost seeds without induction of an associated defense reaction at emergence. Appl Environ Microbiol 76(20):6787–6796 Darwin C (1968 [1859]) On the origin of species by means of natural selection. Murray Google Scholar, London David LA, Maurice CF, Carmody RN, Gootenberg DB, Button JE, Wolfe BE, Biddinger SB (2014) Diet rapidly and reproducibly alters the human gut microbiome. Nature 505(7484):559 Diamond J (2002) Evolution, consequences and future of plant and animal domestication. Nature 418(6898):700 Doornbos RF, van Loon LC, Bakker PA (2012) Impact of root exudates and plant defense signaling on bacterial communities in the rhizosphere. A review. Agron Sustain Dev 32(1):227–243 Fenner M, Thompson K (2005) The ecology of seeds. Cambridge University Press, Cambridge Flandroy L, Poutahidis T, Berg G, Clarke G, Dao MC, Decaestecker E, Sanz Y (2018) The impact of human activities and lifestyles on the interlinked microbiota and health of humans and of ecosystems. Sci Total Environ 627:1018–1038 Garrison VH, Shinn EA, Foreman WT, Griffin DW, Holmes CW, Kellogg CA, Smith GW (2003) African and Asian dust: from desert soils to coral reefs. AIBS Bulletin 53(5):469–480 Germida J, Siciliano S (2001) Taxonomic diversity of bacteria associated with the roots of modern, recent and ancient wheat cultivars. Biol Fertil Soils 33(5):410–415 Glassner H, Zchori-Fein E, Yaron S, Sessitsch A, Sauer U, Compant S (2018) Bacterial niches inside seeds of Cucumis melo L. Plant Soil 422(1–2):101–113 Glick BR, Penrose DM, Li J (1998) A model for the lowering of plant ethylene concentrations by plant growth-promoting bacteria. J Theor Biol 190(1):63–68 Gloria TC, Bonneau S, Bouchez O, Genthon C, Briand M, Jacques MA, Barret M (2018) Functional microbial features driving community assembly during seed germination and emergence. Front Plant Sci 9:902 Gruber K (2017) Agrobiodiversity: the living library. Nature 544(7651):S8–S10 Hanski I, von Hertzen L, Fyhrquist N, Koskinen K, Torppa K, Laatikainen T, Karisola P, Auvinen P, Paulin L, Mäkelä MJ, Vartiainen E, Vartiainen E (2012) Environmental biodiversity, human microbiota, and allergy are interrelated. Proc Natl Acad Sci 109(21):8334–8339 Hardoim PR, Hardoim CC, Van Overbeek LS, Van Elsas JD (2012) Dynamics of seed-borne rice endophytes on early plant growth stages. PLoS One 7(2):e30438 Hardoim PR, Van Overbeek LS, Berg G, Pirttilä AM, Compant S, Campisano A, Sessitsch A (2015) The hidden world within plants: ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiol Mol Biol Rev 79(3):293–320 Harlan RL (1973) Analysis of coupled heat fluid transport in partially frozen soil. Water Resour Res 9(5):1314–1323

98

B. Wassermann et al.

Huang Y, Zhang M, Deng Z, Cao L (2017) Evaluation of probiotic diversity from soybean (Glycine max) seeds and sprouts using Illumina-based sequencing method. Probiotics Antimicrob Proteins 10:293–298 Irizarry I, White JF (2017) Application of bacteria from noncultivated plants to promote growth, alter root architecture and alleviate salt stress of cotton. J Appl Microbiol 122(4):1110–1120 Jack AL, Nelson EB (2018) A seed-recruited microbiome protects developing seedlings from disease by altering homing responses of Pythium aphanidermatum zoospores. Plant Soil 422(1–2):209–222 Jakobsson HE, Abrahamsson TR, Jenmalm MC, Harris K, Quince C, Jernberg C, Andersson AF (2014) Decreased gut microbiota diversity, delayed Bacteroidetes colonisation and reduced Th1 responses in infants delivered by caesarean section. Gut 63(4):559–566 Johnston-Monje D, Raizada MN (2011) Conservation and diversity of seed associated endophytes in Zea across boundaries of evolution, ethnography and ecology. PLoS One 6(6):e20396 Johnston-Monje D, Lundberg DS, Lazarovits G, Reis VM, Raizada MN (2016) Bacterial populations in juvenile maize rhizospheres originate from both seed and soil. Plant Soil 405(1–2):337–355 Khalaf EM, Raizada MN (2016) Taxonomic and functional diversity of cultured seed associated microbes of the cucurbit family. BMC Microbiol 16(1):131 Khalaf EMK, Raizada MN (2018) Bacterial seed endophytes of domesticated cucurbits antagonize fungal and oomycete pathogens including powdery mildew. Front Microbiol 9:42 Klaedtke S, Jacques MA, Raggi L, Préveaux A, Bonneau S, Negri V, Barret M (2016) Terroir is a key driver of seed-associated microbial assemblages. Environ Microbiol 18(6):1792–1804 Laforest-Lapointe I, Paquette A, Messier C, Kembel SW (2017) Leaf bacterial diversity mediates plant diversity and ecosystem function relationships. Nature 546(7656):145 Leck MA, Parker VT, Simpson RL (eds) (2008) Seedling ecology and evolution. Cambridge University Press, Cambridge Lewis SL, Maslin MA (2015) Defining the anthropocene. Nature 519(7542):171 Links MG, Demeke T, Gräfenhan T, Hill JE, Hemmingsen SM, Dumonceaux TJ (2014) Simultaneous profiling of seed-associated bacteria and fungi reveals antagonistic interactions between microorganisms within a shared epiphytic microbiome on Triticum and Brassica seeds. New Phytol 202(2):542–553 Liu Y, Zuo S, Zou Y, Wang J, Song W (2013) Investigation on diversity and population succession dynamics of endophytic bacteria from seeds of maize (Zea mays L., Nongda108) at different growth stages. Ann Microbiol 63(1):71–79 Malinich EA, Bauer CE (2018) The plant growth promoting bacterium Azospirillum brasilense is vertically transmitted in Phaseolus vulgaris (common bean). Symbiosis 76(2):97–108 Mendes R, Garbeva P, Raaijmakers JM (2013) The rhizosphere microbiome: significance of plant beneficial, plant pathogenic, and human pathogenic microorganisms. FEMS Microbiol Rev 37 (5):634–663 Mitter B, Pfaffenbichler N, Flavell R, Compant S, Antonielli L, Petric A, Berninger T, Naveed M, Sheibani-Tezerji R, von Maltzahn G, Sessitsch A (2017) A new approach to modify plant microbiomes and traits by introducing beneficial bacteria at flowering into progeny seeds. Front Microbiol 8:11 Mormile BW (2016) Influence of seed microbiome on fitness of Epichloë infected tall fescue seedlings. Doctoral dissertation, Southern Connecticut State University Nelson EB (2018) The seed microbiome: origins, interactions, and impacts. Plant Soil 422(1–2):7–34 Oerke EC (2006) Crop losses to pests. J Agric Sci 144(1):31–43 Oliver MA, Gregory PJ (2015) Soil, food security and human health: a review. Eur J Soil Sci 66 (2):257–276 Pérez-Jaramillo JE, Mendes R, Raaijmakers JM (2016) Impact of plant domestication on rhizosphere microbiome assembly and functions. Plant Mol Biol 90(6):635–644 Pérez-Jaramillo JE, Carrión VJ, Bosse M, Ferrão LF, de Hollander M, Garcia AA, Ramírez CA, Mendes R, Raaijmakers JM (2017) Linking rhizosphere microbiome composition of wild and domesticated Phaseolus vulgaris to genotypic and root phenotypic traits. ISME J 11(10):2244 Pitzschke A (2016) Developmental peculiarities and seed-borne endophytes in quinoa: omnipresent, robust bacilli contribute to plant fitness. Front Microbiol 7:2

5

Understanding the Indigenous Seed Microbiota to Design Bacterial Seed Treatments

99

Purugganan MD, Fuller DQ (2009) The nature of selection during plant domestication. Nature 457 (7231):843 Qin Y, Pan X, Yuan Z (2016) Seed endophytic microbiota in a coastal plant and phytobeneficial properties of the fungus Cladosporium cladosporioides. Fungal Ecol 24:53–60 Raaijmakers JM, Mazzola M (2016) Soil immune responses. Science 352(6292):1392–1393 Rahman MM, Flory E, Koyro HW, Abideen Z, Schikora A, Suarez C, Cardinale M (2018) Consistent associations with beneficial bacteria in the seed endosphere of barley (Hordeum vulgare L.). Syst Appl Microbiol 41(4):386–398 Remus-Emsermann MN, Schlechter RO (2018) Phyllosphere microbiology: at the interface between microbial individuals and the plant host. New Phytol 218(4):1327–1333 Rezki S, Campion C, Iacomi-Vasilescu B, Preveaux A, Toualbia Y, Bonneau S, Briand M, Laurent E, Hunault G, Simoneau P, Jacques MA (2016) Differences in stability of seed-associated microbial assemblages in response to invasion by phytopathogenic microorganisms. PeerJ 4:e1923 Rezki S, Campion C, Simoneau P, Jacques MA, Shade A, Barret M (2018) Assembly of seedassociated microbial communities within and across successive plant generations. Plant Soil 422 (1–2):67–79 Rybakova D, Mancinelli R, Wikström M, Birch-Jensen AS, Postma J, Ehlers RU, Goertz S, Berg G (2017) The structure of the Brassica napus seed microbiome is cultivar-dependent and affects the interactions of symbionts and pathogens. Microbiome 5(1):104 Sánchez-Cañizares C, Jorrín B, Poole PS, Tkacz A (2017) Understanding the holobiont: the interdependence of plants and their microbiome. Curr Opin Microbiol 38:188–196 Sánchez-López AS, Thijs S, Beckers B, González-Chávez MC, Weyens N, Carrillo-González R, Vangronsveld J (2018) Community structure and diversity of endophytic bacteria in seeds of three consecutive generations of Crotalaria pumila growing on metal mine residues. Plant Soil 422(1–2):51–66 Schiltz S, Gaillard I, Pawlicki-Jullian N, Thiombiano B, Mesnard F, Gontier E (2015) A review: what is the spermosphere and how can it be studied? J Appl Microbiol 119(6):1467–1481 Shade A, Jacques MA, Barret M (2017) Ecological patterns of seed microbiome diversity, transmission, and assembly. Curr Opin Microbiol 37:15–22 Truyens S, Weyens N, Cuypers A, Vangronsveld J (2014) Bacterial seed endophytes: genera, vertical transmission and interaction with plants. Environ Microbiol Rep 7:40–50 Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI (2006) An obesityassociated gut microbiome with increased capacity for energy harvest. Nature 444(7122):1027 Van Der Heijden MG, De Bruin S, Luckerhoff L, Van Logtestijn RS, Schlaeppi K (2016) A widespread plant-fungal-bacterial symbiosis promotes plant biodiversity, plant nutrition and seedling recruitment. ISME J 10(2):389 Vandenkoornhuyse P, Quaiser A, Duhamel M, Le Van A, Dufresne A (2015) The importance of the microbiome of the plant holobiont. New Phytol 206:1196–1206 Vannier N, Mony C, Bittebiere AK, Michon-Coudouel S, Biget M, Vandenkoornhuyse P (2018) A microorganisms’ journey between plant generations. Microbiome 6(1):79 Verma SK, White JF (2018) Indigenous endophytic seed bacteria promote seedling development and defend against fungal disease in browntop millet (Urochloa ramosa L.). J Appl Microbiol 124(3):764–778 Walitang DI, Kim K, Madhaiyan M, Kim YK, Kang Y, Sa T (2017) Characterizing endophytic competence and plant growth promotion of bacterial endophytes inhabiting the seed endosphere of Rice. BMC Microbiol 17(1):209 Wall DH, Nielsen UN, Six J (2015) Soil biodiversity and human health. Nature 528(7580):69 White JF, Kingsley KI, Kowalski KP, Irizarry I, Micci A, Soares MA, Bergen MS (2018) Disease protection and allelopathic interactions of seed-transmitted endophytic pseudomonads of invasive reed grass (Phragmites australis). Plant Soil 422(1–2):195–208 Zachow C, Müller H, Tilcher R, Donat C, Berg G (2013) Catch the best: novel screening strategy to select stress protecting agents for crop plants. Agronomy 3(4):794–815

Part II Seed Endophytes: Ecology, Transmission and Adaptations

6

The Ecology of Seed Microbiota Pablo Hardoim

Contents 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 The Seed Microbiota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.1 Seed Fungal Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.2 Seed Bacterial Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Factors Affecting Seed Microbiota Assembly and Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.1 Seed Microbiota Originated from Host Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.2 Seed Microbiota Originating from Flowers and Fruit Lesions . . . . . . . . . . . . . . . . . . . 6.3.3 Seed Microbiota Assembled During Dispersal Events . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.4 Spermosphere Soil Microbiota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 Mutualistic Functions of Seed Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.1 Solubilization of Inorganic and Organic Phosphorus . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.2 Biosynthesis and Modulation of Phytohormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.3 Secondary Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.4 Host Cell Cycle Machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

104 105 105 108 110 111 112 113 114 115 116 117 119 120 120 121

Abstract

Plants evolved in the presence of microbes, and key functions for the success of these organisms on Earth are attributed to symbionts as source of origin. Therefore, it is not surprising that plants can establish intimate interactions with mutualistic microbes. Beneficial microbial traits might be selected by the host plants to improve their own fitness, and some of these beneficial microbes might even be vertically transmitted via seeds over plant generations, as persistent microbes. However, some seed-borne microbes might be transient and originated from horizontal transmission. In this study, I summarize the results from the literature regarding the diversity of seed-inhabiting microbes, how environmental P. Hardoim (*) Biopromo Agriculture Consulting Business, Praia Grande, São Paulo, Brazil # Springer Nature Switzerland AG 2019 S. K. Verma, J. F. White, Jr (eds.), Seed Endophytes, https://doi.org/10.1007/978-3-030-10504-4_6

103

104

P. Hardoim

and host factors might affect seed microbial assembly and structure, and the putative mutualistic functions of microbes might have during seedling establishment. Because deterministic (i.e., niche theory) and stochastic (i.e., neutral theory) processes are likely to contribute to seed microbiota assembly and structure, seed microbial ecology represents an exciting model to investigate fundamental aspects of ecology. Keywords

Seed endophytes · Seed microbial assembly · Seed microbial structure · Seed microbial functions · Niche theory · Neutral theory · Seed holobiome

6.1

Introduction

Plants are biofactories that provide many important ecosystem services to sustain life on Earth. These functions include maintenance of the atmosphere through photosynthesis, participate actively in biogeochemical cycles by recycling matter, affect the weather by transpiration, protect the soil by mitigating water runoff, create habitats for the majority of land organisms, and provide many products for human well-being. In addition, plants provide the main source of food for herbivores and pollinators as well as seeds are one of the main sources of nutrients for omnivores. All of these beneficial functions are only accomplished because plants coevolved with mutualistic microorganisms (Hardoim et al. 2015). The association of plants with microorganisms has been a dominant feature of plant evolution. For instance, the symbiont arbuscular mycorrhizal fungi (AMF) facilitated the adaptation of plants to the terrestrial environment, allowing the colonization of lands by them (Bidartondo et al. 2011). Therefore, there is a need to understand how sessile plants can cope with biotic and abiotic challenges when associated with mutualistic microorganisms. This is especially important for newly emerged seedlings. At this stage, seedlings no longer have the protection of multiple layers of tissues that function as a physical barrier to withstand adverse biotic and abiotic conditions observed in seeds, and at the same time they also lack biochemical and physical robustness that will be acquired with the proper development of the plant tissues. Therefore, the establishment of a juvenile individual is at the most vulnerable phase in the life cycle of plants. Plant life cycle is regenerated by the synthesis of physiologically independent individuals, which can be accomplished by the production of seeds and through vegetative production of genetically identical offsprings. The establishment of plants from seeds is usually dominant in forest ecosystems, fruit-bearing trees, and most crop species, whereas plant species thriving in limited nutrient and climate conditions, including those of savannahs, grasslands, and arctic/alpine ecosystems, adapt clonal growth as their main mechanism of regeneration (Fenner 2000). These strategies reflect the life history traits of each plant species and also the ecological context that allow the allocation of resources for regeneration. The allocation of resources to the reproductive structures and seed formation is very costly; however

6

The Ecology of Seed Microbiota

105

the success of plants over terrestrial habitats is largely attributed to the formation of flowers. This strategy provides plants with a great deal of independence from water environment, allowing the processes of sexual reproduction and dispersal of seeds to occur in dry land. This mechanism laid the basis for diversification of plants. In this context, plants might form mutualistic association with microorganisms to vertically transmit beneficial traits via seeds and thus increase the chances of establishment of new generation (Ewald 1987).

6.2

The Seed Microbiota

The microbial communities in or on seeds have been detected as earlier as 1840s. Most of these studies reported seed-borne microbes as causal pathogens capable of promoting widespread diseases in crop plants (Munkvold 2009). Today we are aware that plants are colonized by a great diversity of microbial communities, including species of bacteria, fungi, archaea, and protists, both internally and externally as seed endophytes and epiphytes, respectively. These microorganisms play crucial roles in the existence of plants, participating actively or indirectly in the processes of host development, growth, fitness, and even reproduction. The same microbial species are capable to colonize the episphere (i.e., external surface of the plant) and endosphere (i.e., internal tissues of the plant) of the host plant, indicating that a continuum pattern of colonization might be accomplished from outside to inside tissues of the plant and vice versa. Therefore, this classification should be assumed as a rather artificial division that is only valid to determine the colocalization of the symbiont at the moment of the sampling. However more often than it should, this classification has erroneously been used to determine how microorganisms interact with host plants and how this interaction might have the potential to interfere with the host plant growth and development. In general, microorganisms are capable to colonize virtually all tissues of host plants (with exception for meristematic cells), so it is not surprising that some of them are also colonizers of seeds. Those that colonize the aerial tissues of host plants have higher chance to be detected as seed colonizers and therefore be vertically transmitted as endophytes. Among the microbes that interact actively with plants, bacteria and fungi are the most prominent members in seeds, and these groups will be described here.

6.2.1

Seed Fungal Endophytes

As mentioned earlier, plants establish important mutualistic symbiosis with mycorrhizal fungi (MF). These symbionts are obligate biotrophs, in the sense that in the wild there is no record of growth outside of the host plants. They are ubiquitous colonizers of plant root tissues, capable to stretch their hyphae growth extensively outside of rhizosphere zone (up to 15 cm) to fetch nutrients and water to serve the host interests. Although MF are extremely important for seedling development,

106

P. Hardoim

especially under challenged conditions (e.g., Liu et al. 2018), they do not colonize aerial tissues of the host, and therefore they are not vertically transmitted with seeds of host plants. Instead, their propagation occurs strictly in the soil environment via synthesis of spore structures and viable mycorrhizal hyphae. Nevertheless, other types of fungal endophytes also establish mutualistic symbiosis with plants, and those are disseminated within seeds. Indeed, the hereditary symbiosis of fungus species from family Clavicipitaceae with plants of Poaceae taxa is among one of the earliest investigated plant-endophyte system (Petrini 1986). The importance of this group of endophytes for plant ecology, fitness, and evolution was so notorious that fungal endophytes were divided between two major groups, (1) clavicipitaceous endophytes and (2) nonclavicipitaceous endophytes, which reflect their differences in the evolutionary process, taxonomy, and ecological functions. Clavicipitaceous endophytes are fungus species from family Clavicipitaceae (Ascomycota, Hypocreales) capable of infecting only some species of grasses. They are adapted to rapidly colonize the tiller meristems of the host, which is rich in nutrients, but show limited capacity to grow on mature plant tissues. More recently, the ecological significance of fungal endophytes other than MF was classified into four classes based on their host colonization strategy and ecological functions (Rodriguez et al. 2009). Members of classes 1 and 2 are able to colonize extensively both root and shoot tissues of host plants and can be vertically and horizontally transmitted, whereas members of classes 3 and 4 showed limited host colonization capabilities and are restricted to horizontal transmission. Fungi of class 1 are comprised by clavicipitaceous endophytes from strains of the genera Atkinsonella, Balansia, Balansiopsis, Dussiella, Epichloë, Myriogenospora, Parepichloë (Tadych et al. 2009). A multilocus phylogenetic reconstruction of this group revealed that members of mutualistic clavicipitaceous endophytes were close related to members of genera Hypocrella, a pathogen of scale insects and white flies, and Metarhizium, a generalist arthropod pathogen, suggesting a common insect pathogen as an ancestor of this group (Rodriguez et al. 2009; Spatafora et al. 2007). The authors further suggested that some members of clavicipitaceous endophytes were diversified through a dynamic process of inter-kingdom host jumping, in which the initially sap-sucking insect parasites gained access to plant internal tissues through the insect’s stylet or stylet wound and became endophyte by adapting to plant internal environment. This newly formed symbiosis was further reinforced when the fungus symbionts increased their dependency on the host plant to acquire photoassimilates and other nutrients for their growth by reducing the fungus enzymatic machinery involved in metabolism of nutrients (Tadych et al. 2009; Kuldau and Bacon 2008). The nonclavicipitaceous fungal endophytes, class 2, are taxonomically a more diverse group than clavicipitaceous endophytes, and are comprised mostly by ascomycete and basidiomycete fungal and yeast species. Early investigations revealed that certified seeds from cultivated bean varieties (Phaseolus vulgaris) were internally colonized by ascomycete species of Alternaria sp. and Fusarium sp., as well as by the phytophathogens Rhizoctonia solani and Stemphylium sp. (Schnathorst 1954).

6

The Ecology of Seed Microbiota

107

The fungus Undifilum oxytropis (Dothideomycetes, Ascomycota) is detected in all investigated seeds of forb locoweed species of Fabaceae (Ralphs et al. 2011). In other forb plants (species Centaurea cyanus, Centaurea nigra, Papaver rhoeas, Plantago lanceolata, Rumex acetosa, and Senecio vulgaris), seeds are dominated by ascomycetes of the genera Acremonium, Alternaria, Aspergillus, Aureobasidium, Botrytis, Chaetomium, Cladosporium, Colletotrichum, Epicoccum, Fusarium, Geotrichum, Penicillium, Phialophora, Rhabdospora, and Trichothecium (Hodgson et al. 2014). Only members of Alternaria alternata, Aspergillus niger, Chaetomium cochliodes, Cladosporium cladosporioides, Cladosporium sphaerospermum, Colletotrichum coccodes, Colletotrichum dematium, Epicoccum nigrum, Fusarium equiseti, Fusarium merismoides, Fusarium sp., Phialophora verrucosa, and Tricothecium roseum were detected in the seeds with abundance larger than 10% for at least one plant species. In addition, Mucor hiemalis of Mucoromycota phyla was also detected, however, in relatively low abundance (less than 7%). Despite its putative importance for plant growth and development in various environmental challenges, only a small number of studies have investigated the diversity and composition of endophytic fungal communities in seeds. On the other hand, many more studies have reported the seed associated with fungal community, from which we can learn and infer on microbial seed community structure. Spotted knapweed (Centaurea stoebe, Asteraceae) plants growing in North America, an invasive forb plant in this geographic range, have seeds with high abundance of A. alternata (10%), A. tenuissima (16%), Cladosporium herbarum (11%), and Epicoccum sp. (11%) associated with them, whereas plants from its native range (i.e., Euroasia) have seeds dominated mostly by A. alternata (55%) (Shipunov et al. 2008). In another study, the fungal communities associated with 28 plant genotypes mostly affiliated to Brassicaceae family are also dominated by ascomycetes in the classes Dothideomycetes (60.7%), Eurotiomycetes (5.5%), Leotiomycetes (6.3%), and Sordariomycetes (4.9%), as well as by members of basidiomycetes class Tremellomycetes (15.5%) (Barret et al. 2015). Only an unclassified operational taxonomic unit (OTU) from Mycosphaerellaceae was systematically detected in all seed samples, whereas OTUs assigned to Cladosporium, Alternaria, Alternaria infectoria, Cryptococcus, Pleosporaceae, and Filobasidium were considered as core communities detected in at least 90% of all seed samples of 20 Brassicaceae plants investigated. In agreement with this result, the seed fungal microbiota of radish (Raphanus sativus, Brassicaceae) is also dominated by ascomycetes of species from Alternaria, Cladosporium, and Mycosphaerella genera (Rezki et al. 2018). At least five OTUs from this group were consistently detected in high abundance from seeds across three consecutive plant generations. Field-growing dicot plants of common buckwheat (Fagopyrum esculentum, Polygonaceae) have seeds with fungus strains of A. alternata, Aureobasidium pullulans, Botryotinia fuckeliana, Botrytis cinerea, E. nigrum, F. oxysporum, and Stereum hirsutum as endophytes (Kovačec et al. 2016). It is interesting that many of these species are described as ubiquitous endophytes and as opportunistic phytopathogens, which suggest that the interaction outcome between these microbes and their host plants will depend on environmental factors rather than just genetic

108

P. Hardoim

compatibility. It is clear that members of ascomycetes play major role in seed transmission in various plants, whether this consistent selection of, e.g., Alternaria and Cladosporium species is a consequence of fungal dispersal strategy or plant parenting strategy to provide mutualistic microbes for the success of their offspring is far from resolved and certainly deserves greater research attention in the future.

6.2.2

Seed Bacterial Endophytes

Tissues of undamaged and apparently healthy fruits and seeds were generally considered, until six decades ago, to be free of microbes. Nevertheless, empirical studies conducted with seeds and fruits revealed that various parts of these tissues are indeed colonized by diverse bacterial community. In one of the first manuscript on this subject, the authors reported the isolation of 23 bacteria from cultivated bean (P. vulgaris), which was identified by biochemical analysis as Achromobacter sp., Bacillus cereus, B. megaterium, B. subtilis, Klebsiella aerogenes, Lysinibacillus fusiformis, Paenibacillus polymyxa, Pseudomonas sp. (Schnathorst 1954). The authors confirmed that these isolated bacteria were not pathogenic by reintroducing them into five legume plants. No symptom of diseases was observed in this infection assay, suggesting a strict commensal or mutualistic interaction between seed-borne endophytes and the host plants. Since then, few other studies have reported the identification of bacteria from healthy fruits (e.g., Samish et al. 1961) and seeds (e.g., Katznelson et al. 1962), until the seminal study by Mundt and Hinkle on the isolation of endophytic bacterial cells from ovule tissues as well as from recently mature and overwintered seeds (Mundt and Hinkle 1976). Twenty-seven species of woody and herbaceous plants were used, and the most prevalent isolates inside their ovules and seeds were B. cereus, B. megaterium, Flavobacterium devorans, Klebsiella aerogenes, Pantoea ananatis, and Pseudomonas fluorescens, while members from the genera Achromobacter, Acinetobacter, Alcaligenes, Bacillus, Brevibacterium, Corynebacterium, Cytophaga, Erwinia, Flavobacterium, Leuconostoc, Micrococcus, Nocardia, Proteus, Pseudomonas, Serratia, Streptococcus, Streptomyces, and Xanthomonas were less frequently isolated. The percentage of bacterial recovery was greater inside ovules, and it decreased in progression to overwintered seeds, suggesting either loss of bacterial viability or cultivability during the process of seed maturation and dormancy. Throughout the years many more studies were performed to unravel the diversity and community structure of bacterial endophytes inside seeds. The bacterial community isolated from surface-sterilized seeds of 62 species of plants and reported in more than 50 studies are predominately characterized by Gammaproteobacteria, Bacilli, Actinobacteria, and Alphaproteobacteria taxa, while members of Betaproteobacteria, Flavobacteriia, Chitinophagia, Cytophagia, Clostridia, Cyanobacteria, Deinococci, Rubrobacteria, and Sphingobacteriia are detected in less abundance (Truyens et al. 2013; Shahzad et al. 2018). Inside the seeds there is an astonishing diversity of endophytes with a hundred bacterial genera reported. Bacterial species assigned to genera Bacillus, Curtobacterium, Methylobacterium, Microbacterium, Micrococcus,

6

The Ecology of Seed Microbiota

109

Paenibacillus, Pantoea, Pseudomonas, Rhizobium, Sphingomonas, Staphylococcus, and Xanthomonas are the most prominent members, followed by Acidovorax, Acinetobacter, Brevibacillus, Burkholderia, Corynebacterium, Enterobacter, Enterococcus, Erwinia, Flavobacterium, and Stenotrophomonas, all with more than five strains isolated in each genus. Whereas, endophytes assigned to Achromobacter, Actinomyces, Aerococcus, Aeromicrobium, Aeromonas, Afipia, Agrobacterium, Agromyces, Alcaligenes, Arthrobacter, Azospirillum, Bosea, Brachybacterium, Bradyrhizobium, Brevibacterium, Brevundimonas, Caulobacter, Cellulomonas, Chitinophaga, Chryseobacterium, Chryseomonas, Citrobacter, Clavibacter, Clostridium, Comamonas, Cronobacter, Cyanobacterium, Cytophaga, Deinococcus, Delftia, Devosia, Escherichia, Exiguobacterium, Flavimonas, Frigoribacterium, Herbaspirillum, Klebsiella, Kluyvera, Kocuria, Kosakonia, Kurtia, Leclercia, Leuconostoc, Limnobacter, Lysinibacillus, Lysobacter, Massilia, Mesorhizobium, Moraxella, Mucilaginibacter, Mycobacterium, Nocardia, Nocardioides, Ochrobactrum, Paracoccus, Patulibacter, Pectobacterium, Phyllobacterium, Plantibacter, Propionibacterium, Proteus, Pseudolabrys, Pseudonocardia, Pseudorhodoferax, Rahnella, Rhodococcus, Roseomonas, Salmonella, Sanguibacter, Sediminibacterium, Serratia, Sinorhizobium, Streptococcus, Streptomyces, Tukamurella, Undibacterium, Variovorax, and Yersinia are detected inside seed tissues in even less abundance. Culture-independent analysis of seed endophytes corroborates this data, although many bacterial genera identified were unique to each approach, suggesting that both systems can be complementary to discovery unique and previously unrecognized bacterial species in seeds. Sequences of phylogenetic marker genes, mainly 16S rRNA gene, revealed a slight greater diversity in the number of detected bacterial genera than culture-based approach. Members of Gammaproteobacteria, Alphaproteobacteria, Actinobacteria, Betaproteobacteria, and Bacilli classes are predominantly detected. The seed endophytes are largely represented by members of genera Pantoea, Acinetobacter, Enterobacter, Pseudomonas, and Sphingomonas, whereas members of Burkholderia, Erwinia, Serratia, Acidovorax, Flavobacterium, Leclercia, Methylobacterium, Paenibacillus, Roseateles, Staphylococcus, and Stenotrophomonas are also detected in less abundance. In addition, sequences from Acidithiobacillus, Ancylobacter, Anoxybacillus, Bdellovibrio, Carnobacterium, Cellvibrio, Competibacter, Cupriavidus, Curvibacter, Dechloromonas, Dermacoccus, Desemzia, Dokdonella, Enhydrobacter, Facklamia, Finegoldia, Flavisolibacter, Gluconacetobacter, Haemophilus, Hafnia, Halomonas, Jeotgalicoccus, Kineococcus, Lactobacillus, Lentzea, Limnohabitans, Luteibacter, Luteimonas, Marinobacterium, Meiothermus, Microvirga, Mycoplana, Oceanibaculum, Oxalophagus, Peptoniphilus, Planifilum, Propioniciclava, Pseudoxanthomonas, Psychrobacter, Ralstonia, Rhabdochlamydia, Rheinheimera, Ruminococcus, Sediminibacillus, Shigella, Shinella, Sphingobacterium, Sphingopyxis, Sphingosinicella, Tatumella, Tepidimonas, Thermomonas, Thiobacillus, and Varibaculum are only detected by culture-independent approach, although in low numbers. Currently there are 155 bacterial genera detected inside seed tissues of various host plants; however, many of these bacteria are detected sporadically, and only a

110

P. Hardoim

small fraction of these species might be considered part of the core seed bacteriome. Even more surprisingly, some of these seed bacteria are not detected in any other tissue in the host plants, suggesting that seed bacteriome assembly and structure are a combination of vertical (i.e., originating from the host plant tissues) and horizontal (i.e., originating from external environment and colonizing the seed tissues) transmission acting concomitantly.

6.3

Factors Affecting Seed Microbiota Assembly and Structure

The assemblage of seed microbiota and their structure persist unresolved, as for any tissue of the host plants. Community assembly is mediated by four processes: selection, drift, speciation, and dispersal (Vellend 2010), which can be explained mainly by niche and neutral theories. Niche theory predicts that deterministic processes are fundamental for community assembly, whereas the neutral theory relies on the contribution of stochastic processes. Both theories can be complementary, and it is very likely that microbial community assembly is an intricate and interexchange network of processes rather than a contribution from a single process. The combination of these processes has resulted in many theories and ecological models (Vellend 2010). Based on a broad concept of microbial ecology in mind, we might assume that selection and speciation processes are mainly represented by deterministic events, while drift and dispersal processes are largely represented by stochastic events. The microbial community assembly and structure of seeds are originated from (1) internal tissues, microbial community migrating from root and leaf tissues; (2) reproductive organs, external community finds their way via female flowers or fruit lesions; and (3) dispersal agents, microbial community from dispersal agents and from soil where the seed is deposited might ultimately multiply in the spermosphere during seed germination and colonize the new formed seedling as it grows. It is a very common mistake to assume that seed microbiota are only represented by microbes originated from the host plants. Microorganisms labeled with green fluorescent protein (GFP) can be a valuable tool to validate the process of vertical transmission from parental plants to offspring seedlings (Compant et al. 2008). Unfortunately, only a few studies have used this technique, and many more studies are needed to determine the real contribution of vertical transmission to seed microbial assembly. In addition to the source of the microorganisms, it is also known that seed microbial assembly and structure is affected by both biotic and abiotic factors, which might modify host metabolism and the phenotype response to environmental cues (Kristin and Miranda 2013). Therefore, one might also assume that plant physiology is a key selective force contributing to seed microbiota assembly and structure, as we now observe in other parts of the plant (Hardoim et al. 2015; Bulgarelli et al. 2013). The seed microbial assembly can differ considerably for individual seeds originated even from the same cob or pod structure (Rosenblueth et al. 2012). These results corroborate the idea that other factors than just genetics of

6

The Ecology of Seed Microbiota

111

the symbionts might also be involved. Furthermore, the presence of certain microorganisms in the flower structure might also affect the physical and chemical properties of the nectar, and consequently the mutualistic plant–pollinator interactions (Vannette et al. 2013), which will also affect the microbial assembly in the seeds. Moreover, the chronological order of microbe colonization might also affect the final assembly and structure of seed microbiota. For instance, earlier colonization of pear flowers by the biocontrol agent Pseudomonas fluorescens can significantly reduce the potential colonization of the phytopathogen Erwinia amylovora and prevent fire blight infection in pear trees (Wilson and Lindow 1993).

6.3.1

Seed Microbiota Originated from Host Plants

Microbes that are capable to infect gymnosperm and angiosperm plants might vertically be transmitted within seeds. Only those microbes that are originated from the host plant internal tissues can be vertically transmitted. The process of vertical transmission of a given symbiont is likely to persist over generations when the outcome interaction is mutualistic (Ewald 1987). It is assumed that detrimental interactions might not persist for long period of time because it reduces the fitness of the host plant and consequently diminishes the probability of success for the symbiont. Therefore, beneficial traits are faithfully transmitted from parent plants to offspring seedlings. This mechanism of transmission is very common among fungus endosymbionts, with the rate of vertical transmission for some fungus endophytes reaching up to 90% in seeds for some grass species (Ngugi and Scherm 2006). Members of clavicipitaceous endophytes, such as species of Epichloë and their asexual forms Neotyphodium, are vertically transmitted within seeds of grasses (Poaceae) (Clay 1990). Species of Neotyphodium frequently colonize inflorescence primordia, and the mycelium grows within seed tissues. It is interesting that these endophytes also produce conidia on leaf surfaces of the host grasses, and therefore it increases the potential to infect neighbor endophyte-free individual plants by horizontal transmission (Tadych et al. 2009). In addition, nonclavicipitaceous fungal endophytes are also vertically transmitted via seeds. In a greenhouse experiment, nearly all investigated seeds of forb locoweed species of Astragalus mollissimus var. earleii, A. lentiginosus var. araneosus, A. lentiginosus var. wahweapensis, and Oxytropis sericea (Fabaceae) were infected with the endophyte fungus Undifilum oxytropis (Dothideomycetes, Ascomycota), revealing an astonishing rate of near 100% of vertical transmission (Ralphs et al. 2011). In contrast with the fungus community, the total contribution of bacterial endophytes to the mechanism of vertical transmission along host generation is a matter of debate. For instance, seedlings growing in axenic conditions might reveal a greater diversity of bacteria than mature plants growing in the field (Cope-Selby et al. 2017). This suggests that not all of seed-borne bacteria are originated from vertical transmission. There are only few studies providing evidences on the contribution of seed bacterial community originating from the host plant tissues, and these evidences are often debatable. The microbes that are vertically transmitted must

112

P. Hardoim

establish an intimate interaction with the host plants and might be described as competent colonizer of plant tissues. Those that are transmitted to offspring over generations are good candidates to be associated as vertically transmitted endophytes (Johnston-Monje and Raizada 2011; Sánchez-López et al. 2018). For instance, the seed-borne endophyte Methylobacterium sp. Cp3, the most dominant community member over three consecutive seed generations, is capable of systemically colonize the pioneer metallophyte Crotalaria pumila and to promote its seed germination and the development of seedling growing in gnotobiotic conditions with cadmium as metal stressor (Sánchez-López et al. 2018). However, Methylobacterium sp. Cp3 is also able to infect host plants from the soil and thereby be horizontally transmitted. Indeed, Methylobacterium sp. Cp3 has such intimate interaction with the host plant that even when it is inoculated into the soil, it enters the root cortex tissue of C. pumila, then migrates to the vascular cylinder, and finally colonizes the seed tissues to be vertically transmitted. The consistency of Rhizobium in seeds of Arabidopsis thaliana exposed or not to cadmium over 14 plant generations is also remarkable (Truyens et al. 2016). The authors suggested that functional traits rather than bacterial taxonomy are the key factors for plant selection. Perhaps, host selection and bacterial colonization properties are important factors for seed endophytes assembly. In rice plants, up to 45% of the seed-borne bacterial community of endophytes might vertically be transmitted over two consecutive generations (Hardoim et al. 2012). Members of Stenotrophomonas maltophilia, Pseudomonas protegens, and Plantibacter flavus are among the most frequently detected species inside seeds and seedlings growing aseptically in both generations.

6.3.2

Seed Microbiota Originating from Flowers and Fruit Lesions

As seeds develop inside the plant floral structure, microbes that interact casually with the host plant might have the possibility to colonize seeds through floral structures pathway (Darsonval et al. 2009; Shade et al. 2017). It is very likely that bacteria associated with reproductive organs may have an origin distinct from the community encountered in other tissues. A possible explanation would be the mechanism of immigration from external sources to flower tissues and then to seeds. Therefore, the contribution of bacterial species originated from horizontal transmission to seed microbiota assembly and structure might even be greater than that originated from plant tissues. This is the opposite of what was observed for fungus endophytes in seeds. The assessment of bacterial community associated with flowers of apple trees reveals a rich and unexpected diversity with predominance of taxa affiliated with TM7 and Deinococcus–Thermus (Shade et al. 2013), which are rarely encountered to associate with other plant tissues than flowers. The bacterial community assembly patterns from pear blossoms were positively correlated with wind intensity, although pollinators might also play a role in facilitating this mechanism of immigration to flower tissues (Nuclo et al. 1998). Some of these flower’s endophytes might also

6

The Ecology of Seed Microbiota

113

colonize seed tissues and use seeds as a vector to colonize other environmental niches, such as invertebrate and vertebrate guts, during the process of seed dispersal. The detection of microbe species inside seeds, especially in low abundance, that are known to thrive in other natural habitats, such as water, soil, invertebrate, and vertebrate guts, strengthens the idea that these microorganisms are ubiquitous in nature, and given an opportunity to colonize, they can hitchhike on/in the seeds to assess other environmental niches. It is interesting that around 35% of bacterial genera detected in seeds were not previously encountered to associate with any part of the plant tissues (Hardoim et al. 2015), suggesting a weak interaction for these endophytes with the host plants. However, colonization of seeds via flower and fruit tissues is not limited to transient endophytes. This strategy of seed colonization can also be important for microbes that have intimate mutualistic or pathogenic relationships with the host plants. Systemic seed colonization has been empirically validated for the causal agent of bacterial wilt and canker of tomato, the Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis, the cells of this phytopathogen enter tomato fruit through lesions on the exocarp (Tancos et al. 2013). Likewise, for the plant growth-promoting bacteria Paraburkholderia phytofirmans PsJN, once it is introduced to the flowers of parent plants, it can be later detected in high abundance in the offspring seedlings (Mitter et al. 2018). Future investigations are needed to determine the real contribution of each floral trait to facilitate, or even select for, the microbiota that are consistently transmitted to the seeds and perhaps, even more importantly, to determine the ecological relevance of seed microbiota originating from flower structure to seed germination and seedling survival and consequently to plant species fitness.

6.3.3

Seed Microbiota Assembled During Dispersal Events

Seeds are also exposed to dispersal agents, such as wind, water, and animal, which have the potential to modify the seed microbial consortia and therefore to change the fitness of the seedlings. These events, although not included as a life stage in the agricultural context, are very important in natural system because it directly affects the dynamics of plant populations. Wind dispersal agent might not contribute significantly to seed microbiota as the water and animal dispersal agents. This is mostly related to the lack of suitable conditions for atmosphere microbes to interact intimately with seeds. On the other hand, water column containing a rich microbial community specialized in metabolize organic substrates might substantially diminish the survival rate of seedlings, especially if the freshwater environment is colonized by rotting pathogens (Crocker et al. 2015). Perhaps the most important contribution to seed microbiota during dispersal is that mediated by animals. Fruits and seeds consumed by animals are exposed to a rich diversity of microbes during their passage through the oral cavity and intestinal tract of the animal. These gut-inhabiting microbes have great opportunity to colonize seeds in high abundance. Furthermore, when seeds are expelled by either defecation or regurgitation, the intact seeds are deposited on soil with feces or saliva-coated,

114

P. Hardoim

respectively. These are important sources of nutrients, and together with the microbes presented on it, seedlings are provided with additional ecological advance, such as protection against soil-borne pathogens, when compared to seeds without the microbes from the gut and feces (Nelson 2018). Although important, the ecology of seed microbes originated from dispersal agents is highly neglected and definitely deserves future investigation.

6.3.4

Spermosphere Soil Microbiota

In natural system, dispersed seeds are deposited on soil where they can germinate immediately, or degraded by granivorous or by rotten pathogens, or persist in dormant or quiescent state until conditions for germination are met. At this phase of the plant life cycle, seeds are exposed to a rich and highly diverse community of soil microbes, which might impact seed germination and seedling survival and fitness. The frequency of fungal and bacterial infections inside seeds of pioneer tree species deposited in tropical rain forest soil might increase at earlier months of seed burial duration, suggesting that soil microbes might be critical for seed microbial assembly (Zalamea et al. 2015). In addition, the spermosphere produces and exudates carbohydrate, amino acid, and organic acid compounds that attract soil microbial communities to interact with the emerging new host plant. At this critical seed-to-seedling phase of plant development, the soil microbial community may have the strongest impact on overall plant fitness contributing to either improve or diminish the host population (Nelson 2018). The establishment of seedlings might drastically be reduced when germinating seeds are exposed to notorious pathogenic species, such as oomycetes Pythium and Phytophthora, and fungi Alternaria, Cochliobolus, Colletotrichum, Cylindrocarpon, Epicoccum, Fusarium, Pyrenophora, and Rhizoctonia (Nelson 2004). On the other hand, seedling might thrive when germinating seeds are exposed to beneficial microbes, such as members of Azospirillum, Pseudomonas, and Bacillus. The spermosphere soils across various plant species are dominated by members of Proteobacteria (Agrobacterium, Burkholderia, Enterobacter, Klebsiella, Pantoea, Pseudomonas, and Stenotrophomonas), Firmicutes (Bacillus and Paenibacillus), and Actinobacteria (Microbacterium) (Nelson 2004). Species from these genera are frequently encountered in and on surface of plant tissues and also dominate the bacterial community in most soils, suggesting that these taxa might have coevolved with the host plants over a long period of time, thereby creating an intimate and strong interaction between host plants and symbionts. Currently, many of these plant growthpromoting bacteria and fungi are being used as inoculants to increase seed germination uniformity, as well as seedling vigor and protection against soil-borne pathogens species, which might ensure rapid and standard establishment of crops that improves harvest quality and yield (Mahmood et al. 2016).

6

The Ecology of Seed Microbiota

6.4

115

Mutualistic Functions of Seed Endophytes

During seed germination and seedling development, the host plant is in a very vulnerable stage, and all interactions with microbes have the potential to drastically impact plant diversity and composition in natural systems. The literature is replete with studies demonstrating the deleterious effects that microbial pathogens might have in the relative abundance of tree species in tropical forests (Bell et al. 2006; Mangan et al. 2010; Bagchi et al. 2014). In agriculture systems, seed priming is a standard procedure for commercial seeds. This is a seed treatment that triggers pre-germinate metabolism to ensure the preservation of genome integrity, proper uniformity of seed germination, and fast seedling development (Paparella et al. 2015). In addition to priming treatment, conventional disinfectants, natural products with broad-spectrum antimicrobial properties, fungicides, and pesticides might be added to the solution to diminish microbial infection of seed and during earlier seedling development. All of this seed care is done to increase quality and yield in annual crops; however, these seed treatments might also reduce the population of beneficial microbes in and on the seeds, of which we know very little. Many of the seed isolates and taxa assessed with DNA sequencing from uncultured microbes play a major role in various environmental ecosystems including inside plants. Host plant when interacting with mutualistic microbes has been shown to improve seed germination rate, shoot and root growth area and weight, nutrient uptake, chlorophyll content, and tolerance to biotic and abiotic stresses and to increase yield (Hardoim et al. 2015). Beneficial properties provided by microbes to plant growth and development, such as nitrogen fixation (Johnston-Monje and Raizada 2011; Chimwamurombe et al. 2016; Khalaf and Raizada 2016; Puente et al. 2009; Walitang et al. 2017; Xu et al. 2015); biosynthesis and modulation of phytohormones including auxin (Johnston-Monje and Raizada 2011; Truyens et al. 2016; Chimwamurombe et al. 2016; Walitang et al. 2017; Xu et al. 2015; Assumpção et al. 2009; Díaz Herrera et al. 2016; Etesami and Alikhani 2016; Gao and Shi 2018; Hameed et al. 2015; Tchinda et al. 2016; Verma and White 2018; Verma et al. 2017), gibberellin (Shahzada et al. 2017), cytokinins (Goggin et al. 2015), ethylene (Johnston-Monje and Raizada 2011; Truyens et al. 2016; Chimwamurombe et al. 2016; Khalaf and Raizada 2016; Walitang et al. 2017; Xu et al. 2015; Tchinda et al. 2016), polyamines (Kämpfer et al. 2014), and volatile organic compounds (Johnston-Monje and Raizada 2011); and solubilization of iron (Johnston-Monje and Raizada 2011; Chimwamurombe et al. 2016; Khalaf and Raizada 2016; Walitang et al. 2017; Xu et al. 2015; Díaz Herrera et al. 2016; Etesami and Alikhani 2016; Gao and Shi 2018; Hameed et al. 2015; Tchinda et al. 2016; López et al. 2018) and inorganic and organic phosphorus (Johnston-Monje and Raizada 2011; Chimwamurombe et al. 2016; Khalaf and Raizada 2016; Puente et al. 2009; Walitang et al. 2017; Xu et al. 2015; Assumpção et al. 2009; Díaz Herrera et al. 2016; Etesami and Alikhani 2016; Hameed et al. 2015; Tchinda et al. 2016; Verma and White 2018; Verma et al. 2017; López et al. 2018) improve metal tolerance (Truyens et al. 2013; Sánchez-López et al. 2018; Mastretta et al. 2009; Truyens et al. 2014), and antagonistic activities against phytopathogens, including biosynthesis of antibiotic compounds, hydrolytic enzymes, and activation of

116

P. Hardoim

host immune defense response, have been described for seed endophytes (JohnstonMonje and Raizada 2011; Assumpção et al. 2009; Díaz Herrera et al. 2016; Etesami and Alikhani 2016; Hameed et al. 2015; Verma and White 2018; Verma et al. 2017; López et al. 2018). However, there is little empirical evidence to inform our knowledge about seedling–microbial interactions; therefore, this field of science has a great potential for new discoveries.

6.4.1

Solubilization of Inorganic and Organic Phosphorus

On barren volcanic rocks in Baja California, Mexico, giant cardon cactus (Pachycereus pringlei) can only be established with the help of seed-borne bacterial endophytes capable of facilitating rock-weathering process by the release of organic acids (Puente et al. 2009). Inoculated cactus seedlings with seed-borne bacterial endophytes mineralized significant quantities of inorganic phosphorus (P2O5), iron (Fe2O3), potassium (K2O), and magnesium (MgO) than uninoculated seedling, whereas cactus seeds disinfected with antibiotics completely halt seedling development. Adequate supply of phosphorus (P) in early stage of plant development has a long-lasting effect on plant life cycle. Seedling supplied with adequate quantities of P reveals greater plant fitness and suitable development of reproductive tissues than control seedlings supplied with inadequate quantities of P, even if P was provided latter (Sharma et al. 2013). Solubilization of inorganic and organic phosphorus by microbes provides sustainability to natural and agricultural systems. Phosphorus has poor solubility and in organic rich soil it is mostly fixed with organic particles (up to 90% of the total soil P). Generally, P-solubilizing fungi produce and exude various acid compounds, which might have greater P-solubilizing activity than bacteria. Strains of Aspergillus, Penicillium, and Trichoderma have been applied commercially as seed P-solubilizing inoculants to increase P uptake by seedling of various plant species. Dark septate fungus Aspergilus ustus might improve seedling growth and development by solubilizing inorganic phosphorus, which is uptaken by fungus hyphae and transferred to the host plant Fourwing saltbush (Atriplex canescens) (Barrow and Osuna 2002). Among seeds of Zea mays genotypes, solubilization of phosphate is one of the most commonly detected traits of seed-borne bacterial endophytes, with 78% of the isolated population having the ability to solubilize phosphate in vitro (Johnston-Monje and Raizada 2011). In addition, the organic compound phytate is the primary source of the growth factor inositol and the main storage form of P in many plant tissues, especially seeds (up to 72% of P as phytate) and pollen. It is interesting that plants show very limited efficiency to extract P from phytate. This efficiency can be significantly increased when plants are genetically transformed to constitutively express phytase gene (phyA) derived from Aspergilus niger or Escherichia coli (Richardson 2001; Singh and Satyanarayana 2011). The growth efficiency of phytase-expressing plants is often compared to control plants supplied with readily available P form for root uptake. The transgenic maize plants constitutively expressing phyA gene derived from A. niger

6

The Ecology of Seed Microbiota

117

were able to solubilize 95% of the endogenous phytate present in the seeds (Drakakaki et al. 2005). These transformations of plants with phytase-expressing gene suggest that microbes might be in fact a key driver of phytate mineralization both inside seed tissues as well as in the soil (Singh and Satyanarayana 2011). Perhaps not surprisingly, some of the seed-borne microbial community might be able to efficiently mineralize seed-stored phytate during seed germination. This might provide P nutrients for both symbionts and host plants during seedling establishment. Although this hypothesis was not confirmed, evidences from the literature do exist. For instance, all bacterial endophytes isolated from Arabidopsis thaliana seeds showed extracellular phytase activity (Truyens et al. 2013), whereas seeds from bean cultivars (Phaseolus vulgaris) host only few isolates (18%) with extracellular phytase activity (López-López et al. 2010). These data reveals a large variation in the ability of seed-borne bacterial community to mineralize phytate inside seeds. Nevertheless, it does not diminish the importance of these communities to improve P availability to the host plants at the critical growth stage, and therefore it is a valid hypothesis for future investigation.

6.4.2

Biosynthesis and Modulation of Phytohormones

Phytohormones are organic compounds that influence physiological processes, such as growth, differentiation, and development, at low concentrations. There are at least ten different types of phytohormones, many of which are synthesized and modulated by microbes, either directly or indirectly by regulating plant transcription factors involved in biosynthesis, signal transduction, receptor, and response processes of these phytohormones. Therefore, microbes might directly influence plant physiological processes. The phytohormones auxins, gibberellins (GAs), and cytokinins (CKs) are major plant growth-promoting hormones, and they might synergistically act to provide robustness to plant growth and development processes (Schaller et al. 2015; Tsavkelova et al. 2006), whereas the phytohormones abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), and ethylene are major mediators of plant stress response. Auxin can stimulate cell enlargement, cell division, and tissue differentiation and induce the transcription of the plant enzyme 1-aminocyclopropane-1-carboxylate (ACC) synthase that catalyzes the non-ribosomal synthesis of amino acid ACC. ACC is the direct precursor of ethylene, which is synthesized in different tissues in response to various stresses and also acts in the germination of seeds. Ethylene counteracts ABA action in seed to improve dormancy release, thereby triggering seed germination (Arc et al. 2013). After germination, seedling and physiological independent plants are constantly challenged by abiotic and biotic stresses, and the biosynthesis of ethylene are frequently induced, reducing the growth potential of plants. Along with other small molecules, part of the plant ACC is exuded from seed, root, and shoot cells and used by ACC deaminase bacterial endophytes as nitrogen source for their own growth (Penrose et al. 2001). The removal of cell-exuded ACC from plants subjected to a wide range of different kinds of biotic and abiotic stresses

118

P. Hardoim

by ACC deaminase-producing bacteria resulted in improved tolerance of plant to stresses and even promoted plant growth (Glick 2014). Furthermore, when the gene ACC deaminase of P. phytofirmans PsJN was inactivated by mutation, the endophyte lost its ability to improve root elongation in canola seedlings (Sun et al. 2009). These results emphasize the importance of this mutualistic trait for host plant fitness, which was observed in all investigated studies. The phytohormones often act antagonistically to regulate tissue differentiation and development processes. For instance, seed dormancy is an adaptive trait of plant survival in an arrested state until environmental conditions are favorable for seedling growth. Two key hormones, ABA and GAs, are involved in the perception of environmental signals and properly regulate plant responses during complex metabolic process of seed germination. Dormancy is largely maintained by high concentration of ABA, which inhibits water uptake by seed tissues and thereby reduces embryo growth potential. High levels of GAs are stimulated when favorable conditions of light, moisture, and temperature are detected, and the inhibitory effects of ABA over seed germination are released. Although poorly investigated, many plant growth-promoting bacteria, including potential seed-borne members from genera Acinetobacter, Agrobacterium, Arthrobacter, Azospirillum, Azotobacter, Bacillus, Clostridium, Flavobacterium, Micrococcus, Pseudomonas, Rhizobium, Xanthomonas, fungi (Aspergillus, Botryodiplodia, Cercospora, Fusarium, Penicillium, Phaeosphaeria, Rhizopus, Schizophyllum, Sphaceloma, Ustilago, Verticillium), and algae (Chara, Fucus, Porphyra, Tetraselmis), are able to synthesize one or more types of GA compounds (Tsavkelova et al. 2006). It is interesting that many GA-producing bacteria are also able to synthesize auxin, and some of these strains are commercially used as seed inoculants in crop species to improve overall seed germination, seedling development, plant fitness, and crop yield (Tsavkelova et al. 2006). Dormancy of seeds from ornamental plants such as orchid is also released by auxin-producing bacteria (Tsavkelova et al. 2007). Auxin is also important for growth and development of root hair tissues, which are essential for seedling uptake of nutrients and water. Seedlings of orange inoculated with AMF reveal greater root hair growth under drought stress than non-AMF seedlings (Liu et al. 2018). The authors revealed that AMF is able to stimulate auxin biosynthesis and transport in the host tissues, improving root hair density and the uptake of water, thereby enhancing drought tolerance. Although not investigated thoroughly, tall fescue (Festuca arundinacea) and perennial ryegrass (Lolium perenne) grasses infected by seedborne endophytes Neotyphodium and Epichloë species also show an increment in seedling growth not necessarily related to stress alleviation (Clay 1987). This physiological response might be attributed to production of phytohormones, such as auxin compounds, and even the endogenous increment of these in the host plants. In addition, seed germination in Arabidopsis might be activated by the antagonistic interaction between CK and ABA transcription factor regulators (Wang et al. 2011). The phytohormone CKs are synthesized by plant and microbes alike and might promote endosperm growth via promotion of cell division. Biologically active CKs are predominantly detected with sugar conjugates of hydroxylated derivatives cis-zeatin and trans-zeatin when derived from plants, while the free bases and their

6

The Ecology of Seed Microbiota

119

methylthiolated derivatives of isopentenyladenine, cis-zeatin, and trans-zeatin are derived from bacteria (Goggin et al. 2015). Seed-borne endophytes, including pinkpigmented facultative methylotrophs, might stimulate seed germination with the biosynthesis of freebase CKs (Goggin et al. 2015; Lidstrom and Chistoserdova 2002). The role of seed-borne endophytes regarding the other phytohormones, including brassinosteroids, polyamines, signal peptides, and volatile organic compounds, for seed germination and seedling establishment is almost completely absent from the literature and remains to be discerned.

6.4.3

Secondary Metabolites

There are many studies reporting novel and known biological activities of secondary metabolites synthesized by seed-borne endophytic fungi and how these bioactive compounds might improve fitness of plants and symbionts alike. For instance, seedborne clavicipitaceous endophytes improve host plant protection from invertebrate and vertebrate herbivores by increasing the production of secondary metabolites, including ergot, aminopyrrolizidine, pyrolopyrazine, and indole diterpenoid alkaloids (Tadych et al. 2009; Kuldau and Bacon 2008). The seed-borne endophyte fungus U. oxytropis is responsible for the biosynthesis of the toxic alkaloid swainsonine, which confers herbivore resistance by poisoning livestocks (Ralphs et al. 2011). The production and concentration of secondary metabolites correlate directly with the hyphal density and the survival rate of the host plant (Clay 1990), suggesting that grasses infected by these endophytes gained a competitive evolutionary edge over endophyte-free grasses. This mutualistic interaction is further perpetuated by means of fungal dissemination through seeds (Leuchtmann et al. 2000). The beneficial characteristics of clavicipitaceous endophytes for host plant growth go far beyond protection from invertebrate and vertebrate herbivores. Grasses infected by Neotyphodium and Epichloë species are more likely to show resistance to several necrotrophic fungal pathogens (Kuldau and Bacon 2008). The mechanisms of disease suppression by these mutualistic endophytes involve the production of fungal cell wall-degrading enzymes, alkaloid and terpenoid compounds, as well as activation of induced systemic resistance in the host plant by promoting endogenous activation of JA pathway (Bastias et al. 2017). It is interesting that some species of Epichloë are able to repress endogenous SA pathway response, which plays a crucial role defense response against biotrophic, hemi-biotrophic pathogens, and sap-sucking insects as well as the establishment of systemic acquired resistance (Bastias et al. 2017; Bari and Jones 2009). Furthermore, endophyte-infected grasses are more tolerant to stresses induced by drought, low soil pH, heavy metal toxicity, and low levels of soil phosphorus, as well as other abiotic soil stresses (Kuldau and Bacon 2008). The underlying beneficial mechanisms are not very well investigated; however, endophyte infection resulting in biochemical and physiological modifications in the host plants is likely to be decisive. Some alkaloids produced by the mutualistic fungus such as loline alkaloids

120

P. Hardoim

function as allelochemicals responsible for the phenomenon of allelopathy observed in plants and are also reported to affect the osmotic potential in the host and therefore reduce the effects of drought stress (Kuldau and Bacon 2008). This exemplifies the dual role of secondary metabolites produced by mutualistic symbionts on host protection and stress mitigation.

6.4.4

Host Cell Cycle Machinery

Throughout plant life, cell cycle-related processes require the activation of major cell energy-generating pathways to sustain growth, and these cell cycle-related proteins have been implicated in the modulation of energy flux in the cytosol by regulating mitochondrial and chloroplastidial energy-generating pathways (Siqueira et al. 2018). During seedling development, most of the host cells are in heterotrophic stage of growth, and cell division and differentiation processes are directly connected with mitochondrial metabolism. In addition to mitochondrial energygenerating pathway, in the shoot tissues of developing seedling, the functional energy-converting chloroplasts of photoautotrophic cells are added as new source of energy to fuel the processes of plant growth and development. As some endophytic cells have the ability to colonize the host cell internally (Thomas and Sekhar 2014) and considering the theoretical consensus that the chloroplast organelles of plants and protists and the mitochondria of eukaryotic cells were once free-living prokaryotes (Alphaproteobacteria and Cyanobacteria, respectively) that formed endosymbioses with an earlier host cell (Sapp 2004), one might wonder whether or not some of host cell cycle regulatory machineries are also capable to directly regulate the energy metabolism in the symbiont cells. Host cell cycle regulators might synchronistically coordinate the metabolism and metabolic oscillations of both organelles and symbiont cells alike. In addition, some of the symbiont cell metabolism and metabolic oscillator mechanisms and proteins might also modulate host cell cycle-related machinery, impairing cell division when conditions are limited in energy or nutrients (Siqueira et al. 2018). Although this hypothesis, at first, seems farfetching to validate and even out of reasonable comparison, because it still remains unclear how cell cycle regulators connect with the major energy-generating pathways in most plant cells, it is rational and might provide further importance to the concept of holobiome (i.e., host plant and all organisms associated with it functioning as a single unit). These mechanisms of interaction are still poorly understood and represent perhaps the greatest need for research.

6.5

Future Perspectives

Overall, seed microbiota can be an interesting model system because it represents both an endpoint for the plant community to be transmitted within seeds and also a starting point for community assembly in the novel-generated seedling. Seed

6

The Ecology of Seed Microbiota

121

endophyte might be classified as transient, which is a member that use seed as way for dispersion and show casual interaction with the host plants, or as persistent, a member that interact intimately with the host and are transmitted seed-to-seed over many generations and environmental conditions. Many stochastic and deterministic processes are likely to contribute to seed microbiota assembly and structure, and these two types of seed microbial habitants should be distinguished when assessing the seed microbiota. Because both niche and neutral theories are major themes within ecology studies, this should be an exciting avenue of research in the future for investigating fundamental aspects of ecology. Conflict of Interest The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References Arc E, Sechet J, Corbineau F, Rajjou L, Marion-Poll A (2013) ABA crosstalk with ethylene and nitric oxide in seed dormancy and germination. Front Plant Sci 4:63. https://doi.org/10.3389/ fpls.2013.00063 Assumpção LDC, Lacava PT, Dias AC, de Azevedo JL, Menten JOM (2009) Diversity and biotechnological potential of endophytic bacterial community of soybean seeds. Pesq Agrop Bras 44:503–510 Bagchi R, Gallery RE, Gripenberg S et al (2014) Pathogens and insect herbivores drive rainforest plant diversity and composition. Nature 506:85–88 Bari R, Jones JDG (2009) Role of plant hormones in plant defence responses. Plant Mol Biol 69:473–488 Barret M, Briand M, Bonneau S et al (2015) Emergence shapes the structure of the seed-microbiota. Appl Environ Microbiol 81:1257–1266 Barrow JR, Osuna P (2002) Phosphorus solubilization and uptake by dark septate fungi in fourwing saltbush, Atriplex canescens (Pursh) Nutt. J Arid Environ 51:449–459 Bastias DA, Martínez-Ghersa MA, Ballaré CL, Gundel PE (2017) Epichloë fungal endophytes and plant defenses: not just alkaloids. Trends Plant Sci 22:939–948 Bell T, Freckleton RP, Lewis OT (2006) Plant pathogens drive density-dependent seedling mortality in a tropical tree. Ecol Lett 9:569–574 Bidartondo MI, Read DJ, Trappe JM, Merckx V, Ligrone R, Duckett JG (2011) The dawn of symbiosis between plants and fungi. Biol Lett 7:574–577. https://doi.org/10.1098/rsbl.2010.1203 Bulgarelli D, Schlaeppi K, Spaepen S, van Themaat EVL, Schulze-Lefert P (2013) Structure and functions of the bacterial microbiota of plants. Annu Rev Plant Biol 64:807–838 Chimwamurombe PM, Grönemeyer JL, Reinhold-Hurek B (2016) Isolation and characterization of culturable seed-associated bacterial endophytes from gnotobiotically grown Marama bean seedlings. FEMS Microbiol Ecol 92:1–11 Clay K (1987) Effects of fungal endophytes on the seed and seedling biology of Lolium perenne and Festuca arundinacea. Oecologia 73:358–362 Clay K (1990) Fungal endophytes of grasses. Annu Rev Ecol Syst 21:275–297 Compant S, Kaplan H, Sessitsch A, Nowak J, Ait Barka E, Clément C (2008) Endophytic colonization of Vitis vinifera L. by Burkholderia phytofirmans strain PsJN: from the rhizosphere to inflorescence tissues. FEMS Microbiol Ecol 63:84–93 Cope-Selby N, Cookson A, Squance M, Donnison I, Flavell R, Farrar K (2017) Endophytic bacteria in Miscanthus seed: implications for germination, vertical inheritance of endophytes, plant evolution and breeding. GCB Bioenergy 9:57–77

122

P. Hardoim

Crocker EV, Karp MA, Nelson EB (2015) Virulence of oomycete pathogens from Phragmites australis invaded and noninvaded soils to seedlings of wetland plant species. Ecol Evol 5:2127–2139 Darsonval A, Darrasse A, Durand K, Bureau C, Cesbron S, Jacques MA (2009) Adhesion and fitness in the bean phyllosphere and transmission to seed of Xanthomonas fuscans subsp. fuscans. Mol Plant Microbe Interact 22:747–757 Díaz Herrera S, Grossi C, Zawoznik M, Groppa MD (2016) Wheat seeds harbour bacterial endophytes with potential as plant growth promoters and biocontrol agents of Fusarium graminearum. Microbiol Res 186–187:37–43 Drakakaki G, Marcel S, Glahn RP (2005) Endosperm-specific co-expression of recombinant soybean ferritin and Aspergillus phytase in maize results in significant increases in the levels of bioavailable iron. Plant Mol Biol 59:869–880 Etesami H, Alikhani HA (2016) Suppression of the fungal pathogen Magnaporthe grisea by Stenotrophomonas maltophilia, a seed-borne rice (Oryza sativa L.) endophytic bacterium. Arch Agron Soil Sci 62:1271–1284 Ewald PW (1987) Transmission modes and evolution of the parasitism-mutualism continuum. Ann NY Acad Sci 503:295–306 Fenner M (2000) Seeds: the ecology of regeneration in plant communities. CABI, Oxon Gao T, Shi X (2018) Preparation of a synthetic seed for the common reed harboring an endophytic bacterium promoting seedling growth under cadmium stress. Environ Sci Pollut Res 25:8871–8879 Glick BR (2014) Bacteria with ACC deaminase can promote plant growth and help to feed the world. Microbiol Res 169:30–39 Goggin DE, Emery RJN, Kurepin LV, Powles SB (2015) A potential role for endogenous microflora in dormancy release, cytokinin metabolism and the response to fluridone in Lolium rigidum seeds. Ann Bot 115:293–301 Hameed A, Yeh MW, Hsieh YT, Chung WC, Lo CT, Young LS (2015) Diversity and functional characterization of bacterial endophytes dwelling in various rice (Oryza sativa L.) tissues, and their seed-borne dissemination into rhizosphere under gnotobiotic P-stress. Plant Soil 394:177–197 Hardoim PR, Hardoim CCP, van Overbeek LS, van Elsas JD (2012) Dynamics of seed-borne rice endophytes on early plant growth stages. PLoS One 7:e30438 Hardoim PR, van Overbeek LS, Berg G et al (2015) The hidden world within plants: ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiol Mol Biol Rev 79:293–320 Hodgson S, de Cates C, Hodgson J, Morley NJ, Sutton BC, Gange AC (2014) Vertical transmission of fungal endophytes is widespread in forbs. Ecol Evol 4:1199–1208 Johnston-Monje D, Raizada MN (2011) Conservation and diversity of seed associated endophytes in Zea across boundaries of evolution, ethnography and ecology. PLoS One 6:e20396. https:// doi.org/10.1371/journal.pone.0020396 Kämpfer P, Glaeser SP, Mcinroy JA, Busse HJ (2014) Cohnella rhizosphaerae sp. nov., isolated from the rhizosphere environment of Zea mays. Int J Syst Evol Microbiol 64:1811–1816 Katznelson H, Peterson EA, Rouatt JW (1962) Phosphate-dissolving microorganisms on seed and in the root zone of plants. Can J Bot 40:1181–1186 Khalaf EM, Raizada MN (2016) Taxonomic and functional diversity of cultured seed associated microbes of the cucurbit family. BMC Microbiol 16:131 Kovačec E, Likar M, Regvar M (2016) Temporal changes in fungal communities from buckwheat seeds and their effects on seed germination and seedling secondary metabolism. Fungal Biol 120:666–678 Kristin A, Miranda H (2013) The root microbiota: a fingerprint in the soil? Plant Soil 370:671–686 Kuldau G, Bacon C (2008) Clavicipitaceous endophytes: their ability to enhance resistance of grasses to multiple stresses. Biol Control 46:57–71

6

The Ecology of Seed Microbiota

123

Leuchtmann A, Schmidt D, Bush LP (2000) Different levels of protective alkaloids in grasses with stroma-forming and seed-transmitted Epichloë/Neotyphodium endophytes. J Chem Ecol 26:1025–1036 Lidstrom ME, Chistoserdova L (2002) Plants in the pink: cytokinin production by Methylobacterium. J Bacteriol 184:1818–1818 Liu CY, Zhang F, Zhang DJ, Srivastava AK, Wu QS, Zou YN (2018) Mycorrhiza stimulates root-hair growth and IAA synthesis and transport in trifoliate orange under drought stress. Sci Rep 8:1978 López JL, Alvarez F, Principe A et al (2018) Isolation, taxonomic analysis, and phenotypic characterization of bacterial endophytes present in alfalfa (Medicago sativa) seeds. J Biotechnol 267:55–62 López-López A, Rogel MA, Ormeño-Orrillo E, Martínez-Romero J, Martínez-Romero E (2010) Phaseolus vulgaris seed-borne endophytic community with novel bacterial species such as Rhizobium endophyticum sp. nov. Syst Appl Microbiol 33:322–327 Mahmood A, Turgay OC, Farooq M, Hayat R (2016) Seed biopriming with plant growth promoting rhizobacteria: a review. FEMS Microbiol Ecol 92:fiw112 Mangan SA, Schnitzer SA, Herre EA et al (2010) Negative plant–soil feedback predicts tree-species relative abundance in a tropical forest. Nature 466:752–755 Mastretta C, S Taghavi S, van der Lelie D (2009) Endophytic bacteria from seeds of Nicotiana tabacum can reduce cadmium phytotoxicity. Int J Phytoremediation 11:251–267 Mitter B, Pfaffenbichler N, Flavell R et al (2018) A new approach to modify plant microbiomes and traits by introducing beneficial bacteria at flowering into progeny seeds. Front Microbiol 8:11. https://doi.org/10.3389/fmicb.2017.00011 Mundt JO, Hinkle NF (1976) Bacteria within ovules and seeds. Appl Environ Microbiol 32:694–698 Munkvold GP (2009) Seed pathology progress in academia and industry. Annu Rev Phytopathol 47:285–311 Nelson EB (2004) Microbial dynamics and interactions in the spermosphere. Annu Rev Phytopathol 42:271–309 Nelson EB (2018) The seed microbiome: origins, interactions, and impacts. Plant Soil 422:7–34 Ngugi HK, Scherm H (2006) Biology of flower-infecting fungi. Annu Rev Phytopathol 44:261–282 Nuclo RL, Johnson KB, Stockwell VO, Sugar D (1998) Secondary colonization of pear blossoms by two bacterial antagonists of the fire blight pathogen. Plant Dis 82:661–668 Paparella S, Araújo SS, Rossi G, Wijayasinghe M, Carbonera D, Balestrazzi A (2015) Seed priming: state of the art and new perspectives. Plant Cell Rep 34:1281–1293 Penrose DM, Moffatt BA, Glick BR (2001) Determination of 1-aminocycopropane-1-carboxylic acid (ACC) to assess the effects of ACC deaminase-containing bacteria on roots of canola seedlings. Can J Microbiol 47:77–80 Petrini O (1986) Taxonomy of endophytic fungi of aerial plant tissues. In: Fokkema NJ, van den Heuvel J (eds) Microbiology of the phyllosphere, Cambridge University Press, Cambridge Puente ME, Li CY, Bashan Y (2009) Endophytic bacteria in cacti seeds can improve the development of cactus seedlings. Environ Exp Bot 66:402–408 Ralphs MH, Cook D, Gardner DR, Grum DS (2011) Transmission of the locoweed endophyte to the next generation of plants. Fungal Ecol 4:251–255 Rezki S, Campion C, Simoneau P, Jacques MA, Shade A, Barret M (2018) Assembly of seedassociated microbial communities within and across successive plant generations. Plant Soil 422:67–79 Richardson AE (2001) Prospects for using soil microorganisms to improve the acquisition of phosphorus by plants. Funct Plant Biol 28:897–906 Rodriguez RJ, White JF Jr, Arnold AE, Redman RS (2009) Fungal endophytes: diversity and functional roles. New Phytol 182:314–330 Rosenblueth M, López-López A, Martínez J, Rogel MA, Toledo I, Martínez-Romero E (2012) Seed bacterial endophytes: common genera, seed-to-seed variability and their possible role in plants. Acta Hortic 938:39–48

124

P. Hardoim

Samish Z, Etinger-Tulczynska R, Bick M (1961) Microflora within healthy tomatoes. Appl Microbiol 9:20–25 Sánchez-López A, Pintelon I, Stevens V et al (2018) Seed endophyte microbiome of Crotalaria pumila unpeeled: identification of plant-beneficial methylobacteria. Int J Mol Sci 19:291 Sapp J (2004) The dynamics of symbiosis: an historical overview. Can J Bot 82:1046–1056 Schaller GE, Bishopp A, Kieber JJ (2015) The yin-yang of hormones: cytokinin and auxin interactions in plant development. Plant Cell 27:44–63. https://doi.org/10.1105/tpc.114.133595 Schnathorst WC (1954) Bacteria and fungi in seed and plants of certified bean varieties. Phytopathology 44:588–592 Shade A, McManus PS, Handelsman J (2013) Unexpected diversity during community succession in the apple flower microbiome mBio 4:e00602–e00612 Shade A, Jacques MA, Barret M (2017) Ecological patterns of seed microbiome diversity, transmission, and assembly. Curr Opin Microbiol 37:15–22 Shahzad R, Khan AL, Bilal S, Asaf S, Lee IJ (2018) What is there in seeds? Vertically transmitted endophytic resources for sustainable improvement in plant growth. Front Plant Sci 9:24 Shahzada R, Waqasab M, Khan AL (2017) Seed-borne endophytic Bacillus amyloliquefaciens RWL-1 produces gibberellins and regulates endogenous phytohormones of Oryza sativa. Plant Physiol Biochem 106:236–243 Sharma SB, Sayyed RZ, Trivedi MH, Gobi TA (2013) Phosphate solubilizing microbes: sustainable approach for managing phosphorus deficiency in agricultural soils. SpringerPlus 2:587 Shipunov A, Newcombe G, Raghavendra AKH, Anderson CL (2008) Hidden diversity of endophytic fungi in an invasive plant. Am J Bot 95:1096–1108 Singh B, Satyanarayana T (2011) Microbial phytases in phosphorus acquisition and plant growth promotion. Physiol Mol Biol Plants 17:93–103 Siqueira JA, Hardoim P, Ferreira PCG, Nunes-Nesi A, Hemerly AS (2018) Unraveling interfaces between energy metabolism and cell cycle in plants. Trends Plant Sci 23:731–747 Spatafora JW, Sung GH, Sung JM, Hywel-Jones NL, White JF Jr (2007) Phylogenetic evidence for an animal pathogen origin of ergot and the grass endophytes. Mol Ecol 16:1701–1711 Sun Y, Cheng Z, Glick BR (2009) The presence of a 1-aminocyclopropane-1-carboxylate (ACC) deaminase deletion mutation alters the physiology of the endophytic plant growth-promoting bacterium Burkholderia phytofirmans PsJN. FEMS Microbiol Lett 296:131–136 Tadych M, Torres MS, White JF Jr (2009) Diversity and ecological roles of clavicipitaceous endophytes of grasses. In: White JF Jr, Torres MS (eds) Defensive mutualism in microbial symbiosis. CRC Press, Boca Raton Tancos MA, Chalupowicz L, Barash I, Manulis-Sasson S, Smart CD (2013) Tomato fruit and seed colonization by Clavibacter michiganensis subsp. michiganensis through external and internal routes. Appl Env Microbiol 79(22):6948–6957 Tchinda RAM, Boudjeko T, Simao-Beaunoir AM, Lerat S, Tsala E, Monga E, Beaulieu C (2016) Morphological, physiological, and taxonomic characterization of actinobacterial isolates living as endophytes of cacao pods and cacao seeds. Microbes Environ 31:56–62 Thomas P, Sekhar AC (2014) Live cell imaging reveals extensive intracellular cytoplasmic colonization of banana by normally non-cultivable endophytic bacteria. AoB Plants 6. https://doi.org/ 10.1093/aobpla/plu002 Truyens S, Weyens N, Cuypers A, Vangronsveld J (2013) Changes in the population of seed bacteria of transgenerationally Cd-exposed Arabidopsis thaliana. Plant Biol 15:971–981 Truyens S, Jambon I, Croes S et al (2014) The effect of long-term Cd and Ni exposure on seed endophytes of Agrostis capillaris and their potential application in phytoremediation of metalcontaminated soils. Int J Phytoremediation 16:643–659 Truyens S, Beckers B, Thijs S, Weyens N, Cuypers A, Vangronsveld J (2016) Cadmium-induced and trans-generational changes in the cultivable and total seed endophytic community of Arabidopsis thaliana. Plant Biol 18:376–381 Tsavkelova EA, Klimova SY, Cherdyntseva TA, Netrusov AI (2006) Microbial producers of plant growth stimulators and their practical use: a review. Appl Biochem Microbiol 42:117–126

6

The Ecology of Seed Microbiota

125

Tsavkelova EA, Cherdyntseva TA, Klimova SY, Shestakov AI, Botina SG, Netrusov AI (2007) Orchid-associated bacteria produce indole-3-acetic acid, promote seed germination, and increase their microbial yield in response to exogenous auxin. Arch Microbiol 188:655–664 Vannette RL, Gauthier MPL, Fukami T (2013) Nectar bacteria, but not yeast, weaken a plant– pollinator mutualism. Proc R Soc Lond B Biol Sci 280:20122601 Vellend M (2010) Conceptual synthesis in community ecology. Q Rev Biol 85:183–206 Verma SK, White JF Jr (2018) Indigenous endophytic seed bacteria promote seedling development and defend against fungal disease in browntop millet (Urochloa ramosa L.). J Appl Microbiol 124:764–778 Verma SK, Kingsley K, Irizarry I, Bergen M, Kharwar RN, White JF Jr (2017) Seed-vectored endophytic bacteria modulate development of rice seedlings. J Appl Microbiol 122:1680–1691 Walitang DI, Kim K, Madhaiyan M, Kim YK, Kang Y, Sa T (2017) Characterizing endophytic competence and plant growth promotion of bacterial endophytes inhabiting the seed endosphere of Rice. BMC Microbiol 17:209 Wang Y, Li L, Ye T et al (2011) Cytokinin antagonizes ABA suppression to seed germination of Arabidopsis by downregulating ABI5 expression. Plant J 68:249–261 Wilson M, Lindow SE (1993) Interactions between the biological control agent Pseudomonas fluorescens A506 and Erwinia amylovora in pear blossoms. Phytopathology 83:117–123 Xu C, Liberatore KL, MacAlister CA et al (2015) A cascade of arabinosyltransferases controls shoot meristem size in tomato. Nat Genet 47:784–792 Zalamea PC, Sarmiento C, Arnold AE, Davis AS, Dalling JW (2015) Do soil microbes and abrasion by soil particles influence persistence and loss of physical dormancy in seeds of tropical pioneers? Front Plant Sci 5:799. https://doi.org/10.3389/fpls.2014.00799

7

Programming Plants for Climate Resilience Through Symbiogenics Rusty Rodriguez, Alec Baird, Sang Cho, Zachery Gray, Evan Groover, Roman Harto, Marian Hsieh, Katie Malmberg, Ryan Manglona, Malia Mercer, Natalie Nasman, Tia Nicklason, Melissa Rienstra, Alex Van Inwegen, Andy VanHooser, and Regina Redman

Contents 7.1 7.2 7.3 7.4

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endophyte-Conferred Abiotic Stress Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Symbiotic Lifestyle Switching by Fungal Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endophyte Commercialization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4.1 BioEnsure® Field Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 Climate Mitigation and the Future of Poverty, Food Security, and Political Stability . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

128 129 130 131 132 135 136

Abstract

All plants in natural ecosystems are thought to be symbiotic with fungal endophytes, some of which confer abiotic stress tolerance (drought, temperature, salinity). Recently, some of these fungal endophytes were commercialized as a R. Rodriguez (*) Adaptive Symbiotic Technologies, Seattle, WA, USA Symbiogenics, Seattle, WA, USA University of Washington, Seattle, WA, USA e-mail: [email protected] A. Baird University of California, Los Angeles, CA, USA S. Cho · Z. Gray · R. Harto · M. Hsieh · K. Malmberg · R. Manglona · M. Mercer · N. Nasman · T. Nicklason · M. Rienstra · A. Van Inwegen · A. VanHooser Adaptive Symbiotic Technologies, Seattle, WA, USA E. Groover University of California, Berkeley, CA, USA R. Redman Adaptive Symbiotic Technologies, Seattle, WA, USA Symbiogenics, Seattle, WA, USA # Springer Nature Switzerland AG 2019 S. K. Verma, J. F. White, Jr (eds.), Seed Endophytes, https://doi.org/10.1007/978-3-030-10504-4_7

127

128

R. Rodriguez et al.

product, BioEnsure®, to confer abiotic stress tolerance to food crops (www. adsymtech.com, Redman and Rodriguez, Functional importance of the plant endophytic microbiome: implications for agriculture, forestry and bioenergy, Springer, 2017). These endophytes enhance crop production on marginal lands and diminish the impacts of high temperatures on crop fertilization. Yield results from endophyte-colonized monocot and eudicot plants are remarkable and directly proportional to stress levels. Under low stress, BioEnsure® yield averages are 3% above control plants and increase to 26% under high stress. This was best exemplified in Rajasthan, India, where BioEnsure® was applied to pearl millet and mung bean seeds for 400 small landholding farmers. Under the hot, dry growing conditions that are typical in this part of India, the resulting average yield increases were 29% and 56%, respectively, compared to untreated plants. This translated to improved food security, animal fodder, carry-over seed, and revenues. Interest in the USA is growing with BioEnsure® treated seeds planted in 300,000 acres in 2017 and 600,000 acres in 2018, and more than 2,000,000 acres are projected for 2019. Keywords

Climate mitigation · Food security · Endophyte commercialization · Crop production · Symbiotic lifestyles

7.1

Introduction

One of the greatest needs in agriculture this century is to mitigate the impacts of abiotic stress (drought, temperature, salinity) on crop plants. The frequency and severity of drought, extreme temperatures, and soil salinization are increasing as a result of climate change (https://climate.nasa.gov/effects/). Considering the fact that the majority of agricultural effort globally involves small landholders and dryland cultivation, climate impacts on crop yields are already increasing poverty, famine, human migration, and political instability (Skøt et al. 2016; Brinkman and Hendrix 2011; Deaton and Lipka 2015; Simmons 2017). One solution to this problem is to generate abiotic stress-tolerant crops. In the last 40 years, there has been a tremendous effort to generate abiotic stress-tolerant crops via breeding or genetic modification. Unfortunately, these efforts have not been very fruitful because the underlying strategies incorrectly assumed plants adapt themselves to stress in natural ecosystems (Nuccioa et al. 2018; Gurian-Sherman 2012; Komives and Kirlay 2017). It is now clear that plants in high-stress habitats commonly adapt to environmental stresses by establishing symbiotic associations with fungal endophytes (Lugtenberg et al. 2016; Singh et al. 2011). In fact, without the fungal partners, native plants are no more adapted to abiotic stress than agricultural crops. Based on more than 25 years of research studying how plants in nature adapt to stress, Adaptive Symbiotic Technologies (AST) developed the product line BioEnsure®, a novel seed treatment comprising fungal endophytes that form symbiotic associations with a diverse group of crop plants and confer significant levels of

7

Programming Plants for Climate Resilience Through Symbiogenics

129

abiotic stress tolerance (www.adsymtech.com). Field tests have demonstrated that during periods of high drought and salinity stress, BioEnsure® increased crop yields on average from 26 to 60%. During growing seasons with little to no stress, BioEnsure® has been shown to increase yields an average of 3%. This chapter will cover the science behind BioEnsure®, results from 6 years of field testing, and efforts to bring this technology to poor rural farmers in India to help stabilize their future of agriculture and, in doing so, political security.

7.2

Endophyte-Conferred Abiotic Stress Tolerance

There are at least four classes of fungal endophytes associated with plants (Rodriguez et al. 2009) that can be differentiated based on host range and colonization, mode of transmission, symbiotic function, and genetic diversity. Collectively, these endophytes can have profound impacts on plant health, fecundity, survival, adaptation, and ecology. In fact, these symbiotic associations are one of the few universal components of plant ecology, and it appears that many, if not most, endophytes confer some level of drought tolerance to plants. It has been suggested that symbiotically conferred drought tolerance may be an evolutionarily conserved property of endophytes stemming back to the movement of plants onto land 450 million years ago (Redecker et al. 2000; Krings et al. 2007). More intriguing is the fact that many endophytes confer tolerance to other environmental stresses including temperature, salinity, heavy metals, toxic chemicals, and low nutrients (Figs. 7.1 and 7.2). The ecological ramifications of endophyte-conferred stress tolerance are significant and may occur in a habitat-specific manner (Rodriguez et al. 2008). Remarkably, plants that have resided in high-stress habitats for hundreds or thousands of years are not themselves stress-adapted. For example, Dichanthelium lanuginosum (tropical panic grass) thrives in geothermal soils where temperatures can reach 60
C

BioEnsure Yield (%) Response Relative to Abiotic Stress Severity

30 20 10 0 1

2

3

4

5

6

7

8

9

10

11

-10 -20 -30

Increasing Abiotic Stress (Low to High)

Fig. 7.1 Hypothetical relationship between endophyte conferred abiotic stress tolerance and yield

130

R. Rodriguez et al.

Yield Difference of BE vs Check (%)

40 35 30 25 20 15 10 5 0

1

2 Irrigation Level

3

Fig. 7.2 Percentage yield difference between untreated and BioEnsure-treated corn under varying levels of irrigation. Study was performed by an independent third party, RD4Ag in Yuma, AZ. Untreated and BioEnsure-treated seeds of a commercial corn hybrid were grown under three different irrigation regimes (1 ¼ 100%, 2 ¼ 75%, 3 ¼ 50% of recommended levels of water). Four replicate plots (4 rows  30 ft) of each biological treatment were planted per each water regime. Plants were exposed to high heat and variable water stress for the duration of the growing season. The percentage differences between untreated and BioEnsure-treated plants at both 100 and 75% irrigation levels were statistically significant ( p < 0.1). The difference at 50% irrigation was not statistically significant, and all plants were negatively impacted by the high level of stress

during the summer. The heat tolerance of this plant is dependent on a symbiotic interaction with the fungal endophyte Curvularia protuberata. Neither the plant nor the fungus is heat tolerant when grown axenically, so it is the symbiotic communication that is responsible for the fitness and survival of both partners (Redman et al. 2002). To make this story more complicated, there is a third member of the association, a double-stranded RNA virus in the fungus that is required for heat tolerance of the plant. This three-way symbiosis provides heat tolerance to both eukaryotic partners, and the association is not obligate because both partners can establish symbioses with genetically unrelated partners (Márquez et al. 2007). Even though symbioses are a requirement for plants in nature, the vast majority of plant-fungal symbioses do not involve obligate species-species interactions. The reasons for this are not known, but it is tempting to hypothesize that the lack of obligate associations reflects an evolutionary need for adaptive flexibility that allows plants to colonize complex landscapes that vary in environmental stresses.

7.3

Symbiotic Lifestyle Switching by Fungal Endophytes

Since many mutualistic benefits are based on symbiotic communication rather than direct biochemical contributions, it is not surprising that both partners have the ability to alter the outcome of the association. For example, individual fungal isolates can switch between mutualism and parasitism based on the genetic and/or ecological

7

Programming Plants for Climate Resilience Through Symbiogenics

131

environment they are exposed to (Redman et al. 2001; Alvarez-Loayza et al. 2011; Lofgren et al. 2018). Good examples of how host genetics can influence the outcome of symbioses have been reported for species of Colletotrichum and Fusarium. Several plant-pathogenic Colletotrichum species are able to colonize and express mutualistic lifestyles in asymptomatic host plants. For example, individual isolates of Colletotrichum magna are virulent pathogens of cucurbit species but are able to asymptomatically colonize tomato plants and express a mutualistic lifestyle. In this study, plants were separated into disease hosts, non-disease hosts, and nonhosts based on fungal colonization, disease expression, and plant stress tolerance. Mutualism was confirmed by enhanced plant biomass, drought tolerance, and disease protection (Redman et al. 2001). Studies with Fusarium in native prairie grasses revealed that F. graminearum asymptomatically colonizes plant tissues and seeds (Lofgren et al. 2018). F. graminearum is a common pathogen of agricultural grain crops such as wheat where it colonizes seeds, produces the toxin DON (vomitoxin), and causes head blight disease. Surprisingly, there was no evidence for vomitoxin production in any of the native grasses analyzed even though the fungus was present. However, when commercial wheat varieties were exposed to the F. graminearum isolates from native grasses, the plants were colonized and vomitoxin produced. Although the symbiotic lifestyle of the isolates in native grasses has not been assessed, it is clear that a genetically induced lifestyle shift occurs in commercial wheat. A fantastic example of how environmental conditions can result in a symbiotic lifestyle switch was demonstrated with palm trees and the endophyte Diplodia mutila. This plant-fungal symbiosis is common in tropical habitats, and the fungus switches symbiotic lifestyle in response to light. When the plants are growing in shade, the D. mutila expresses either a mutualistic or commensal lifestyle. However, when the plants are shifted into direct light, D. mutila expresses a pathogenic lifestyle (Alvarez-Loayza et al. 2011). Collectively, these studies and others revealed a new aspect of plant-fungal symbioses: symbiotic lifestyle switching. This can be viewed as a form of “symbiotic plasticity” that may have influence over both partners’ ability to establish and compete for resources in natural habitats. More importantly, it is an indication that there is still much to learn about symbiosis and its significance to life on earth.

7.4

Endophyte Commercialization

Since the inception of agriculture over 10,000 years ago, abiotic stress has been a major concern of farmers, and in modern times, this concern has not abated. As postindustrialization levels of atmospheric CO2 have continued to increase, global temperatures have increased, and concern over climate impacts on agriculture is now the major concern in many countries. The need for climate-resilient crops is at an all-time high but is considered a technological barrier to food security. To overcome this agricultural barrier, AST began testing fungal endophytes for the ability to confer abiotic stress tolerance to food crops (Redman and Rodriguez 2017,

132

R. Rodriguez et al.

www.adsymtech.com). In 2012, AST began field testing formulations and working to overcome the many hurdles to commercialization including scale-up production, product shelf life, environmental vulnerabilities, regulatory approval, and market penetration. During 2013–2018, field testing has expanded each year to encompass 40 US states and many locations in South America, Africa, Europe, Australia, and India. In 2017, AST commercialized its first product BioEnsure® as a microbial seed treatment to generate climate-resilient crops by virtue of enhanced drought, salt, and temperature stress tolerance. In 2018, AST expanded testing onto more than 30,000 acres in the USA with both liquid and powder formulations used to treat seeds, apply in furrow during planting, or apply directly to plants via foliar spray or fertigation.

7.4.1

BioEnsure® Field Performance

There are many ways to assess the effects of endophytes on crop species including seedling emergence, stand, seedling vigor, above- and belowground biomass, chlorophyll levels, spectral reflectance patterns, fertilization, grain fill, time to maturity, etc. However, the bottom line for farmers is the abiotic stress tolerance, yield, and yield quality of crops. Since 2012, AST has been working with independent cooperators to test the efficacy of BioEnsure® in field trials with a diversity of crops including some of the major crops that sustain human life (Table 7.1). As anticipated from previous studies (Redman and Rodriguez 2017), the BioEnsure® endophytes conferred abiotic stress tolerance similarly in monocot and eudicot crop plants. Moreover, the formulation of endophytes was designed to generate climate-resilient crops with enhanced tolerance to multiple abiotic stresses (water, temperature, salinity, low nutrient). Field test results have allowed AST to better understand the relationship between endophytes, abiotic stress, and crop production (Fig. 7.1). The yield differences between BioEnsure® treated and untreated plants is small when stress levels are low. However, as stress levels increase, the yield differences continually increase until the stress levels reach a point where all plants are impacted. There is a transition phase where control plants are not affected by the stress and maintain comparative yield levels to treated plants, but there is a point where the stress exceeds untreated plant tolerance levels and yields decrease. During the phase of increasing stress when untreated plant yields decrease, BioEnsure® plants continue to increase yield potential. For example, when BioEnsure® benefits were assessed in relation to recommended irrigation levels, corn yields of treated plants were higher at 75% of recommended irrigation levels compared to the 100% level (Fig. 7.2). The temperature during the irrigation trial was very hot and dry which resulted in significant yield differences even at 100% irrigation. Previous studies on the ability of fungal endophytes to confer abiotic stress tolerance revealed that the symbiotic communication responsible for stress tolerance was conserved across genetically distant monocots and eudicots (Rodriguez et al. 2008). These plant lineages diverged more than 145 million years ago (Wolfe et al. 1989; Yang et al. 1999; Chaw et al. 2004), suggesting that symbiotic communication had been established long before that. The evolutionary conservation of endophyte-

7

Programming Plants for Climate Resilience Through Symbiogenics

Table 7.1 Observed effects of BioEnsure treatment on crops in relation to plant biomass, stress tolerance, and overall yield

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Crop Alfalfa Barleya Blueberries Canola Corna Cotton Cucumber Dry beansa Field peas Guar Leafy greens Lentilsa Milleta Mung bean Okra Onion Pasture grass Potatoa Ricea Sesame Sorghum Soybeana Sugar beets Wheata

Biomass + + + + + + + + + + + + + + + + + + + + + + + +

Stress tolerance + NYD NYD + + + NYD NYD + + NYD + + + + + + NYD + + + + + +

133

Yield + + NYD NYD + + NYD NYD + + NYD + + + + + + + + + + + + +

NYD not yet determined Some of the 20 major crops that sustain human life

a

conferred stress tolerance is represented by the relationship between crop yields and abiotic stress in corn and cotton (Fig. 7.3). The data were generated from field tests in various locations in the USA and India where different levels of either water or temperature stress occurred during the growing season. These results are in line with the concept portrayed in Fig. 7.1. As part of a grant from USAID, AST was asked to accelerate the development of BioEnsure® and bring this climate mitigation technology to small landholding farmers living below poverty levels. Field testing was coordinated with 300 farmers in a remote region of Rajasthan, India. In this part of India, dryland cultivation dominates and is dependent on annual monsoon rains. However, in recent times rainfall has become less abundant and inconsistent resulting in drier and very hot growing seasons with temperatures commonly above 38
C. In 2016 and 2017, AST staff traveled to Rajasthan to treat seeds and work with farmers to design comparative field tests with BioEnsure® treated and untreated seeds. The crops treated were

134

R. Rodriguez et al.

Corn

Cotton 90 80

50

Yield Difference vs Checks (%)

Yield Difference vs Checks (%)

60

40 30 20 10

70 60 50 40 30 20 10

0

0 1

2

3

4 5 6 Field Test

7

8

1

3

5

7 9 11 13 15 17 Field Test

Fig. 7.3 BioEnsure yield increases as abiotic stress increases (left to right in each graph). Each bar represents a field comparison of treated versus untreated (check) plants. Field plots varied from 1 to 50 acres with a single replicate for each treatment. Testing was performed by farmers on their properties

pearl millet and mung bean, the major staple crops of the region, which allowed us to compare the relationship between stress and yield in a monocot and eudicot simultaneously in the same soil types. In addition, comparisons were made between BioEnsure® treated carry-over seeds from previous growing seasons and untreated fresh market seeds. This was important because there is not enough replacement seed produced annually in India, and fresh market seed is 4–8 times the cost of carry-over seed and beyond the economic capacity of most farmers. The yield results were greater than farmers expected with average BioEnsure® yield increases of 29% in pearl millet and 56% in mung bean (Fig. 7.4). There were several important outcomes of BioEnsure® conferred benefits to these plants: 1. The yield of BioEnsure® treated carry-over seeds was equal to or higher than yields of fresh market seeds. 2. The biomass of pearl millet was increased by 25–50%. 3. Stress tolerance delayed plant senescence sufficiently to allow for secondary fertilization of mung bean.

7

Programming Plants for Climate Resilience Through Symbiogenics

Mung Bean

2000

750

1900

700

1800

650

1700

Average Yield (lb/acre)

Average Yield (lb/ac)

Pearl Millet

1600 1500 1400 1300

600 550 500 450

1200

400

1100

350

1000

135

300 Check

BioEnsure

Check

BioEnsure

Fig. 7.4 BioEnsure enhanced yield results for two staple crops in Rajasthan, India. Cultivation was dryland with water derived from the annual monsoon. Temperatures were typically above 38
C during the days. The bars represent field tests on 300 farms with test plots varying in size from 0.5 to 5 acres. Each farm had one treated plot and one untreated plot. The yield differences were statistically significant (T test, p value 80% of the plantassociated communities. In the cultivated agave, Enterobacteriales represent nearly 43% of the rhizosphere and Flavobacteriales 27.6% of the phyllosphere. Drought apparently does not influence the microbial communities, since they are similar between the species, so it was suggested that these microorganisms participate in plant fitness (Coleman-Derr et al. 2016). These reports suggest that adaptation of Agave species to arid areas, extreme environments, and nutritionally limited soils could be mediated not only by their inherent characteristics (CAM metabolism, morphology adapted to arid environments) but also by their symbiotic microbes.

8.4.1

The Culturables Microbes

Martínez-Rodríguez et al. (2014) reported cultivable endophytic bacteria and evaluated their agronomic potentials and antifungal effects. Three hundred cultivable endophytic bacteria were isolated from leaf bases of A. tequilana. In plant tissue bacteria occurred at mean population densities of 3 million CFU/g of fresh plant. Bacteria were grouped into eight different taxa that shared high homology with other known sequences and were proved to be plant growth-promoting bacteria (PGPB) for their ability to fix N, producing auxins, solubilizing phosphates, and possessing antagonistic activity against a pathogenic strain of Fusarium oxysporum AC132, demonstrating that endophytic bacteria are a promising alternative as biofertilizers for agave cultures, or to replant microorganisms lost in agave seedlings originated by micropropagation where native endophytes may have been eliminated. Endophytic microbes become excellent candidates as bio-inoculants to improve the productivity

8

Agave Seed Endophytes: Ecology and Impacts on Root Architecture, Nutrient. . .

147

Table 8.1 Endophytic bacteria isolated or characterized from agave plants Agave species A. tequilana

A. americana A. salmiana a

Bacterial genera (isolates or aOTUs) Endophytic Acinetobacter sp., A. baumannii, A. bereziniae, Acidobacteria Gp4, Alcaligenes faecalis, Acidovorax facilis, Achromobacter spanius, Arthrobacter roseus, Bacillus sp., B. litoralis, B. tequilensis, B. mycoides, B. oshimensis, B. safensis, B. siralis, Brevundimonas diminuta, Burkholderia caribensis, B. gladioli, B. phenazinium, Cellulosimicrobium cellulans, Citrobacter freundii, Clostridium, Cronobacter sakazakii, Chryseobacterium gleum, Corynebacterium glutamicum, Enterobacter, E. cancerogenus, E. cloacae, E. cowani, E. hormaechei, Enterococcus casseliflavus, Erwinia pyrifoliae, Gluconobacter oxydans, Klebsiella oxytoca, K. pneumonia, Lactobacillus fructivorans, L. paracasei, L. plantarum, Leuconostoc mesenteroides, Micrococcus luteus, Myroides odoratus, Pseudomonas sp., P. aeruginosa, P. libanensis, P. stutzeri, P. syringae, Reynarella, Solibacillus silvestris, Staphylococcus capitis, S. warneri, Streptomyces, Stenotrophomonas sp., S. maltophilia, S. acidaminiphila, Rhizobium radiobacter, Paenibacillus amylolyticus, Pantoea agglomerans, P. terrea, Ochrobactrum gallinifaciens, O. grignonense, Weissella halotolerans Rhizobium, Pseudomonas Acidobacteria Gp4, Acidobacteria Gp6, Streptomyces, Clostridium Reynarella, Enterobacter, Stenotrophomonas, Bacillus

Bacterial genera presented in bold were identified by independent culture method Fonseca-Sepulveda (2017)

b

of this domesticated Agave. In Table 8.1, we summarize those endophytic bacteria identified as part of the Agave microbiome using independent and dependent-culture methods.

8.5

Core Microbiome of Agave Seeds: Who Is There?

In recent decades it has been discovered that seeds bear microbial communities comprised of bacteria and fungi. Seed transmission of endophytic microbes appears to be important in shaping the endophyte community in the mature plant and consequently acts as the initial inoculum for the plant microbiota (Barret et al. 2015). Seed-associated microbes should be regarded as very intimate microbial partners of higher plants, with the potential to connect successive plant generations (Shade et al. 2017). Those microbes participate in seedling growth, favor intake of nutrients and resistance to abiotic and biotic stress, and, in some cases, can be used as “food” for the plants (Beltran-Garcia et al. 2014). It is well-known that endophytes transmitted by seed were first discovered in ryegrass in 1898, and the importance of seed bacteria has gradually been realized in recent decades. One hundred and thirty genera from 4 different phyla of 25 different plants species have been reported as naturally occurring seed endophytes (Truyens et al. 2014). The most predominant seed endophytes belong to the γ-proteobacteria class, Proteobacteria phyla, followed by the Actinobacteria, Firmicutes, and

148

A. Martinez-Rodriguez et al.

Bacteroidetes phyla. In general, Bacillus, Pseudomonas, Paenibacillus, Micrococcus, Staphylococcus, Pantoea, and Acinetobacter are often detected in seeds. To investigate endophytic and seed-endophytic populations, researchers used the culture-dependent and culture-independent methods. Currently the newly developed high-throughput sequencing technique has been applied to assess plant seed microbiomes. Actually, one of the difficulties to studying plant endophytic microbiomes is the ability to determine a plant’s core microbiome (Zhang et al. 2018). Discovery of the core microbiome is important for understanding the stable, consistent components across complex microbial assemblages. A core microbiome is typically defined as the suite of members shared among microbial consortia from similar habitats and is represented by the overlapping areas of circles in Venn diagrams, in which each circle contains the membership of the sample or habitat being compared (Shade and Handelsman 2012). Yet, their cultivation in the laboratory is required to appreciate microbial physiology and to conduct experiments to evaluate the role that microbes play in plant fitness.

8.5.1

What Microbes Are Inside Agave Seeds?

To know the microbial ecology of agave seeds, three Agave species were selected under an ecological criterion to show the composition of the cultivable seed microbiome, according to domestication state and the economic tendency to create a monoculture: (a) A. tequilana Weber or “blue agave” was the first domesticated species, and it is no longer found in a wild condition. A. tequilana is in monoculture in five Mexican states that make up the appellation of origin “Tequila.” The crop shows low genetic variability; its cultivation depends on heavy chemical fertilization applications and high use of pesticides to control insects and pathogens. In addition, it is susceptible to low temperatures. (b) A. angustifolia Haw, also known as “agave espadin,” is semidomesticated and in progress toward being a monoculture, but it is still possible to find wild varieties in seasonal dry scrubs and forests of Quercus-Pinus, from Mexico to Central America. Its maturation takes from 8 to 12 years and is the source of production of the “Bacanora” and mezcal in the states of Sonora and Oaxaca; it is considered as an ancestor of both blue agave and henequen agave. (c) A. marmorata Roezl is an endemic species of lowland forests and scrub ends of the states of Oaxaca and Puebla. However, it is already in the process of domestication. Reports indicate that it is an endangered agave. “Mezcal tepextate” is produced exclusively from wild individuals that take 18–25 years to mature. It is found on sunny and rocky hillsides, sometimes clinging directly to the rock, on the top of a rock, or on almost vertical slopes with minimal or no topsoil to sustain it; however, its roots extend affirming and avoiding erosion in rainy seasons. There are no protection programs for this species to safeguard its genetics and populations (Fig. 8.2).

8

Agave Seed Endophytes: Ecology and Impacts on Root Architecture, Nutrient. . .

149

Fig. 8.2 Seeds of Agave species. Seeds are flat and black in color and showed a considerably variation in size: shiny A. marmorata seeds (0.5 cm width  0.5 cm length), opaque A. tequilana seeds (0.9 cm width  0.7 cm length), and opaque A. angustifolia (0.8 cm width  0.7 length). Seeds were collected directly from fruits of plants located in Mexican states of Oaxaca, Jalisco, and Sonora, respectively

The colony-forming unit (CFU) of bacteria recovered from each Agave seed ranged from Log10 3.36–6.54 CFU per gram of seeds, where seeds of A. angustifolia had the highest number of bacteria recovered and A. tequilana the lowest bacterial abundance. Significant difference was observed between A. tequilana and the other Agave species, but these differences were not significant between semidomesticated A. angustifolia and wild A. maximiliana and A. marmorata. Colonies of different morphologies and colors were picked for isolation and identification. Using MALDI-TOF mass spectrometry as the first option for ID, all bacteria were grouped. Two isolates of each group were selected and then identified by the partial sequencing analysis of 16SrDNA. A total of 500 seed bacteria were identified. The number of seed-endophyte bacterial genera was diverse among Agave species. Microbial isolates of A. maximiliana was grouped in 8 genera, 16 genera in A. angustifolia, 14 genera in A. tequilana, and 19 genera in A. marmorata. Further classification showed that bacteria belonged to four phyla: Proteobacteria, Actinobacteria, Firmicutes and Bacteriodetes. The most commonly seed-endophytic bacteria on Agave belong to the phylum Firmicutes and constituted 54%, 50%, 47%, and 44% of the total species types of A. angustifolia, A. tequilana, A. maximiliana, and A. marmorata, respectively (Fig. 8.3). Species of the phylum Firmicutes included Bacillus altitudinis, B. amyloliquefaciens, B. cereus, B. circulans, B. endophyticus, B. galactosilyticus, B. gibsonii, B. licheniformis, B. megaterium, B. mojavensis, B. nealsoni, B. pseudomycoides, B. pumilus, B. rhizosphaerae, B. safensis, B. siralis, B. sonorensis, Bacillus sp., B. subtilis, B. tequilensis, B. thuringiensis, B. vallismortis, B. weihenstephanensis, Enterococcus casseliflavus, Lactobacillus crispatus, L. kalikensis, Listeria grayi, Lysinibacillus fusiformis, Solibacillus silvestris, Staphylococcus chromogenes, S. cohnii, S. epidermidis, S. haemolyticus, S. hominis, S. pasteuri, S. warneri, S. xylosus, Paenibacillus sp., and P. taiwanensis. Other phyla identified included Proteobacteria, Actinobacteria, and Bacteroidetes. Proteobacteria

150

A. Martinez-Rodriguez et al.

Agave angustifolia

Agave maximiliana 4%

3% 9%

34%

49%

47%

54%

Actinobacteria

Agave tequilana

Agave marmorata

Firmicutes Proteobacteria Bacteroidetes

8% 27%

29%

42% 50%

44%

Fig. 8.3 Relative abundance of cultured seed endophytes of the Agave species

was the second most abundant phylum and contained 28 species including Acinetobacter radioresistens, A. lwoffi, Achromobacter denitrificans, A. spanius, Aeromonas molluscorum, Alcaligenes faecalis, Brevundimonas diminuta, Enterobacter asburiae, E. cloacae, E. cowanni, E. hormachei, E. kobei, Klebsiella pneumoniae, K. variicola, Kosakonia oryzae, Novosphingobium aromaticivorans, Pantoea agglomerans, P. terra, Pseudomonas aeruginosa, P. lutea, P. monteilii, P. putida, Pseudomonas sp., P. stutzeri, Sphingomonas thalpophilum, Stenotrophomonas acidaminiphila, S. maltophilia, and S. rhizophila. The phylum Actinobacteria included nine genera such as Brevibacterium, Curtobacterium, Kytococcus, Kocuria, Microbacterium, Micrococcus, Rhodococcus, Streptomyces, and Tsukamurella, and finally the phylum Bacteroidetes had the lowest abundance and included 13 isolates of Sphingobacterium from A. angustifolia seeds. Interestingly, the distribution pattern within seed-endophytic isolates of four agaves showed that 14 bacterial species comprised 48% of the total isolates, with the most abundant being B. pumilus, B. subtilis, A. faecalis, and E. cloacae all of them with 28 isolates and 26 isolates of B. safensis. The second most abundant species were Sphingobacterium thalpophilum, from Bacteroidetes phylum that solely appear in A. angustifolia seeds (13), P. aeruginosa (10), E. cowanni (10), and S. maltophilia (10). The third group including K. marina (7), B. thuringiensis (7), B. sonorensis (7), and B. tequilensis (7). The concept of core microbiome was firstly established for human microbiome and further expanded to other host-associated microbiomes such as plants (Shade and Handelsman 2012). The composition and function of plant core microbiomes

8

Agave Seed Endophytes: Ecology and Impacts on Root Architecture, Nutrient. . .

151

Fig. 8.4 Venn diagram of the culturable seed-endophyte core microbiome based on shared composition of bacterial taxa of three Agave species. The major shared genera were Pseudomonas, Enterobacter, Bacillus, and Stenotrophomonas. Different circles represent different Agave species, and the intersections of the circles showed the shared bacterial genera in their seeds

have been achieved for several model plants, such as Arabidopsis, maize, and rice (Johnston-Monje and Raizada 2011; Lundberg et al. 2012; Edwards et al. 2015). The bacterial communities identified of the four Agave species were used to create the Venn diagram shown in Fig. 8.4. We found enriched core taxa (genera) overlap among these agave seeds, which include bacterial genera Bacillus (Firmicutes), Staphylococcus (Firmicutes), Micrococcus (Actinobacteria), Acinetobacter (γ-proteobacteria), Enterobacter (γ-proteobacteria), Pantoea (γ-proteobacteria), Pseudomonas (γ-proteobacteria), and Stenotrophomonas (γ-proteobacteria). These findings suggested an interesting coevolution among those bacterial taxa and Agave. Besides the major shared microbial phylum of the agave seeds reported here, they also have been found in other plant seeds such as maize, bean, rice, Brassica, salvia, and soybean. These bacterial taxa and their host seeds are summarized in Table 8.2. As we show in Table 8.2, the abundant taxa of seed bacteria of agave are also conserved in other plant species. Functionality of Bacillus and Pseudomonas taxa range from plant growth promotion to disease protection. These genera in the seed microbiomes are likely to be important reservoirs of rhizosphere or endosphere microbiome. The ability of Enterobacter to increase seed germination and seedling

152

A. Martinez-Rodriguez et al.

Table 8.2 Endophytic microbes found in seeds of different plant species Taxa Bacillus

Staphylococcus Micrococcus Acinetobacter Enterobacter Pantoea

Pseudomonas

Stenotrophomonas

Plant species Triticum aestivum, Oryza sativa, Lycopersicon esculentum, Tylosema esculentum, Zea mays, Cucurbita pepo, Vitis vinifera, Glycine max, Coffea arabica, Brassica napus, Medicago sativa Z. mays, V. vinifera, Phaseolus vulgaris, M. sativa O. sativa, C. arabica, M. sativa, Z. mays, G. max, P. vulgaris, O. sativa Z. mays, Nicotiana tabacum, Salvia miltiorrhiza, O. sativa, T. aestivum Tylosema esculentum, Z. mays, O. sativa, C. arabica, T. aestivum, Hordeum vulgare Phragmites australis, O. sativa, C. pepo, N. tabacum, B. napus, H. vulgare, M. sativa O. sativa, N. tabacum, C. arabica, M. sativa

Function Plant growth promotion, antifungal, IAA production, metabolite production, osmotic stress tolerance

Plant growth promotion, antimicrobial Plant growth promotion Phytate solubilizing, plant growth promotion, ACC deaminase Plant growth promotion Antibiotic production, IAA production, antifungal, plant growth promotion Plant growth promotion, antifungal, IAA production, metabolite production, mitigating metal toxicity, protease production Antifungal, plant growth promotion, mitigating biotic and abiotic stress

References: Rybakova et al. (2017), Verma et al. (2017), White et al. (2018), Yang et al. (2017), Rahman et al. (2018), Shahzad et al. (2018), Chen et al. (2018)

growth has been reported (Shahzad et al. 2018). In recent years, some studies found Pantoea spp. isolated from rice, maize, wheat, and Brassica seeds and showed antagonistic activities against pathogens and plant-growth promoting traits (Rybakova et al. 2017). However, Pantoea is well-known as a plant pathogen that can be transmitted by seed (Barret et al. 2016). Therefore, the function of Pantoea as seed-associated microbe needs to be further evaluated. In conclusion, contrary to other seed microbiomes, the Firmicutes phylum dominates over other bacterial phyla, with predominance of Bacillus spp. In the next section, we will discuss this finding in relation to functionality of Firmicutes.

8.6

Dynamics of Seed-Endophytic Bacteria During Emergence Until Formation of the Seedling: The Case of Wild Agave marmorata

Seeds represent the initial microbial inoculum of the plant microbiome and could subsequently have a significant impact on plant health and productivity. Indeed, the composition of the seed microbiome can contribute to seed preservation, release of seed dormancy, and increase or decrease in the germination rate. Moreover, seed

8

Agave Seed Endophytes: Ecology and Impacts on Root Architecture, Nutrient. . .

153

transmission of phytopathogenic agents is a major means of dispersal and is therefore significant in the emergence of any disease. Few studies have focused on community diversity and dynamic succession of endophytic bacteria during different seed developmental stages (Johnston-Monje and Raizada 2011; Liu et al. 2013; Barret et al. 2015; Pitzschle 2018). Microbial community dynamics of the wild A. marmorata was assessed from seed to mature seedlings using MALDI-TOF/16S rDNA sequencing, and we found significant variability in the seeds and the subsequent stages of emergence at 10, 20, and 60 days, which include seedling development up to a 1-year-old plant. In the “intact seeds or stage 0,” 95 strains were isolated and grouped into 20 genera and 35 species. The phylum Firmicutes (44%) dominated the number of total isolates of the seed, followed by Actinobacteria (30%) and phylum Proteobacteria (26%). The largest number of isolates belonged to the genera Bacillus, Staphylococcus, Kocuria, Stenotrophomonas, and Micrococcus, which are commonly recognized as beneficial. An interesting question to be answered is, why do Agave seeds host Firmicutes and Actinobacteria? To our knowledge, Firmicutes include bacteria that are generally more resistant to drying and UV radiation, which might provide an advantage in their capacity to survive on/in seeds. Bacillus is an aerobic endospore former; endospore formation may protect bacteria from changes within seeds (to tolerate storage, desiccation, seed maturation, seed germination). Also, Bacillus spp. use a wide range of substrates and have a large number of enzymes that degrade complex polysaccharides such as extracellular cellulases. It has been hypothesized that plants have selected Bacillus as part of their core seed microbiome to assist with proteases, nitrogen uptake, and assimilation following their migration to roots (Khalaf and Raizada 2016). As we will show later, Bacillus species alter root architecture of agave seedlings and serve as “food” in nutrient limited soils. Bacillus safensis, a seed endophyte of maize, upregulates genes involved in remodeling cell walls, the antioxidant responses, and the inorganic N-uptake in the maize roots (Prieto et al. 2017). Recently, Irizarry and White (2018) showed how cotton seedling roots respond to inoculation with B. amyloliquefaciens. They observed an overexpression of genes involved in auxin transport (WAT1), auxin homeostasis (IAA synthetase GH3.1), and lateral root formation by IAA14 gene expression. Thus, that upregulation impacts root architecture. Also, they found the upregulation of genes encoding enzymes involved for carbohydrate metabolism, nitrogen acquisition, and genes to prevent fungal disease. In seeds, reactive oxygen species (ROS) production is beneficial for seed germination and seedling growth by regulating cellular growth and has important roles for endosperm degradation, mobilization of seed reserves, and protection against pathogens. Some Bacillus act as part of an antioxidant system indirectly by expression of genes encoding catalases, peroxidases, and SOD, which work to prevent oxidative damage to the seedlings. Micrococcus luteus (Actinobacteria) was identified as a seed endophyte of agaves; this microbe has been considered to be an Rpfs producer (resuscitationpromoting factors). The Rpf is a 16–19 kDa protein with muralytic activity, which can facilitate cell division and regrowth at very low picomolecular concentrations by

154

A. Martinez-Rodriguez et al.

Fig. 8.5 Ring charts showing composition endophytic bacteria and their dynamics from seed of A. marmorata to seedlings. Seeds contain a selected core of microbes whose functions are indispensable for seedling emergence. The ring shows phyla, genus, and species, and colors represent Firmicutes (black), Actinobacteria (orange), and Proteobacteria (yellow)

remodeling the cell envelope of viable but not culturable (VBNC) cells. The release of Rpf by an actively growing individual into the environment wakes up neighboring cells, resuscitating them from a dormant state. Because Rpf is released outside of the cell, it has been proposed that it can potentially wake up neighboring cells from different lineages, increasing the competition for new available resources. The abundance of Firmicutes and Actinobacteria in agave seeds at stage 0, and the information published of their endophytic members, create a possible scenario of the function of these microbes in agave and maybe other plants from arid and semiarid environments. Recently, we found strains of M. luteus producing Rpf in our collection derived from agave seeds. However, further studies are necessary to prove microbial functionality on seeds. Independently on the initial composition of the A. marmorata seed’s microbiota, germination affects both microbial diversity and the number of isolates. Ten days after radicle appearance, seedlings undergo dramatic shifts in their microbial composition. Proteobacteria were the most abundant phylum at this stage (57%), indeed Gammaproteobacteria becomes the most abundant bacterial class associated to seedlings, but alpha- and β-Proteobacteria also appear (Fig. 8.5). Actinobacteria was the second most abundant (35%), and the Firmicutes decay up to 8%. As was previously reported, during germination the dominant taxa decrease with a marked increase in relative abundance of Proteobacteria (Barret et al. 2015; Torres-Cortés

8

Agave Seed Endophytes: Ecology and Impacts on Root Architecture, Nutrient. . .

155

et al. 2018). During emergence the embryo takes nutrient released from endosperm, so the disposition of amino acids and simple carbohydrates may select for fastgrowing microorganisms. However, the difference in growth rate is not the only fact that can argue the abundance of Proteobacteria and the exclusion of Firmicutes. According to publications about plant growth promotion activities of the abundant taxa such as Stenotrophomonas, Pseudomonas, Achromobacter, and Ochrobactrum, we can suggest that microbes will be selected because of their influence in seedling growth and development, including nutrient acquisition, suppression of pathogenic microorganisms, and promotion of resistance to biotic and abiotic stress (Alavi et al. 2013; Singh and Jha 2017; Zhang et al. 2018; White et al. 2018; Rahman et al. 2018). The predominance of phylum Proteobacteria is maintained until 60 days (60% vs. Actinobacteria 20% and Firmicutes 20%), reaching its maximum level at 20 days (74%). New bacterial genera such as Aeromonas, Sphingomonas, Alcaligenes, and Burkholderia appear in seedling tissues (Fig. 8.5). After 60 days, seedlings were transferred from phytagel to a microcosm with sand free of organic material for plant support. Seedlings were incubated for 10 months under conditions of 12 h light–12 h darkness at 32
C, watered with 1 ml of H2O every 5 days. The composition of A. marmorata microbiome returned to have a greater abundance of the phylum Firmicutes (60%), while Proteobacteria was 35%, and Actinobacteria had the lower abundance (5%). The Log10 was 5.204 CFU/gram of tissue. Strains of B. pumilus, B. safensis, P. barcinonensis, L. fusiformis, and Staphylococcus were isolated from plantlets after 365 days . From phylum Proteobacteria we found species such as A. xylosoxydans, A. insolitus, Burkholderia gladioli, S. maltophilia, and A. faecalis. As has been proposed by Barret et al. (2016), seeds disperse pathogenic microorganisms. Here we found phytopathogenic strains of Pseudomonas caricapapayae, P. stutzeri, and P. monteilii. Some endophytes leave roots to colonize the surrounding soil, establishing a communication that benefits dispersion and colonization of largest areas.

8.7

Agave “Eats” Microbial Endophytes to Survive in Soils Without Nitrogen

Diazotrophic endophytes could provide nutrients to plants even though they lack nodules, under a process called associative nitrogen fixation (Carvalho et al. 2016). Some work has been done to explain how the bacterial nitrogen moves to the plant directly from microbes. Paungfoo-Lonhienne et al. (2010) showed how plants consume microbes internalized into root cells. They named this microbe consumption process “rhizophagy” since in the process, roots consumed microbes (Lonhienne et al. 2014). White et al. (2012) proposed oxidative nitrogen scavenging (ONS) as a mechanism for transfer of organic nitrogen from microbe to plant (White et al. 2014). ONS involves plant secretion of reactive oxygen species (e.g., H2O2) onto microbes and their secreted enzymes; microbes and their protein content is oxidatively degraded; later plants secrete proteases that further degrade denatured enzymes into peptides that may be absorbed by plants and associated bacteria. We also observed that in some plants, intracellular bacterial endophytes in root

156

A. Martinez-Rodriguez et al.

Fig. 8.6 Plants without bacterial inoculation did not show intracellular bacteria in the root hairs (left side). Two days after inoculation with the A. tequilana seed-endophyte Enterobacter cloacae, presence of H2O2 (brown coloration) is observed in the tips of root hairs. Brown spots within root hairs indicate sites of microbial degradation/exposure by reactive oxygen (right side)

epidermis cells exit plant roots at the tips of developing root hairs to acquire additional soil nutrients and then reenter the root at the root tip meristem and are subjected to reactive oxygen as the root cells differentiate (Fig. 8.6). Because of the cyclic nature of this process, it has been denominated as “rhizophagy cycle” and has been suggested to sustainably provide nutrients (micronutrients) from the symbiotic rhizobacteria (Kakar et al. 2018; Prieto et al. 2017). We hypothesize that nutrients like iron and other difficult-to-acquire soil micronutrients may be obtained by plants from microbes that have siderophores to sequester micronutrients and transport them back to plant roots where they may be oxidatively extracted and absorbed by root cells in the rhizophagy cycle. In plant tissues reactive oxygen secreted onto endophytic microbes may induce electrolyte leakage from bacteria that result in loss of electrolytes from bacterial cells. Electrolytes, including macro- and micronutrients, may then be absorbed by root cells. Beltran-Garcia et al. (2014) conducted isotopic nitrogen tracking experiments to evaluate nitrogen transfer from bacterial cells into the plant. A seed-endophytic B. tequilensis was labeled with 15N by cultivation in M9 broth containing 15N-labeled nitrogen. 15N-labeled bacteria were applied to plants of Agave tequilana over multiple months. 15N-labeled nitrogen was measured in chlorophyll molecules using mass-spec analysis. Detection of 15N in plant molecules demonstrated that nitrogen in the bacteria passed to the plant. In a second experiment comparing absorption of 15N-labeled live bacteria to absorption of nitrogen in labeled but heat-killed bacteria, it was found that more nitrogen moved into the plants when live bacteria were used than when heatkilled bacteria were used. This suggested that movement of nitrogen from microbe to

8

Agave Seed Endophytes: Ecology and Impacts on Root Architecture, Nutrient. . .

157

plant was more efficient from living endophytic microbes and thus may not simply be the result of mineralization of bacterial proteins in soils around plant roots.

8.7.1

Seed-Endophytic Fungi May Also Transfer Organic Nitrogen

Behie et al. (2012) showed the potential role of fungal endophytes for organic nitrogen transfer to plants. They used Metarhizium robertsii a soil-dwelling insectpathogenic fungi and are able to form close symbiotic associations with plants as endophyte. To probe the movement of nitrogen, an insect larva was enriched with 15 NH4Cl and then infected with mycelia in a plant microcosm. They traced the 15N incorporation in the amino acids of the two plant species. The fungus had the ability to infect larva and colonize roots at the same time, creating a bridge where the insectderived nitrogen is translocated to plants. In our lab we isolated Diaporthe sp. as endophytic fungus from seeds of A. tequilana. Diaporthe (Phomopsis) species have often been reported as plant pathogens, nonpathogenic endophyte, or saprobes, commonly isolated from a wide range of hosts. Agave plantlets were inoculated with Diaporthe and irrigated with water and Mineral Medium (MM) as negative and positive source of nutrients, respectively. Leaves of water-irrigated plants were chlorotic, developed a reddish-black pigmentation, and then dried; plants irrigated with MMN produced new leaves and appeared new roots, but at the end of the experiment, biomass decreased. Fungal-treated plants developed more new leaves than other treatments and also increased root size, but finally did not influence the growth of the plants, senescence was delayed, and no plants died. To evaluate 15N transfer to plants, fungal mycelium was grown in modified Czapek-Dox broth enriched with 15NH4Cl and the inoculated into plant microcosm. The incorporation of the 15N label into plants inoculated with 15NDiaporthe is consistent with a scenario where N is transferred to plants when plants are nutrient limited. According to Fig. 8.7, our results show incorporation of 15N into pheophytin, a molecule derived from plant chlorophylls. The percentage of relative abundance of isotopomers shows 15N label into some of the four nitrogen atoms of tetrapyrrole (1N ¼ 873.57, 2N ¼ 874.57, 3N ¼ 875.57, and 4N ¼ 876.57).

8.7.2

Seed Endophytes Can Shape Root Architecture

Root architecture is often defined as the spatial configuration of the root system in growth media and determines the three-dimensional distribution of different root types in the root system across the soil profile. Variation in root system architecture plays a key role in crop nutrient efficiency (Parada et al. 2016; Prieto et al. 2017). The plant root system is obviously essential for plant growth and serves a wide range of functions, including nutrient and water acquisition, anchorage, and symbiosis with beneficial microbiota for enhancing the efficiency of nutrient absorption. Many endophytic and rhizospheric bacteria can indeed synthesize ethylene, gibberellins, cytokinins, and auxins (Raheem et al. 2018; López-Bucio et al. 2007). Among these

158

A. Martinez-Rodriguez et al.

15N-enriched Diaporthe sp.

60

Relative abundance of phephytin isotopomers (%)

50 40 30 20 10 0 871.58

872.58

873.58 874.58 m/z

875.58

876.58

875.58

876.58

Control

100 90 80 70 60 50 40 30 20 10 0

871.58

872.58

873.58 874.58 m/z

Fig. 8.7 The endophytic fungus Diaporthe sp. transfers organic-N to Agave. Plants were watered monthly with 15N-labeled Diaporthe sp. for a 3-month period. We tracked incorporation of 15N into pheophytin by mass-spec analysis and demonstrated that fungal cells passed nitrogen plants. We observed an increment of isotopomers at 874.58, 875.58, and 876.58 m/z, compared to nutrient solution as control. This indicates incorporation of 15N into the four nitrogen atoms of pheophytin. However, B. tequilensis was most effective, because 871.58 m/z (theoretical value of pheophytin) was not totally reduced to negative values as we showed previously (Beltran-Garcia et al. 2014). Also, we could not recover Diaporthe cells from within agave roots, suggesting that the fungus was degraded to obtain nutrients to favor plant growth in absence of nutrients

phytohormones, auxins, and in particular indole 3-acetic acid (IAA) coordinate modifications of root-system architecture, including functionality and morphology (e.g., root formation, apical dominance, and tropism). Root architecture influences

8

Agave Seed Endophytes: Ecology and Impacts on Root Architecture, Nutrient. . .

159

on the ability of the plant to acquire nutrients and water, which in turn also impacts the development of organs that grow above ground. IAA production by microbes varies greatly between different species and strains (Sukumar et al. 2013). Bacillus safensis, a seed-transmitted bacteria isolated from maize, are able to alter root morphology and architecture in host seedlings, including accumulation of N-transporters, including organic N in the membranes of roots (Prieto et al. 2017). Here, seed endophytes from agave were inoculated in corn seedlings to evaluate their functionality. We used maize plantlets, because agaves are slow-growing plants. We did not find significant difference in plant weight, leaves number, length, and roots number compared with controls (sucrose 0.05% and 50% Murashige and Skoog solution). Slight significant differences were observed in parameters as roots number and plant height caused by B. subtilis, B. altitudinis, and B. pumilus from A. angustifolia and E. cloacae of A. tequilana (Fig. 8.8). Not all bacteria are auxin producers but have in common the production of ACC deaminase and fixing nitrogen; therefore agave bacteria cannot be considered as biofertilizers for corn plants. It is well demonstrated those endophytic bacteria play an important role for plant growth promotion by production of auxins and IAA-like molecules. Figure 8.9 shows the effect on root architecture of two selected endophytes of A. tequilana (B. tequilensis and Enterobacter cloacae) and P. aeruginosa from A. marmorata; the Bacillus and Pseudomonas are IAA producers. It is well-known that IAA at low concentration causes an elongation of roots, whereas a high concentration reduces root length, increases root diameter, triggers lateral root emergence, and increases root hair density. Each endophyte is associated with a different level of alteration of the primary root defined as size, root hair number, and lateral root formation (Fig. 8.8). More specifically, E. cloacae induced longer roots. Agave roots included in B. tequilensis and E. cloacae treatments and showed root length and lateral roots comparable to the non-inoculated plantlets, respectively. Nevertheless, the emergence of lateral roots was greatest in P. aeruginosa followed by B. tequilensis (Fig. 8.9). A strong increase in the length and number of root hairs was induced by P. aeruginosa in which plantlets displayed longer and more branched roots; similar results were founded by Zamioudis et al. (2013) using Arabidopsis-Pseudomonas spp. model. The highest number of root hairs was observed in plantlets treated with B. tequilensis; however, these root hairs were shorter than those of the control. The E. cloacae treatment showed the most dramatic change, because seedlings developed shorter and smaller root hairs. Finally, the colonization of root hairs apparently was highest in plants treated with E. cloacae and was less in plants treated with P. aeruginosa; nevertheless it was difficult to distinguish clearly, mainly due to root exudates. The appearance of root hairs shown in Fig. 8.9 is similar to the hairs presented in Fig. 8.6. At the end of our analysis on changes in root architecture induced by auxin-producers endophytes, we may discern promising candidates as potential growth promoters derived from agave seeds.

160

A. Martinez-Rodriguez et al.

Fig. 8.8 Agave bacteria in maize seedlings. (a) Maize plantlets (after 2 weeks of growing) were inoculated with seed endophytes from A. tequilana (letters in blue), A. marmorata (red), and A. angustifolia (green). (b) Statistical analysis of one of each growth parameter

8

Agave Seed Endophytes: Ecology and Impacts on Root Architecture, Nutrient. . .

161

Fig. 8.9 Seed-endophytic bacteria change root architecture. A. tequilana roots were stained with DAB-aniline blue after 30 days post-inoculation to evaluate colonization. The lower four microphotograph show root hairs with internal oxidizing bacteria in bacteria-treated plants

8.8

Seed Endophytes Confer Fitness to Cold Stress on Agave Plantlets: A Metabolomic Approach

Temperature is an important environmental factor that determines plant growth and development. Plants are continuously exposed to changes in diurnal or seasonal temperatures and consequently must adjust their metabolism and physiology to improve or maintain their performance at the new growth temperature. When the temperature deviates from the range of optimal survival values, plants can experience a severe degree of physiological, cellular, metabolic, and molecular dysfunction that can lead to growth cessation and ultimately to death. Cold stress, which occurs annually with winter, is among the most intimidating forms of abiotic stress. Cold stress can be classified as either chilling stress (0–15
C or freezing stress (45a 0.22 0 2.33 0 + 80.6 0.31 2.68 0 0 0 0.02 0 0.01 0.55 0 0 24.9 0 0.91 0.41 3.42 0 0.05

0, absent; +, viridiol detected, but not quantifiable From a previous analysis

a

However, within this microbiome, there is also competition, competition among the microorganisms, between fungi and bacteria, and between fungi and fungi. In all of these interactions, secondary metabolites are involved. The examples in this chapter have only dealt with those between two organisms. In planta, there are not just bipartite, but multipartite interactions. What are the outcomes of these? Multiple equilibriums exist between the antagonisms of the plant residents, resulting in balanced associations between all members of the holobiont (Fig. 9.6a). Multiple abiotic and biotic factors can disturb this balance, including the virulence of an alien pathogen, e.g., Hymenoscyphus fraxineus in Fraxinus excelsior (Fig. 9.6b). The hope for the future survival of F. excelsior lies either in the introduction of an endophyte to F. excelsior that can sufficiently control the pathogen (Fig. 9.6c), in breeding of

Chemical Warfare in the Plant Microbiome Leads to a Balance of Antagonisms. . .

185

antifungal metabolites

Endophytic fungi

antibacterial metabolites

antifungal metabolites

Endophytic fungi

Phytohormones, phytotoxic & signaling metabolites antifungal & signaling metabolites mechanical defence

Endophytic bacteria antibacterial metabolites

_______________competition for resources________________

9

Endophytic bacteria

a Endophytic fungi

Endophytic fungi Hymenoscyphus fraxineus

Endophytic bacteria

Endophytic bacteria

b Endophytic fungi

Endophytic fungi Hymenoscyphus fraxineus

Endophytic bacteria

Endophytic bacteria

c

Fig. 9.6 (a) Microbiome and host interact metabolically, resulting in multiple equilibriums between members of the plant holobiont. Involved are not only phytotoxic, antibacterial, antifungal, hormonal, and signaling metabolites, but also mechanical defense of the host and competition for

186

B. J. Schulz et al.

relatively resistant clones or in evolution, i.e., that the host itself or members of the microbiome will evolve to control virulence of the alien pathogen. Acknowledgments We would like to thank Drs. Simone Bergmann, Sophie de Vries, and Christine Boyle for critical comments and excellent suggestions for improving the manuscript and Nicole Andrée and Patrick Bork for the permission to use photos and results from their dual culture experiments and express our gratitude to Dr. Frank Surup for drawing the structures in Figs. 9.1 and 9.2.

References Adame-Álvarez R-M, Mendiola-Soto J, Heil M (2014) Order of arrival shifts endophyte-pathogen interactions in bean from resistance induction to disease facilitation. FEMS Microbiol Lett 355:100–107 Andersson PF, Johansson SBK, Stenlid J et al (2010) Isolation, identification and necrotic activity of viridiol from Chalara fraxinea, the fungus responsible for dieback of ash. For Path 40:43–46 Araújo WL, Maccheroni W, Aquilar-Vildoso CI et al (2001) Variability and interactions between endophytic bacteria and fungi isolated from leaf tissues of citrus rootstocks. Can J Microbiol 47:229–236 Arnold AE, Maynard Z, Gilbert GS et al (2000) Are tropical fungal endophytes hyperdiverse? Ecol Lett 3:267–274 Babikova Z, Gilbert L, Bruce TJA et al (2013) Underground signals carried through common mycelial networks warn neighbouring plants of aphid attack. Ecol Lett 16(7):835–843. https:// doi.org/10.1111/ele.12115 Baral H-O, Queloz V, Hosoya T (2014) Hymenoscyphus fraxineus, the correct scientific name for the fungus causing ash dieback in Europe. IMA Fungus 5(1):79–80 Bennett JW, Hung R, Lee S et al (2012) Fungal and bacterial volatile organic compounds: an overview and their role as ecological signaling agents. In: Hock B (ed) Fungal associations, The Mycota IX, 2nd edn. Springer, Heidelberg, pp 373–393 Bloemberg GV, Camacho Carvajal MM (2006) Microbial interactions with plants: a hidden world? In: Schulz B, Boyle C, Sieber T (eds) Microbial root endopyhtes, Soil biology, vol 9. Springer, Berlin, pp 321–336 Blumenstein K, Albrectsen BR, Martín JA et al (2015) Nutritional niche overlap potentiates the use of endophytes in biocontrol of a tree disease. BioControl 60:655–667 Brian PW, McGowan JC (1945) Viridin: a highly fungistatic substance produced by Trichoderma viride. Nature 156:144–145 Brundett MC (2002) Coevolution of roots and mycorrhizas of land plants. New Phytol 154:275–304 Cao Y, Halane MK, Gassmann W et al (2017) The role of plant innate immunity in the legumerhizobium symbiosis. Annu Rev Plant Biol 68:535–561 Carroll GC (1995) Forest endophytes: pattern and process. Can J Bot 73(S1):1316–1324 Carroll GC (1999) The foraging ascomycete. In: 16th International Botanical Congress, Abstracts, 309 Chagas FO, Dias LG, Pupo MT (2013) A mixed culture of endophytic fungi increases production of antifungal polyketides. J Chem Ecol 39:1335–1342

Fig. 9.6 (continued) resources among members of the microbiome. (b) Virulence of an alien pathogen, e.g., Hymenoscyphus fraxineus in Fraxinus excelsior, can disturb these equilibriums. (c) Introduction of an endophyte to F. excelsior could sufficiently control the pathogen

9

Chemical Warfare in the Plant Microbiome Leads to a Balance of Antagonisms. . .

187

Chanclud E, Morel J-B (2016) Plant hormones: a fungal point of view. Mol Plant Pathol 17 (8):1289–1297 Citron C, Junker C, Schulz B et al (2014) A volatile lactone of Hymenoscyphus pseudoalbidus, pathogen of European ash dieback, inhibits host germination. Angew Chem Int Ed 53:4346–4349 Combès A, Ndoye I, Bance C et al (2012) Chemical communication between the endophytic fungus Paraconiothyrium variabile and the phytopathogen Fusarium oxysporum. PLoS One 7(10): e47313 Courty P-E, Walder F, Boller T et al (2011) Carbon and nitrogen metabolism in mycorrhizal networks and mycoheterotrophic plants of tropical forests: a stable isotope analysis. Plant Physiol 156:952–961 Demain A (2000) Microbial biotechnology. TIBTECH 18:26–31 Drenkhan R, Hanso M (2010) New host species for Chalara fraxinea. New Dis Rep 22:16 Drenkhan R, Adamson K, Hanso M (2015) Fraxinus sogdiana, a central Asian ash species, is susceptible to Hymenoscyphus fraxineus. Plant Protect Sci 51(3):150–152 Egamberdieva D, Wirth SJ, Alqarawi AA (2017) Phytohormones and beneficial microbes: essential components for plants to balance stress and fitness. Front Microbiol 8:2104. https://doi.org/10. 3389/fmicb.2017.02104 Frasz SL, Walker AK, Nsiama TK et al (2014) Distribution of the foliar fungal endophyte Phialocephala scopiformis and its toxin in the crown of a mature white spruce tree as revealed by chemical and qPCR analyses. Can J For Res 44:1138–1143 Gross A, Holdenrieder O, Pautasso M et al (2014) Hymenoscyphus pseudoalbidus, the causal agent of European ash dieback. Mol Plant Pathol 15(1):5–21 Gunatilaka AAL (2006) Natural products from plant-associated microorganisms: distribution, structural diversity, bioactivity, and implications of their occurrence. J Nat Prod 69:509–526 Halecker S, Surup F, Kuhnert E et al (2014) Hymenosetin, a 3-decalinoyltetramic acid antibiotic from cultures of the ash dieback pathogen, Hymenoscyphus pseudoalbidus. Phytochemistry 100:6–91 Hardoim PR, van Overbeek LS, Berg G et al (2015) The hidden world within plants: ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiol MolBiol R 79:293–320 Hassani MA, Durán P, Hacquard S (2018) Microbial interactions within the plant holobiont. Microbiome 6:58 Hiscox J, Boddy L (2017) Armed and dangerous – chemical warfare in wood decay communities. Fungal Bio Rev 31:169–184 Höller U, Wright AD, Matthée GF et al (2000) Fungi from marine sponges: diversity, biological activity and secondary metabolites. Mycol Res 104:1354–1365 Junker C, Mandey F, Pais A et al (2014) Hymenoscyphus pseudoalbidus and Hymenoscyphus albidus: viridiol concentration and virulence do not correlate. For Path 44:39–44 Klein T, Siegwolf RTW, Koerner C (2016) Belowground carbon trade among tall trees in a temperate forest. Science 352(6283):342–344 Krohn K, Schulz B (2013) Antifungal metabolites of endophytic fungi. In: Antifungal metabolites from plants. Springer, Berlin, pp 243–262 Lahrmann U, Zuccaro A (2012) Opprimo ergo sum – evasion and suppression in the root endophytic fungus Piriformospora indica. Mol Plant Microb Interact 26:727–737 Lee S, Bennett J, Behringer G et al (2018) Effects of fungal volatile organic compounds on Arabidopsis thaliana growth and gene expression. Fungal Ecol 37:1–9. https://doi.org/10.1016/ j.funeco.2018.08.004 Maillet F, Poinsot V, André O et al (2011) Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza. Nature 469:58–64 Margulis L (1991) Symbiogenesis and symbionticism. In: Margulis L, Fester R (eds) Symbiosis as a source of evolutionary innovation: speciation and morphogenesis. MIT, Cambridge MA, pp 1–14L

188

B. J. Schulz et al.

Marmann A, Aly AH, Lin W et al (2014) Co-cultivation – a powerful emerging tool for enhancing the chemical diversity of microorganisms. Mar Drugs 12:1043–1065 Matusova R, Rani K, Verstappen FWA et al (2005) The strigolactone germination stimulants of the plant-parasitic Striga and Orobanche spp. are derived from the carotenoid pathway. Plant Physiol 139:920–934 McKinney LV, Nielsen LR, Collinge DB et al (2014) The ash dieback crisis: genetic variation in resistance can prove a long-term solution. Plant Pathol 63:485–449 McMullan M, Rafiqi M, Kaihakottil G et al (2018) The ash dieback invasion of Europe was founded by two genetically divergent individuals. Nat Ecol Evol 2:1000–1008 Moran-Diez E, Rubio B, Domínguez S et al (2012) Transcriptomic response of Arabidopsis thaliana after 24 h incubation with the biocontrol fungus Trichoderma harzianum. J Plant Physiol 169:614–620 Morath SU, Hung R, Bennett JW (2012) Fungal volatile organic compounds: a review with emphasis on their biotechnological potential. Fungal Biol Rev 26:73–83 Mousa WK, Raizada MN (2013) The diversity of anti-microbial secondary metabolites produced by fungal endophytes: an interdisciplinary perspective. Front Microbiol 4:65. https://doi.org/10. 3389/fmicb.2013.00065 Ola ARB, Thomy D, Lai D et al (2013) Inducing secondary metabolite production by the endophytic fungus Fusarium tricinctum through coculture with Bacillus subtilis. J Nat Prod 76:2094–2095 Oláh B, Brière C, Bécard G et al (2005) Nod factors and a diffusible factor from arbuscular mycorrhizal fungi stimulate lateral root formation in Medicago truncatula via the DMI1/ DMI2 signalling pathway. Plant J 44:195–207 Parniske M (2008) Arbuscular mycorrhiza: the mother of plant root endosymbiosis. Nat Rev Microbiol 6:763–775 Patkar RN, Naqvi NI (2017) Fungal manipulation of hormone-regulated plant defense. PLoS Pathog 13(6):e1006334. https://doi.org/10.1371/journal.ppat.1006334 Petrini O (1991) Fungal endophytes of tree leaves. In: Andrews JH, Hirano SS (eds) Microbial ecology of leaves. Brock/Springer series in contemporary bioscience. Springer, New York, NY, pp 179–197 Rao RP, Hunter A, Kashpur O et al (2010) Aberrant synthesis of indole-3-acetic acid in Saccharomyces cerevisiae triggers transition, a virulence trait of pathogenic fungi. Genetics 185:211–220 Schäfer P, Pfiffi S, Voll LM et al (2009) Manipulation of plant innate immunity and gibberellin as factor of compatibility in the mutualistic association of barley roots with Piriformospora indica. Plant J 59:461-A Schulz B (2006) Mutualistic interactions with fungal root endophytes. In: Schulz B, Boyle C, Sieber TN (eds) Microbial root endophytes. Springer, Berlin, pp 261–279 Schulz B, Boyle C (2005) The endophytic continuum. Mycol Res 109:661–686 Schulz B, Sucker J, Aust H-J et al (1995) Biologically active secondary metabolites of endophytic Pezicula species. Mycol Res 99:1007–1015 Schulz B, Römmert A-K, Dammann U et al (1999) The endophyte-host interaction: a balanced antagonism. Mycol Res 103:1275–1283 Schulz B, Boyle C, Draeger S et al (2002) Review: Endophytic fungi: a source of novel biologically active secondary metabolites. Mycol Res 106:996–1004 Schulz B, Boyle C, Sieber TN (eds) (2006) Microbial root endophytes. Springer, Berlin Schulz B, Haas S, Junker C et al (2015) Fungal endophytes are involved in multiple balanced antagonisms. Curr Sci India 109:39–45 Simard SW, Beiler KJ, Bingham MA et al (2012) Mycorrhizal networks: Mechanisms, ecology and modeling. Fungal Biol Rev 26:39–60 Singh S, Parniske M (2012) Activation of calcium- and calmodulin-dependent protein kinase (CCaMK), the central regulator of plant root endosymbiosis. Curr Opin Plant Biol 15:444–453 Stacey G, McAlvin CB, Kim S-Y et al (2006) Effects of endogenous salicylic acid on nodulation in the model legumes Lotus japonicus and Medicago truncatula. Plant Physiol 141:1473–1481

9

Chemical Warfare in the Plant Microbiome Leads to a Balance of Antagonisms. . .

189

Stringlis IA, Zhang H, Corné MJ et al (2018) Microbial small molecules – weapons of plant subversion. Nat Prod Rep 35(4):410–433. https://doi.org/10.1039/c7np00062f Sumarah MW, Miller JD, Adams GW (2005) Measurement of a rugulosin-producing endophyte in white spruce seedlings. Mycologia 97:770–776 Sumarah MW, Puniani E, Blackwell BA et al (2008) Characterization of polyketide metabolites from foliar endophytes of Picea glauca. J Nat Prod 71(8):1393–1398 Surup F, Halecker S, Nimtz M et al (2018) Hyfraxins A and B, cytotoxic ergostane-type steroid and lanostane triterpenoid glycosides from the invasive ash dieback ascomycete Hymenoscyphus fraxineus. Steroids 135:92–97 Tan RX, Zou WX (2001) Endophytes: a rich source of functional metabolites. Nat Prod Rep 18:448–459 Terhonen E, Sipari N, Asiegbu FO (2016) Inhibition of phytopathogens by fungal root endophytes of Norway spruce. Biol Control 99:53–63. https://doi.org/10.1016/j.biocontrol.2016.04.006 Thomas DC, Vandegrift R, Ludden A et al (2016) Spatial ecology of the fungal genus Xylaria in a tropical cloud forest. Biotropica 48:381–393 Ujor VC, Adukwu EC, Okonkwo CC (2018) Fungal wars: the underlying molecular repertoires of combating mycelia. Fungal Biol 122:191–202 Verma VC, Kharwar RN, Strobel GA (2009) Chemical and functional diversity of natural products from plant associated endophytic fungi. Nat Prod Commun 4(11):1511–1532 Wang X-M, Yang B, Ren C-G et al (2014) Involvement of abscisic acid and salicylic acid in signal cascade regulating bacterial endophyte-induced volatile oil biosynthesis in plantlets of Atractylodes lancea. Physiol Plant 153(1):30–42. https://doi.org/10.1111/ppl.12236 Zhao Y-J, Hosoya T, Baral H-O et al (2012) Hymenoscyphus pseudoalbidus, the correct name for Lambertella albida reported from Japan. Mycotaxon 122:25–41 Zhou JY, Zhao XY, Dai CC (2014) Antagonistic mechanisms of endophytic Pseudomonas fluorescens against Athelia rolfsii. J Appl Microbiol 117(4):1144–1158. https://doi.org/10.1111/jam.12586

Fungal and Bacterial Maize Kernel Interactions with the Vertically Transmitted Endophytic State of Fusarium verticillioides

10

Charles W. Bacon and Dorothy M. Hinton

Contents 10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Evolution of Core Microbial Endophytes and Vertical Transmission in Seed . . . . . . . . . 10.3 Anatomy of Maize Kernel Infection by Fusarium verticillioides . . . . . . . . . . . . . . . . . . . . . . 10.4 Co-endophytic Infections of Maize by Fusaria and Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5 Quorum Sensing and Maize Seed Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

192 195 198 200 202 205 206

Abstract

The genus Fusarium consists of 100 or more valid species, although it is anticipated that additional cryptic species will be defined. We are concerned with one species F. verticillioides (Saccardo) Nirenberg, the sexual stage of which is Gibberella moniliformis Wineland. Historically, this fungus was based on the broad concept of Fusarium nomenclature, and the common synonyms F. moniliforme or Gibberella fujikuroi, mating population A, were used. In this review, both synonyms will be used depending upon the author’s earlier citations. F. verticillioides under its various synonyms is associated with well over 32 plant families and is known to be seed-borne on at least 10 of these, where it causes seedling blights, root rots, stem rots, and pre- and post-harvest kernel rots. Thus, this species and other Fusarium species are nonhost-specific or host generalists. F. verticillioides, however, is a noted parasite of maize and is found throughout the world wherever maize is grown. Of considerable importance is that this fungus infects maize as a symptomless endophyte, where it produces several homologues of the fumonisin mycotoxins, primarily fumonisin B1 and B2. The C. W. Bacon (*) · D. M. Hinton USDA, ARS, US National Poultry Research Center, Toxicology & Mycotoxin Research Unit, Russell Research Center, Athens, GA, USA e-mail: [email protected] # Springer Nature Switzerland AG 2019 S. K. Verma, J. F. White, Jr (eds.), Seed Endophytes, https://doi.org/10.1007/978-3-030-10504-4_10

191

192

C. W. Bacon and D. M. Hinton

fumonisins produce toxicity syndromes in livestock, poultry, and is implicated in several human diseases. Surveys indicate that approximately 95% of the world’s isolates are capable of producing this group of mycotoxins apparently resulting from the endophytic infection. In addition to the fumonisins, F. verticillioides produces several other metabolites of varying toxicities to plants and animals. Fusaric acid is one such toxin although it is produced by this and all other fusaria examined. The role of fusaric acid within the association now includes activity as a quorum sensing inhibitor. Quorum sensing is crucial to the proper performance and ecological fitness of most microbial populations, including fungi. Inhibiting this mechanism dooms most organisms to low competitive interspecific and intraspecific competition, while allowing the fusaria to dominate the intercellular spaces of maize. Thus, importance of quorum sensing is briefly reviewed along with the emphasis that it might play in maintaining dominant interactions among other F. verticillioides as a means of control or regulating competing organisms. Keywords

Fumonisins · Fusaric acid · Mycotoxins · Reduced virulence · Quorum sensing inhibitor

10.1

Introduction

Seeds are the very essence of a productive agricultural system as they are the reproductive organ of economically important crops, housing and expressing the genetic expressions for cultivars. Seeds also harbor many beneficial microbes that assist the host plant in maintaining environmental sufficiency. In addition to carriers of beneficial microbes, they are also carriers of pathogens, many of which can affect crop plants but also animals. The microbes are found as both surface- and endophyteborne seed dwellers, and the latter offer an immense protection of which we only now understand. Endophytic pathogenic fungi and bacteria are found in equal abundance, as are those that are beneficial. Due to the inadequacy of research, we are not aware of the extent of how many of the plus 5.1 million fungal species can be referred to as obligate seed-borne nor do we know how many of the one nonillion bacterial species are obligate seedborne. Most species of microbes are, however, facultative in their endophytic interactions with plants and seed. Of particular importance are the Fusarium species, most of which are seed-borne, either superficially or endophytically. Fungi of the genus Fusarium are a large genus consisting of 100 or more valid species (Leslie and Summerell 2006), although several other cryptic species exist, particularly those that are associated with insects. The diseases on economically important crops caused by the fusaria are numerous. We are concerned with one species, F. verticillioides (Saccardo) Nirenberg, the sexual stage of which is Gibberella moniliformis Wineland. This fungus has more common synonyms of F. moniliforme or Gibberella fujikuroi, mating population A (Leslie et al. 1992), and in this review both synonyms will be used depending upon that authors’

10

Fungal and Bacterial Maize Kernel Interactions with the Vertically. . .

193

citation. Thus, F. verticillioides under its various synonyms is associated with well over 32 plant families and is known to be seed-borne on at least 10 of these, where it is the cause of seedling blights, root rots, stem rots, and pre- and post-harvest fruit rots (Bacon and Nelson 1994). Thus, this species and probably other Fusarium species are host generalists. The fungus, however, throughout most of its association with maize is nonpathogenic and is found throughout the world wherever maize is grown as a symptomless endophyte. As an endophyte, this species is a biotrophic symbiont since it dwells within the living tissue of plants, usually throughout the life of the host. Further, strains of this species appear highly compatible within maize, remaining in the association throughout the maize life cycle. Apparently, this non-specific host association has also confounded its biogeographic origin, which is due to the origins of its non-specific hosts. Nevertheless, its origin is considered tropical but not necessarily African, which is tacitly assumed (Summerell et al. 2010; O’Donnell et al. 1998). Further, F. verticillioides’ ability to exist as a biotrophic pathogen as well as a saprophyte confounds studies designed to prevent its infection into most agronomically important plants. The universal symptomless infection of maize by F. verticillioides is of economic importance due to the production of several homologues of the fumonisin mycotoxins of which fumonisin B1 is the most active homologue. This class of mycotoxins produces toxicity syndromes in livestock, poultry, and in humans (Riley et al. 1994; Broomhead et al. 2002), and surveys indicate that approximately 95% of the world’s isolates of F. verticillioides are capable of producing these toxins (Thiel et al. 1986; Shephard et al. 1996). Plant-fungal symbioses are broadly divided into either balanced or non-balanced association. In the non-balanced association, the symbiosis moves either in the direction of a negative and often pathogenic relationship induced by the fungus in which the plant is killed. On the other hand, if the association remains balanced, the association remains symptomless and perhaps even mutualistic. Further, depending on the fungus and plant response, the relationship may evolve into either a facultative or obligatory relationship. In the balanced association, the relationships can be long-term and compatible for both partners. Lewis (1974) presents several choices for such association, but here we are concerned with the balanced and non-balanced concept. In symptomless symbiotic associations, genetic interactions are implicated. Thus, in addition to the plant’s genome, there are also the microbial genomes, which function within the rhizosphere, intercellular niche of roots and leaves. The highly interactive nature of populations within the intercellular niche requires a coordination of organisms, each expressing its genome while interacting with others. It is agreed upon that the plant selects bacteria for fitness interactions on and within the association by releasing specific compounds as attractants. Such assemblages of microorganisms are the beginning of symbionts for distribution within plants and for subsequent vertical transmission in seeds. Such associations further imply that there are mechanisms that coordinate and communicate the genetic expressions of the holobionts for successful interactions. Thus, the impact of our economically important plants depends on the seed microbiome, which is based on its origins and interactions (Nelson 2017). A recent discovery of control or communication among populations of organisms is that of quorum sensing and quorum inhibitions or quenching (Fuqua et al. 1994;

194

C. W. Bacon and D. M. Hinton

Sheiner et al. 2005). Quorum sensing generally is a mechanism through which microbial communities coordinate activities. These activities are arranged via the production of chemical signals that are either a universal small molecule or other small molecules that are produced constitutively synthesized in small amounts. When an amount is synthesized at critical limits of concentration within a certain habitat such as the intercellular space, various genes are expressed inducing order to specific biochemical events relating environmental conditions. On the other hand, other microbes and plants have the ability to destroy signaling activity, although they may have signaling mechanisms themselves. This repressing of quorum signaling is referred to as quorum quenching or inhibition. Thus, many biochemical pathways are not produced, microbial organelles are not produced, and cellular structures are not produced, which produce either latent or population decline. Quorum signaling is universal along with quorum quenching or inhibition indicating the essentiality of both mechanisms. In the past decades, several studies have indicated the importance that endophytic fungi play in the overall ecological success of vascular plant. Fungal and bacterial are co-endophytes of plants, occurring throughout the developmental period of most plant species, including the seed. Emphasizing the importance of seed-borne endophytic association is the indication that such associations are ancient (Saikkonen et al. 2004). Considerable attention has been placed on the benefits derived from endophytes during their vegetative stages, but little is known of their effects during the seed stages. However, little is known about the co-occurrence of the endophytic state of facultative symbionts such that F. verticillioides and other endophytic symbionts as bacteria and other fungi in seed ecology. Seed-borne endophytes represent the beginning and ending of the life cycle of plants, and such endophytes represent the process of passing successive plant and microbes from one generation to the next. The relations of each are based on the genetics of both, and one of several genotypes within a seed is capable of expression phenotypes depending on the many environmental constraints. In this review we are concerned with those seed-borne organisms that are not opportunistic pathogens, rather those that are symptomless in their relationship although not obligatory in the requirement of being seed-borne which is in contrast to those obligate mutualist found in some of the grasses and other plants. Notably, such fungi are very versatile since in some hosts or conditions, they are pathogenic and oftentimes saprophytic. Thus, the relationships are complex, and studies of such organisms are behind those based on the obligate mutualists associated with grasses, but which there are some similarities and we will draw on these when the occasions arise. In this review, we will describe the nature, anatomy of maize kernels along with the importance of vertical transmission of seed-borne microbes, and the possible signaling involving quenching activity produced by F. verticillioides in maize.

10

Fungal and Bacterial Maize Kernel Interactions with the Vertically. . .

10.2

195

Evolution of Core Microbial Endophytes and Vertical Transmission in Seed

Maize (Zea mays ssp. mays) originated in southern Mexico approximately 9000 years ago, which is based on DNA analysis from a single domestication, presumably from closely related teosinte species and diversified into highland and lowland races (Matsuoka et al. 2002). In efforts to follow that single line of domestication, Desjardins et al. (2000) made a study of the origin and distribution of the Fusarium species within the Gibberella fujikuroi, mating population A complex, which includes F. verticillioides relative to their association with Mexican maize and wild teosinte species. That study showed the infection rate was much lower in the teosinte species (4%) as opposed to 100% infection in the Mexican maize (Z. mays spp. mays) (Desjardins et al. 2000). Both the Mexican maize and the wild teosinte species were present as symptomless seed-borne infections. Further, the teosinte and F. verticillioides isolates were also capable of producing a similar spectrum of fumonisin mycotoxins and other secondary metabolites. Maize and teosinte species however do have completely different types of seed, which may account for the infection percentages. Maize has an exposed or necked seed imbedded on a cob, while teosinte is completely enclosed with a hard, almost impenetrable, shell. That research indicates that F. verticillioides and F. subglutinans are present as symptomless endophytes in five of the wild species and subspecies of teosintes collected from Mexico and Central America, especially Zea mays ssp. parviglumis, the very large Balsa teosinte considered by most to be the closest relative to domesticate maize. The conclusion from these studies relative to our objective is that the symptomless association of the fusaria with maize and its wild relatives is perhaps more ancient than maize domestication data indicate. Using the lowest calibration point of Taylor and Berbee (2006), both land plants and fungi such as the fusaria originated roughly the same time, well over 100 million years ago. Although maize was domesticated at least 700 years ago using data based on starch grains (Piperno et al. 2009), which is earlier than the 900 years indicated above based on years indicated by DNA analysis (Matsuoka et al. 2002). However, the age of establishment of endophytic associations of fungi with maize and other plants is completely unknown. Direct and indirect evidence from the fossil records suggest that fungal endophytes were associated with plants following the emergence of vascular land plants in the late Devonian period, about 400 million years ago (Taylor and Krings 2005; Taylor and Berbee 2006; Krings et al. 2007a, b). Similar to the discoveries of fungi as endophytic with plant is the fossil symbiosis of endophytic cyanobacteria and other organisms (Taylor and Krings 2005; Krings et al. 2005). An important component of these fossil discoveries is the absence of direct evidence for the nature of those physiological and biochemical features that we currently know are combined with what we attribute to symbiosis and mutualisms, along with their specific benefits. The vertical transmission of endophytic F. verticillioides as a compatible association was demonstrated in field plantings at two different locations in Georgia, USA. Yates et al. (1999), and Yates and Jaworski (2000) used F. verticillioides transformed with

196

C. W. Bacon and D. M. Hinton

gusA reporter gene for beta-glucuronidase to demonstrate the cyclical nature of the symptomless endophytic infection that began with surface transformed-F. verticillioides infected seed, followed to the seedling, and mature plant as an endophyte, and the seed harvested from these mature plants, tested for transformed fungi and these planted for yet another cycle and gusA-transformants recovered in the third generation from surface disinfected seed. Under ideal conditions, there was no evidence of pathogenesis, although the production of the fumonisin mycotoxins was detected. These studies indicated the vertical transmission of this species in maize and suggested the intimate associated with maize, expressed by statistically the same germination as non-infected controls. Further, a faster rate of lignification was observed in the infected seed. However, under severe stresses such as drought, transformed plants produced a larger amount of the fumonisins, due probably to the larger amount of dying tissue, i.e., there is no balance between endophyte and host. Strain differences in isolates of F. verticillioides from maize kernels are indicated, as maize strains from seed do not possess the extracellular enzymes that are attributed to pathogens including amylases, cellulases, proteases, and lipases (Abe et al. 2015). The symptomless infection by F. verticillioides may be attributed to the loss of activity of these hydrolytic enzymes demonstrated during the endophytic state. This aspect of infection by seed-borne strains of F. verticillioides indicates that this symbiosis is balanced by the fungus and maize plants, while during any stressful condition, invading dead and dying tissue, the fungus progresses from a biotroph to a weak pathogen and to saprophyte. There are numerous theories with examples for the evolution and genetics of symbiotic systems. The balance concept expressed by the theory of Lewis (1974) while somewhat modified may have some application to the symptomless infections by facultative pathogens. What is not considered in the concept of Lewis (1974) and the genetic and evolutionary discussions by Margulis (1976) is the origin and fate of the many symptomless endophytic microbes within maize kernels. That is, are all holobionts similarly affected, implying that the origin any hydrolytic enzymes may originate from these, while F. verticillioides is now an opportunist? Regardless of the plant and microbe combinations, the physiological or biochemical basis for the long- or short-term compatibility of the symbiosis is unknown. Experiments conducted by Redman et al. (2001) using the pathogen Colletotrichum and its mutants indicated that the lifestyle of this pathogen is controlled by the plant and suggested that the pathogenic lifestyle is either expressed or repressed. Furthermore, Redman et al. (2001) state that the lifestyle shown by a fungal endophyte is the results of its habit in the initial host, which is maintained in subsequent hosts. Therefore, this study indicates that a fungus may be a pathogen in one host, a mutualist in another, or a saprophyte in a dead or dying host, which is maintained as strains of the basic species. Thus, this is an added result and possibly an important benefit for vertical transmission. This appears to fit the lifestyles of F. verticillioides, although no one has made such fungus-host transfer. We are aware, however, of a few pathogenic stains of F. verticillioides to maize, and the pathogenicity is apparently stable in strains in culture. Such strains however are vertically transmitted as the seedling maize plant usually dies before it develops kernels.

10

Fungal and Bacterial Maize Kernel Interactions with the Vertically. . .

197

The seed-borne origin of most maize F. verticillioides infections suggests a biocontrol where the fungus is replaced by a biocontrol organism such as endophytic bacteria. Bacterial endophytes offer several advantages since they too are seed-borne or seed inoculated from which they infect the seedling, growing along with it. Further, they can provide similar nutritional and other non-defined benefits. The genera used include both Gram-negative and Gram-positive bacteria. Most bacterial endophytes are ancient associations in plants and can be widely distributed into the plant tissue after seed germination and growth occur (Kandel et al. 2017). JohnstonMonje and Raizada (2011) showed that a core of maize microbiota is conserved in evolution that persists across continental migration and human genetic selections. Further, closely related species are known to consist of similar endophytic species. For example, maize and rice cultivars do occupy different locations and habitats but consist of similar microbial endophytes (Liu et al. 2013; Mukhopadhyay et al. 1996). However, in the majority of studies, the endophytes were obligate endophytes and most if not all were not identified to species, rather they were identified by 16S rRNA gene sequencing, so no definite conclusions can be reached on the essentiality of specific species as they are transferred from generation to generation. The similarity of an obligate endophyte with a fungus such as F. verticillioides is unknown, and we know of no known benefits to the association derived from and during vertical transmission, only that it occurs. Those benefits derived from other vertically transmitted organism might be implied as described by benefits indicated above. The question of benefits needs to be examined in more detail. In the absence of data, the alternative conclusion is that F. verticillioides is getting a free ride from and to very highly nutritional and protective host generations such as maize. The seed offers a very secure habitat for endophytic microbes, especially the endophytes that produce dormant spores in the seed endosphere or even the spermosphere. However, at these locations, the transmission of an obligate endophyte is dependent on completion of the basic life of the host. That is, most endophytes die if their hosts are not planted within a year or two without cold storage; their hosts do remain viable. The natural cycle usually involves development of mature seed, requiring or not requiring a brief cold dormancy period, followed by germination usually the following fall or spring. However, there are seeds where dormancy can be extended usually under frigid conditions, and this may extend the life expectancy of both microbe and seed embryo indefinitely. Some microbes may be capable of true cryptobiotic states, and their life can be extended if the seed is similarly maintained. In maize kernels, the life expectancy is expected to be somewhat seasonal although this can be extended by various refrigerated temperatures. The resistant fungal or bacterial stage within the seed is usually dormant spores although some non-sporulating and inactive bacteria are known to reside in the seed for an extended period of years. Among endophytic microbes that are associated with maize kernels are those that offer some protection against infections by other pathogenic and nonpathogenic organisms. These organisms, usually bacteria, offer some biological control potential. The usual concepts based on their operation are one of simple productions of antibiotics that affect pathogens. The objective of this review is to survey the

198

C. W. Bacon and D. M. Hinton

literature on the biological control of seedling diseases. We are interested in detailing aspects of possible interactions occurring during kernel imbibition and infection processes by the biocontrol agent and native seed-borne microbes that allow for the endophytic symptomless expression of that agent and alternatively those processes leading to the disease process presumably preventing the activity of the biocontrol agent.

10.3

Anatomy of Maize Kernel Infection by Fusarium verticillioides

Bacon et al. (1992) provided the first visual evidence of endophytic infections in asymptomatic kernels of field maize using scanning electron microscopy. They further demonstrated that if such kernels are allowed to imbibe for a short period and these kernels are sections longitudinally and microscopically examined, the identity of the fungus was easily determined as F. verticillioides (Fig. 10.1a). This approach reveals the characteristic production from monophialides of long chains of nonseptate oval-shaped microconidia typical of this fungus on half of the kernel, which is typical of conidiation when allowed to grow on laboratory media (Leslie and Summerell 2006). The maize kernels used for this work were known to produce leukoencephalomalacia when fed to horses, and other toxicities when fed to laboratory animals indicated that although symptomless and capable of germinating (Fig. 10.1b), they were contaminated with the fumonisin toxins (Voss et al. 1989). Thus, such kernels indicated that at some point in time, the endophytic state was biochemically active. Further, while the infection is symptomless relative to disease expression, the infection did result in increased gross morphological and histological development, as well as early lignin deposition in maize seedling germinated from F. verticillioides-infected kernels (Yates and Jaworski 2000). Planting of symptomless kernels and examination of seedlings showed that although symptomless infections persisted beyond the seedling stage, mycotoxin contamination occurred in the kernel from such plants some 90 days post-germination (Bacon and Hinton 1996). There are, however, natural variations from the symptomless expressions. One study demonstrated that a transformed and non-transformed virulent strain of F. verticillioides was capable of producing symptoms of infection in both vegetative and reproductive tissues. This strain was isolated from field-grown maize seedling showing Fusarium stalk rot. The authors concluded that tissue type, maturity, and physical conditions of maize plants are factors responsible for differences in mycelial proliferations and the subsequent disease expression (Yates and Jaworski 2000). Obviously, this strain also has the potential for production of pathotoxins and other enzymeinciting disease components. The occurrences of such strains are either rare or maize cultivar dependent. Development of the symptomless endophytic infection by F. verticillioides as described here is the result of vertical transmission, although we have no evidences of any similarity to symptomless kernel infections resulting from horizontal infections that are also common in maize by this and other Fusarium species. Maize insects and pests

10

Fungal and Bacterial Maize Kernel Interactions with the Vertically. . .

199

Fig. 10.1 Seed-borne infection of Fusarium verticillioides in a sound maize kernel. (a) A surfacesterilized sound maize kernel produced on a symptomless infected plant, germinating on water agar showing a maize seedling germinating, and concurrently a colony of F. verticillioides growing out of that kernel onto water agar. (b) Scanning micrograph of Fusarium verticillioides developing from a symptomless kernel longitudinally sectioned that was covered overnight in a Petri dish to induce fungal growth to show the long chains of microconidia (arrow) characteristic of the species (Bacon et al. 1992)

serve as vectors for transferring inoculants onto maize, invading wounds made by pests. Another avenue of infection is through either age-induced or stressed-induced tissue senescence, and in this case F. verticillioides has been demonstrated to produce an abundance of the fumonisin mycotoxin. A major concern is at initiation of reproduction resulting in kernel development since kernel infection is the initiation of seedling infection in maize by this species oftentimes leading to ear and stalk rot. However, F. verticillioides is well known for a symptomless infection of maize, but when infection occurs via wounded or senescence tissue, it might not necessarily produce a disease, but always it produces the fumonisins and other secondary metabolites some of which are toxic to humans and livestock.

200

10.4

C. W. Bacon and D. M. Hinton

Co-endophytic Infections of Maize by Fusaria and Bacteria

The question is now asked: Are all similarly vertical disseminated microbes from the same plant and kernel symptomless? Since most studies are based on cultural organisms, what about those that are not culturable? Are seed endophytes all active or quiescent and only in time or space? As a symptomless endophyte in maize, F. verticillioides coexists with a variety of other endophytic microbes, each within its own population although all populations of a species are assumed to affect its host. Evidence for plant selection for desirable endophytic species is unavailable. Several studies have indicated that there is a broad spectrum of bacterial endophytes associated with maize and other seed, in which when the fusaria are indicated, the endophytic ecology of such associations is complex. Along with these associations, we must also factor in the interactions of the maize plant while serving as the host of such a complex association of species. Such multiple associations have only been recently recognized, the extent is probably underestimated, and the factors governing their interaction are not understood. Data indicate that such complex endophytic species are probably derived from the early relatives of maize. Thus, maize has served as hosts for endophytic microbes throughout its evolution. These endophytic associations apparently coevolved millions of years ago. Quorum mechanisms are envisioned as confined to the small-structured and compartmentalized cavity of a seed. Competition among the seed-borne endophytic microbes, particularly those with quorum inhibitors such as F. verticillioides, is able to successfully colonize while possibly suppressing the populations of competing microbes. It is proposed that maize seed has a core of bacteria within the space and that is conserved across maize evolution, domestication, and migration from one of the wild grasses of maize progenitors such as teosinte. The study of Matsuoka et al. (2002) indicates that there was only one domestication in maize from which all maize types arose, which is estimated to have started about 900 years ago in southern Mexico. The study was not designed to determine if seed endophytic species were conserved during the domestication of maize by humans from its wild relatives to modern lines of maize. However, Johnston-Monje and Raizada (2011) indicate that during evolution, a core microbiota was indeed conserved but with only a fraction of primal seed endophytes persisting during human migration and presently associated with diversifications due to modern breeding and farming practices. Bacterial endophytes of maize vary from the location, cultivar, and stage of germination ranging from the proembryo, milk, and the late dough stage of kernel development. In all instances, there was a succession of populations depending on dormancy and germination (Mundt and Hinkle 1976; Rijavec et al. 2007; Johnston-Monje and Raizada 2011; Liu et al. 2013). Thus, endophytic bacterial strains isolated from maize kernels and identified by 16S rRNA gene sequencing ranged from species belonging to classes in the phyla γ- and α-Proteobacteria, Firmicutes, Bacteroides, and Actinobacteria. By far the most common genera are from the Proteobacteria represented by the genera Enterobacter, Pseudomonas, and Pantoea. Bacterial endophytes of the phyla Firmicutes are dominated by species of genus Bacillus. Most of kernel endophytes are indicated as

10

Fungal and Bacterial Maize Kernel Interactions with the Vertically. . .

201

having biocontrol potential; however only a few have exhibited strong biocontrol of target plant pathogens under field conditions. Most of the biocontrol traits identified are attributable to fungicidal compounds produced by the bacterium, such as that by Bacillus mojavensis (Snook et al. 2009; Blacutt et al. 2016; Ongena et al. 2007) although several of Pseudomonas strains used as biocontrol agents are also plant pathogens. Recent interpretation of the fungicidal compounds produced by the bacteria, particularly the lipopeptides, is assigned additional roles as quorum inhibitors (Lopez et al. 2009; Verbeke et al. 2017). Possibly these as well as other endophytic genera can disrupt other compatible and positive plant microbial endophytes, thus affecting plant health (Andreote et al. 2009). We interpret this as a possible indication of a quorum inhibiting mechanism, which will be discussed in sections below. Mousa et al. (2015) made a study comparing the bacterial endophytes from wild teosintes and modern maize genotypes for their ability to antagonize F. graminearum as well as for their ability to suppress the production of the mycotoxin deoxynivalenol by this fungus. The results indicated that contrary to the modern genotypes of maize, the wide teosintes contained bacterial endophytes that reduced the production of this mycotoxin as well as suppressing a broad spectrum of F. graminearum and other fungal pathogens (Mousa et al. 2015). The results indicated that within all the wild types examined, only three antifungal species, two strains of Paenibacillus polymyxa, and a species of Citrobacter were responsible for the observed inhibition. Their work indicated the importance of endophytes in the evolution of crop domestication systems during human selection and breeding relative to plant diseases. In addition, this study points the way for perhaps a better format for selection of biocontrol species. The seed endophytes also consist of fungi, perhaps virus, and not all are beneficial but most are fungi isolated from the seed belonging to Ascomycota (Fisher et al. 1992; Shahzad et al. 2018). Most of these are considered ecologically detrimental without being tested (Nguyen et al. 2016). However, some fungi are consistent seed endophytes in certain plant groups or families of plants that have been established as being highly beneficial. Outstanding in this respect are the clavicipitalean species of endophytes such as the Balansia and Epichloe species that are associated only with grasses and their allies. In an attempt to categorize endophytes, Rodriguez et al. (2009) established four classes, and for each class various characteristics were identified. In terms of this review, class one and two might have some relevance for our discussion since they are transmitted vertically and/or horizontally. The clavicipitalean species indicated above are included in class one due primarily to their ecological role in the association and their narrow host range. The Fusarium species are included in class two due to their wide host range and extensive colonization of tissue throughout the plant axis and are transmitted both vertically and horizontally, and the endophytic fusaria have a poorly defined and unknown ecological role to the plant. A direct ecological role for endophytic infection to the seed has not been established for any class. Compartmentalized within the seed is the manner by which reproduction and disseminations of the infections are maintained. During the process of germination, all ecological roles are established early in the developing seedling for both facultative and obligate seed-borne microbes as seed-

202

C. W. Bacon and D. M. Hinton

borne organisms are distributed throughout the plant (Rijavec et al. 2007; Hardoim et al. 2012; Yang et al. 2013).

10.5

Quorum Sensing and Maize Seed Endophytes

Initially, quorum sensing was discovered in Gram-negative bacteria and later in all Gram-positive bacteria. The mechanism apparently is universally present in all bacteria. In addition to bacteria, quorum signaling is also found in several yeast species, a few fungi, and other organisms (Rasmussen et al. 2005b; Helman and Chernin 2014; Rodrigues et al. 2015). Quorum sensing is the result of dynamic activities of populations and its growth, requiring a variety of biochemical processes, and none of which can occur in varying degrees of the cryptobiotic states of a seed. Considering the high diversity of endophytic microbes in maize seed, especially during germination (Yang et al. 2013), quorum mechanisms should be operational shortly after germination if indeed there is need for coordinating the various bacterial populations (Hyun-Soo et al. 2007; Schikora et al. 2016). However, little is known of such activities except for the embryonic development and its activity. Quorum activities might not include all seed-borne organisms, but a perusal of those reported as seed-borne approximately 20% is known to utilize quorum mechanism, while a high percentage of the unknown species is related to those scored as positive. Considering the essentiality of the bacteria and fungi that enter mutualistic relationships with plants, it is expected that they are targeted for quorum activity, including quorum inhibition. Quorum metabolites are produced in response to population size; it is therefore likely that quorum metabolites will be produced during seed development stage, perhaps as late as the emergence of the embryonic maize root. However, during the germination, there are population busts within several seed-borne microbes, including and the inclusion of soil-borne organisms that enter the seed via cracks produced by the germinating seedling. Competition at this time should be high as there are those metabolites that are rather quorum quenchers or inhibitors becoming of consequence. Quorum inhibitors prevent the ordered behavior of populations of microbes, resulting in the disruption of several basic developmental activities of specific microbes. An important aspect of a quorum inhibitor is that it does not inhibit the growth of the producer nor the competing organisms. Most assays of quorum inhibition may not take this into account resulting in a dubious assignment to specific metabolites that is affecting growth and not specifically one of the quorum mechanisms indicated below (Defoirdt et al. 2013; Subramoni et al. 2014). The characteristic of all quorum sensing mechanisms involves three stages. First, there is signal production, which is followed by signal accumulation and finally signal detection. A quorum inhibitor may silence a signaling mechanism through four mechanisms. First, an inhibitor can simply inhibit via the production of inhibitory compounds. Secondly, there may be a degradation of a quorum signal by enzymes. Third, there may be an inhibition of the biosynthesis of the quorum signal, and fourth, there may be the production of an interfering signal detection system. All bacterial quorum inhibitors act on one of

10

Fungal and Bacterial Maize Kernel Interactions with the Vertically. . .

203

these three systems (LaSarre and Federle 2013). Fungi do not have an acknowledged immune system, but they appear to rely on various chemical defense mechanisms such as the production of inhibitory compounds. As fungi are highly adept at the synthesis of many novel compounds, the potential list of compounds that may prove inhibitory to quorum sensing systems of bacteria might prove extensive. Although not established within the seed, the quorum inhibitor used by endophytes such as F. verticillioides might involve its production of inhibitory compounds, and this might include any produced by the maize plant. One compound produced by Fusarium verticillioides and all other Fusarium species is fusaric acid (Bacon et al. 1996), which was recently shown to be a quorum inhibitor along with several of its analogs (Tung et al. 2017). Fusaric acid was earlier considered a phytotoxin and is rated as a weak mycotoxin although other pharmacological activities have been assigned to it since its initial isolation. Other weak mycotoxins produced by fungi include patulin that is produced by species of Aspergillus and penicillic acid produced by species of Penicillium, Aspergillus, and Byssochlamys (Rasmussen et al. 2005a, b). The almost universal production of fusaric acid suggests that it may be considered universal quorum sensor inhibitors for the fusaria since it is inhibitory to both Gramnegative and Gram-positive bacterial systems. Its universal occurrence can be used as the answer to the equivalents of universal quorum sensors produced by Gram-negative and Gram-positive bacteria. However, the mechanisms involved in inhibition of each have not been established, although it appears to relate simply to the inhibition by these substances. Identification and demonstrating a suspect compound that has quorum inhibitory activity are conveniently done with the use of a series of biosensor bacteria. A major biosensor bioassay includes the inhibition of pigment violacein accumulation by the biosensor bacterium Chromobacterium violaceum. This bacterium is commonly used for carbon compounds within a relatively small molecular weight and, by following their use with well-established chemical protocol (McLean et al. 2004, and references cited therein; Tan et al. 2013), should make the case for inhibition. Using such assays, quorum inhibitory activity has been detected in cell-free extracts of F. graminearum, Fusarium sp., Lasiodiplodia sp., Trichoderma sp., Aspergillus sp., Alternaria sp., and Aspergillus sp. (Rasmussen et al. 2005a, b; Martin-Rodriguez et al. 2014; Mookherjee et al. 2017). Similarly, several plants have produced quorum inhibitors (Helman and Chernin 2014, Mookherjee et al. 2017). As correctly indicated within the seed, there is very little evidence for the existence of quorum mechanisms, although after the inhibition process the developing seed along with increases in activity with the cadre of seed-borne microbes, the seed internal structures provide ample habitats including the rhizosphere, phyllosphere, and endosphere for quorum mechanisms. There is ample evidence that the universal quorum sensors of Gram-negative bacteria are also effective with eukaryotic plants (Hartman and Schikora 2012). The inside cavity of a maize kernel is highly structured and small, allowing the diverse bacterial and fungal populations to rapidly reach their quorum levels, while allowing the fungus to produce its quorum inhibitor such as fusaric acid. Further, not all seed-borne microbes are active quorum producers. Faure et al. (2009) estimated that only 10–20% of the endophytic

204

C. W. Bacon and D. M. Hinton

and rhizospheric bacteria are quorum producers, and of these most are capable of communicating with the seedling plant. Such diverse inter- and intraspecies communication provides the population of microbes within the developing seedling the need for a biocontrol to be effective for the several pathogenic organisms in the developing seedling. As a result, varied phenotypes may be observed within seedlings during their initial germination. The environment is another factor in the equation allowing for the successful growth of the developing plant. We now present evidence that the maize seed and seedlings are highly effective in producing a class of quorum inhibitors. During the early stages of seedling development, there is a release of preformed metabolites that are mainly nutritional, while others are inhibitory. In maize the inhibitor or protective metabolites include the derivatives of the benzoxazinoids: 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA), 2-benzoxazolinone (BOA), 2,4-dihydroxy-7- methoxy-1,4-benzoxazin-3-one (DIMBOA), MDIBOA (2-hydroxy-4,7-methoxybenzoxazin-3-one), and 6-cloro-2-benzoxazolinone (CDHB) (Zhang et al. 2000; Glenn et al. 2001; Guo et al. 2016). These are cyclic hydroxamic acids found also in other members of the family Poaceae (Niemeyer 1988, 2009). Since they are preformed and exist at physiological significant concentrations, they are regarded as phytoanticipins and are highly active although short-lived during their reactive phase of early seedling development. The use of the decomposition product of DIMBOA, the major compound in germinating maize and seedling, is MBOA, a rather inactive product that, higher than physiological concentrations, is toxic to F. verticillioides, other fungi, and maize seedling. However, endophytic and seedborne bacteria such as B. mojavensis can transform MBOA further to the stable and highly toxic product to F. verticillioides; 2-amino-3H-phenoxazin-3-one (APO) in culture studies indicates that F. verticillioides and other species can rapidly degrade the cyclic hydroxamic acids (Yue et al. 1998; Bacon et al. 2007). Thus, these two provide a negative example of a quorum antagonism. At higher than physiological concentrations, the benzoxazinoids and their biotransformation products are not only toxic to most microbes and insects (Niemeyer 1988; Bacon et al. 2007) but to maize plants. Therefore, bioassays should consider their physiological concentrations for a target species. Further, these compounds are also allelopathic (Yue et al. 1998), affecting the germination of non-Poaceae plants. Regardless of this cascade of decomposition products, the benzoxazinoids, especially DIMBOA and MDIBOA, at their physiological concentrations are quorum inhibitors (Subramoni et al. 2014). Since this class is preformed, seed metabolism is not necessary although slight seed injury as done during germination or predation is required for their release of such metabolites from their inactive glucoconjugated form (Cambrier et al. 1999; Park et al. 2004). In addition to this class, other metabolites, the seedling, and kernel are also indicated as having quorum inhibitory functions including IAA and its degradation products, as well as γ-amino butyric acid and salicylic acid (Zuniga et al. 2013). Although these actually activate the quorum quenching mechanisms resulting in a reduction in the quorum signals, they nevertheless are inhibiting quorum activity, representing one of the mechanisms indicated earlier (Defoirdt et al. 2013; Subramoni et al. 2014). It is important to note that the cyclic hydroxamic acids are only present

10

Fungal and Bacterial Maize Kernel Interactions with the Vertically. . .

205

during the seed and early seedling stage, 9–11 and less 9 (Cambrier et al. 2000), representing a seed-borne regulatory mechanism interactive with quorum mechanisms.

10.6

Summary

Maize has a long history with humankind, as it was influential in the domestication of livestock, poultry, as well as a major contributor to the establishment of civilizations. Fungal endophytes exhibit a wide spectrum of plant associations. Maize and other plants are infected with a titer of microbial endophytes. In general, data indicates that these endophytes are consistent, and instances exist that seeds of modern maize cultivars contain similar titers as those of the wild relatives. The microbiome is still transferred vertically in modern cultivars and wild relatives although the seed of each is different. The fossil records indicate that fungi became associated with plants millions of years ago resulting currently in almost a uniform association, and a high percentage of the associations is endophytic infection. We reviewed two vertically transmitted endophytic groups, those that are mutualist and those that are facultative asymptomatic infections. F. verticillioides belongs to the latter group, although it is also a very common non-endophytic pathogen of several hosts. In maize, it has a cyclical biotrophic phase that can last season long as the vertically transmitted symptomless infection remains. Evidence is reviewed indicating the anatomical association of F. verticillioides within kernels of maize, along with its ecological and evolutionary history, which followed the evolution of maize domestication by humans. Emphasis is placed on the nature of the endophytic biome as well as the interaction and control process using quorum mechanism along with controls by competing organisms and the seedling maize using quorum inhibitors. Maize has a class of natural quorum sensing inhibitors that are only found in germinating seed and young seedling that are capable of inhibiting quorum sensors of both Gram-negative and Gram-positive bacteria, although one of its decomposition products is also fungicidal but not at physiological and natural concentrations. The information available to date indicates that quorum inhibitor might be interactive with the seed-borne microbiome and that there are complex interactions between the endophytic holobionts of maize seed, many of which have yet to be analyzed sufficiently in order for a valid conclusion to be made. The review nevertheless should stimulate future studies on this complex interaction, involving the vertical transmission of the seed containing multiple organisms. Organisms, which we now know consist of apparent helpful and genetically diverse groups of organisms that are vertically transmitted with maize seed with similar species, passed from generation to generation. Thus, we presently defined maize seed as an organ that, in addition to its genetics, consists of several genomes derived from a diversity of organisms, containing among others a symptomless infection, F. verticillioides. The entire genetics of maize and the endophytic biome constitute an organism of great economic importance. The picture emerging is that within the intercellular spaces, is the need for systematic completion. Regulating this symbiotrophic organism are simpler metabolites. Maize thus consists of a microbial supernumerary

206

C. W. Bacon and D. M. Hinton

genome similar to the B-chromosome characteristic of the host maize. However, many aspects of the maize seed endophytic biome, their genomes, including the many inter- and intra-kingdom assemblages, and their interactions currently remain a priori.

References Abe CAL, Faria CB, Castro FF, de Souza SR, Santos FC, da Silva CN, Tessmann DJ, BarbosaTessmann IP (2015) Fungi isolated from maize (Zea mays L.) grains and production of associated enzyme activities. Int J Mol Sci 16:15328–15346 Andreote FD, de Araujo WI, de Azevedo JL, van Elas JD, da Rocha UN et al (2009) Endophytic colonization of potato (Solanum tuberosum L) by a novel competent bacterial endophyte, Pseudomonas putida strain P9, and its effect on associated bacterial communities. Appl Environ Microbiol 75:3396–3406 Bacon CW, Hinton DM (1996) Symptomless endophytic colonization of maize by Fusarium moniliforme. Can J Bot 74:1195–1202 Bacon CW, Nelson PE (1994) Fumonisin production in maize by toxigenic strains of Fusarium moniliforme and Fusarium proliferatum. J Food Protect 57:514–521 Bacon CW, Bennett RM, Hinton DM, Voss KA (1992) Scanning electron microscopy of Fusarium moniliforme within asymptomatic maize kernels and kernels associated with equine leukoencephalomalacia. Plant Dis 76:144–148 Bacon CW, Porter JK, Norred WP, Leslie JF (1996) Production of fusaric acid by Fusarium species. Appl Environ Microbiol 62:4039–4043 Bacon CW, Hinton DM, Glenn AE, Macias FA, Marin D (2007) Interaction of Bacillus mojavensis and Fusarium verticillioides with a benzoxazolinone (BOA) and its transformation product, APO. J Chem Ecol 33:1885–1897 Blacutt AA, Mitchell TR, Bacon CW, Gold SE (2016) Bacillus mojavensis RRC101 lipopeptides provoke physiological and metabolic changes during antagonism against Fusarium verticillioides. Mol Plant Micro Inter 29(9):713–723 Broomhead JN, Ledoux DR, Bermudez AJ, Rottinghaus GE (2002) Chronic effects of fumonisin B1 in broilers and turkeys fed dietary treatments to market age poultry. Science 81:56–611 Cambrier V, Hance T, de-Hoffmann E (1999) Non-injured maize contains several 1,4-benzoxazin3-one related compounds but only as glucoconjugates. Phytochem Anal 10:119–126 Cambrier V, Hance T, de-Hoffmann E (2000) Variation of DIMBOA and related compounds content in relation to the age and plant origin in maize. Phytochemistry 53:223–229 Defoirdt T, Brackman G, Coenye T (2013) Quorum sensing inhibitors: how strong is the evidence? Trends Microbiol 21:619–624 Desjardins AE, Plattner RD, Gordon TR (2000) Gibberella fujikuroi mating population A and Fusarium subglutinans from teosinte species and maize corn from Mexico and Central America. Mycol Res 104:865–872 Faure D, Vereecke D, Leveau JYH (2009) Molecular communication in the rhizosphere. Plant Soil 321:279–303 Fisher PJ, Petrini O, Scott HML (1992) The distribution of some fungal and bacterial endophytes in maize (Zea mays L). New Phytol 122:299–305 Fuqua WC, Winans SC, Greenburg EP (1994) Quorum sensing in bacteria: the LuxR-LuxI family of cell density-dependent transcriptional regulators. J Bacteriol 176:269–275 Glenn AE, Hinton DM, Yates IE, Bacon CW (2001) Detoxification of corn antimicrobial compounds as the basis for isolating Fusarium verticillioides and some other Fusarium species from corn. Appl Environ Microbiol 67:2973–2981

10

Fungal and Bacterial Maize Kernel Interactions with the Vertically. . .

207

Guo B, Zhang Y, li S, Lai T, Chen J, Ding W (2016) Extract from maize (Zea mays L.): antibacterial activity of DIMBOA and its derivatives against Ralstonia solanacearum. Molecules 21:1396–1419 Hardoim PR, Hardoim CP, van Overbeek LS, van Elsas JD (2012) Dynamics of seed-borne rice endophytes on early plant growth stages. PLoS One 7(2):e30438. https://doi.org/10.1371/journalpone.0030438 Hartman A, Schikora A (2012) Quorum sensing of bacteria and tans-kingdom interaction of N-acyl homoserine lactone with eukaryotes. J Chem Ecol 38:740–713 Helman Y, Chernin L (2014) Silencing the mod: disrupting quorum sensing as a means to fight plant disease. Mol Plant Pathol 16(3):316–329. https://doi.org/10.1111/mpp.12180 Hyun-Soo C, Park S-Y, Ryu C-M, Kim JF, Kim J-G, Park S-H (2007) Interference of quorum sensing and virulence of the rice pathogen Bukholderia glumae by an engineered endophytic bacterium. FEMS Microbiol Ecol 60:14–23 Johnston-Monje D, Raizada MN (2011) Conservation and diversity of seed associated endophytes in Zea across boundaries of evolution, ethnography, and ecology. PLoS One 6:1–22 Kandel SL, Joubert PM, Doty SL (2017) Bacterial endophyte colonization, and distribution within plant. Microorganisms 5:77–103 Krings M, Hass H, Kerp H, Taylor TN, Agerer R, Dotzler N (2005) Endophytic cyanobacteria in a 400-million -yr.-old plant: a scenario for the origin of a symbiosis? Rev Palaeobot Palyno 153:62–69 Krings M, Taylor TN, Hass H, Kerp H, Dotzler N, Hermen EJ (2007a) Fungal endophytes in a 400-million-yr-old land plant: infection pathways, spatial distribution and host responses. New Phytol 174:648–657 Krings M, Taylor TN, Hass H, Kerp H, Dotzler N, Hermsen EJ (2007b) An alternative mode of early land plant colonization by putative endomycorrhizal fungi. Plant Sig Behavior 2:125–126 LaSarre B, Federle MJ (2013) Exploiting quorum sensing to confuse bacterial pathogens. Microbiol Mol Biol Evol 77:73–111 Leslie JF, Summerell BA (2006) The Fusarium laboratory manual. Blackwell, Iowa Leslie JF, Plattner RD, Desjardins AE, Llitich CJR (1992) Fumonisin B1 production and vegetative compatibility of strains from Gibberella fujikuroi mating population “A” (Fusarium moniliforme). Mycopathologia 117:37–45 Lewis DH (1974) Micro-organisms and plants: the evolution of parasitism and mutualism. Symp Soc Gen Microbiol 24:374–392 Liu Y, Zuo S, Zou Y, Wand J, Song W (2013) Study on diversity of endophytic bacterial communities in seed of hybrid maize and their parental lines. Arch Microbiol 194:1001–1012 Lopez D, Vlamakis H, Losick R, Kolter R (2009) Paracrine signaling in a bacterium. Genes Dev 23:1631–1638 Margulis L (1976) Genetic and evolutionary consequences of symbiosis. Exp Parasitol 39:277–349 Martin-Rodriguez AJ, Reyer F, Martin J, Perez-Yepez J, Leon-Barrios M, Couttolene A, Espinoza C, Trigos A, Martin VS, Norte M, Pernandez JJ (2014) Inhibition of bacterial quorum sensing by extracts from aquatic fungi: first reports from marine endophytes. Mar Drugs 12:5503–5526 Matsuoka Y, Vigouroux Y, Dermastia M, Goodman MM, Sanchez GJ, Buckler E, Doebley J (2002) A single domestication for maize shown by multilocus microsatellite genotyping. Proc Natl Acad Sci USA 99:6080–6084 McLean RJC, Pierson LS, Fuqua C (2004) A simple screening protocol for the identification of quorum signal antagonists. J Microbiol Methods 58:351–360 Mookherjee A, Singh S, Maiti MK (2017) Quorum sensing inhibitors: can endophytes be prospective sources? Arch Microbiol 200(2):355–369. https://doi.org/10.1007/s00203-017-1437-3 Mousa WK, Shearer CR, Limay-Rios V, Zhou T, Raizada MN (2015) Bacterial endophytes from maize suppress Fusarium graminearum in modern maize and inhabit mycotoxin accumulation. Front Plant Sci 6:805–824 Mukhopadhyay K, Gassison NK, Hinton DM, Bacon CW, Khush GS, Peck HD Data N (1996) Identification and characterization of bacterial endophytes of rice. Mycopathologia 134:151–159 Mundt JO, Hinkle NF (1976) Bacteria within ovules and seeds. Appl Environ Microbiol 32:694–698

208

C. W. Bacon and D. M. Hinton

Nelson EK (2017) The seed microbiome: origins, interactions, and impacts. Plant Soil 422:7–34 Nguyen NH, Song Z, Bates ST, Branco S, Tedersoo L, Menke J et al (2016) FUNGuild: an open annotation tool for parsing fungal community datasets by ecological guild. Fungal Ecol 20:241–248 Niemeyer HM (1988) Hydroxamic acids (4-hydroxy-1,4-benzoxazin-3-ones), defense chemical in the Gramineae. Phytochemistry 27:3349–3358 Niemeyer HM (2009) Hydroxamic acids derived from 2-hydrox-2H-1,4-benzoxazin-3(4H)-one: key defense chemicals of cereals. J Agric Chem 57:1677–1696 O’Donnell K, Cigelnik E, Nirenberg HI (1998) Molecular systematic and phylogeography of the Gibberella fujikuroi species complex. Mycologia 90:465–493 Ongena M, Jourdan E, Adam A, Paquot M, Brana A, Joris B, Arpignyy J-L, Thonart P (2007) Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants. Environ Microbiol 9:1084–1090 Park WJ, Hochholdinger F, Giel A (2004) Release of the benzoxazinoids defense molecules during lateral and crown rot emergency in Zea mays. J Plant Physiol 161:981–985 Piperno DR, Ranere AJ, Holst I, Iriarte J, Dickau R (2009) Starch grain and phytolith evidence for early ninth millennium B.P. maize from the central Balsas River Valley, Mexico. Proc Natl Acad Sci USA 106:5019–5024 Rasmussen TB, Bjarnsholt T, Skindersoe ME, Hentzer M, Kristoffersen P, Kote M, Nielsen J, Eberl L, Givskov M (2005a) Screening for quorum-sensing inhibitors (QSI) by use of a novel genetic system, the QSI selector. J Bacteriol 187:1799–1814 Rasmussen TB, Skindersoe ME, Bjarnsholt T, Phipps RK, Christensen KB, Jensen PO, Andersen JB, Koch B, Larsen TO, Hentzer M, Eberl L, Hoib N, Givskov M (2005b) Identity and effects of quorum-sensing inhibitors produced by Penicillium species. Microbiology 151:1325–1340 Redman RS, Dunigan DD, Rodriguez RJ (2001) Fungal symbiosis from mutualism to parasitism: who controls the outcome, host or invader? New Phytol 151:705–716 Rijavec T, Lapanje A, Dermastia M, Rupnik M (2007) Isolation of bacterial endophytes from germinated maize kernels. Can J Microbiol 53:802–808 Riley RT, Voss KA, Yoo H-S, Gelderblom WCA, Merrill AH (1994) Mechanisms of fumonisins toxicity and carcinogenesis. J Food Protec 57:638–645 Rodrigues AC, de Oliveria BD, de Silva ER, Sacramento NTB, Bertoldi MC, Pinto UM (2015) Food Sci Technol Campinas 36:337–343 Rodriguez RJ, White Jr JF, Arnold AE, Redman RS (2009) Fungal endophytes: diversity and functional roles. New Phytol 182:314–330 Saikkonen K, Wali P, Helander M, Faeth SH (2004) Evolution of endophyte-plant symbioses. Trends Plant Sci 9:275–280 Schikora A, Schenk ST, Hartmann A (2016) Beneficial effects of bacteria-plant communication based on quorum sensing molecules of the N-acyl homoserine lactone group. Plant Mol Biol 90:605–612 Shahzad R, Khan AL, Bilal S, Lee I-J (2018) What is there in seeds? Vertically transmitted endophytic resources for sustainable improvement in plant. Growth Front Plant Sci 9:24. https://doi.org/10. 3389/fpls.2018.00024 Sheiner EK, Rumbaugh KP, Williams SC (2005) Inter-kingdom signaling: deciphering the language of acyl homoserine lactones. FEMS Microbiol Rev 29:935–947 Shephard GS, Thiel PG, Stockenström S, Sydenham EW (1996) Worldwide survey of fumonisin contamination of corn and corn-based products. J Assoc Off Anal Chem Int 79:671–687 Snook ME, Mitchell T, Hinton DM, Bacon CW (2009) Isolation and characterization of Leu-7 surfactin from the endophytic bacterium Bacillus mojavensis RRC 101, a biocontrol agent for Fusarium verticillioides. J Agric Food Chem 57:4287–4292 Subramoni S, Nathoo N, Klimov E, Vuan A-C (2014) Agrobacterium tumefaciens responses to plant-derived signaling molecules. Front Plant Sci 5:1–12 Summerell BA, Laurence EB, Liew ECY, Leslie JF (2010) Biogeography and phylogeography of Fusarium: a review. Fungal Divers 44:3–13

10

Fungal and Bacterial Maize Kernel Interactions with the Vertically. . .

209

Tan SY, Chua SL, Chen Y, Rice SA, Kjelleberg S, Nielsen TE, Yang L, Gibskov M (2013) Identification of five structurally unrelated quorum-sensing inhibitors of Pseudomonas aeruginosa from a natural-derived database. Antimicro Agents Chemother 57:5629–5641 Taylor JW, Berbee MI (2006) Dating divergence in the fungal tree of life: review and new analyses. Mycology 98:838–849 Taylor TN, Krings M (2005) Fossil microorganisms and land plants: associations and interactions. Symbiosis 40:119–135 Thiel PG, Gelderblom WCA, Marasas WFO, Nelson PE, Wilson TM (1986) Survey of fumonisin production by Fusarium species. Appl Environ Microbiol 57:1098–1093 Tung TT, Jakobsen TH, Dao TT, Fuglsang AT, Givskov M, Christensen SB, Nielsen J (2017) Fusaric acid and analogues as gram-negative bacterial quorum sensing inhibitors. Eur J Med Chem 126:1011–1020 Verbeke F, Craemer SD, Debuune N, Janssens Y, Wynendaele E, Van de Wiele C, Spiegleer BD (2017) Peptides as quorum sensing molecules: measurement techniques and obtained levels in vitro and in vivo. Front Neuro Sci 11:183–201 Voss KA, Norred WP, Plattner RD, Bacon CW (1989) Hepatotoxicity and renal toxicity in rats of corn samples associated with field cases of equine leukoencephalomalacia. Food Chem Toxicol 27:89–96 Yang L, Zuo S, Zou Y, Wang J, Song W (2013) Investigation on diversity and population succession dynamics of endophytic bacteria from seeds of maize (Zea mays L, Nogda 108) at different growth stages. Ann Microbiol 63:71–79 Yates IE, Jaworski AJ (2000) Differential growth of Fusarium moniliforme relative to tissues from ‘Silver queen’, a sweet maize. Can J Bot 78:472–480 Yates IE, Hiett KL, Kapczynski DR, Smart W, Glenn AE, Hinton DM, Bacon CW, Meinersmann R, Liu S, Jaworski AJ (1999) Gus transformation of the maize fungal endophyte Fusarium moniliforme. Mycol Res 103:129–136 Yue Q, Bacon CW, Richardson MD (1998) Biotransformation of BOA and 6-methoxybenzoxazolinone by Fusarium moniliforme. Phytochemistry 48:451–454 Zhang J, Boone L, Kocz R, Zhang C, Binns AN, Lynn DG (2000) At the maize/Agrobacterium interfaces natural factors limiting host transformation. Chem Biol 7:611–621 Zuniga A, Poupin MJ, Donso R, Ledger T, Guiliani N, Gutierrez RA, Gonzalez B (2013) Quorum sensing and indol-3-acitic acid degradation play a role in colonization and plant growth promotion of Arabidopsis thaliana by Burkholderia phytofirmans PsJN. Mol Plant Microbe Interact 26:546–553

Part III Seed Endophytes: Biology and Functional Roles in Plant Development

Functional Roles of Seed-Inhabiting Endophytes of Rice

11

Gaurav Pal, Kanchan Kumar, Anand Verma, James Francis White, Jr, and Satish K. Verma

Contents 11.1 11.2 11.3 11.4

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diversity and Distribution of Rice Seed Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transmission of Seed Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional Role of Rice Seed Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.4.1 Seed Endophytes as Plant Growth-Promoting Agents . . . . . . . . . . . . . . . . . . . . . . . . 11.4.2 Nutrient Acquisition Facilitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.5 Seed Endophytes as Biocontrol Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.5.1 Synthesis of Allelochemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.5.2 Antibiotic Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.5.3 Lytic Enzyme Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.5.4 Quorum Sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.5.5 Induced Systemic Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.6 Seed Endophytes for Improving Phytoremediation of Soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.7 Metagenomic Studies on Rice Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

214 215 216 220 220 221 225 226 227 227 228 228 229 230 230 231

Abstract

Endophytic microbes including bacteria and fungi inhabiting in seed tissues have recently gained significant importance owing to a diversity of roles that they play and eventually resulting in improved plant growth as well as plant fitness. Some of the major roles played by seed endophytic microbes include plant growth promotion by enhanced nutrient acquisition or production of growth hormones, nitrogen fixation, phosphate solubilisation, and protection against pathogens as well as abiotic stresses. Since, rice is one of the important staple crop across the G. Pal · K. Kumar · A. Verma · S. K. Verma (*) Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi, UP, India J. F. White, Jr (*) Department of Plant Biology, Rutgers University, New Brunswick, NJ, USA # Springer Nature Switzerland AG 2019 S. K. Verma, J. F. White, Jr (eds.), Seed Endophytes, https://doi.org/10.1007/978-3-030-10504-4_11

213

214

G. Pal et al.

globe, there is a great need to explore and decipher the roles of the endophytic community present inside it. This chapter focuses on the diversity and distribution of rice seed endophytes, their transmission along with the various functional roles that they play inside the plants with an aim to provide deep insights on rice seed endophytes as plant growth promoting and biocontrol agents. Keywords

Endophytic microbes · Rice · Plant growth promotion · Biocontrol agents

11.1

Introduction

The term ‘endophyte’ originally was coined by de Bary in 1866 for all those microbes which live inside plants. Endophytes are often bacteria and fungi, which grow and reside for all or part of their life cycles inside the living host plant without showing disease symptoms or negative effect to the plant tissues (Wilson 1995). Bacterial and fungal endophytes have been found to be associated with nearly all plants that have been examined. It is important to discover the various impacts and roles that endophytes have on the plant development (Arnold et al. 2000). Endophytes have been isolated from roots (Murphy et al. 2015), shoots (Rojas et al. 2016), flowers, leaves (Yumlembam and Borkar 2014), fruits (Krishnan et al. 2012), seeds (Truyens et al. 2015; Verma et al. 2017a, b) and meristematic tissues of the plants (Pirttilä et al. 2003). The bacterial community of seeds, either endophytic or an epiphytic, has been shown to contribute to rhizospheric community development (Johnston-Monje et al. 2016). Seed endophytes have been reported from numerous crops including rice (Verma et al. 2018), wheat (Larran et al. 2016), cotton (Lopez 2015; Irizarry and White 2017) and corn (Gond et al. 2015; Shehata et al. 2016). Seed-vectored endophytic bacteria are indigenous in plants and have been shown to be transmitted vertically from generation to generation. Endophytes are known to have positive impacts on plants starting from germination and seedling establishment, since they play important roles in nutrient mobilisation (Li et al. 2012; White et al. 2015), nitrogen fixation (Elbeltagy et al. 2001), phosphate solubilisation (Barrow and Osuna 2002; Verma et al. 2017a, b) and plant growth promotion (Compant et al. 2010) and provide tolerance against biotic and abiotic stresses (Kuldau and Bacon 2008; Gond et al. 2015). Bacterial seed endophytes are also found to reduce fungal disease infection during seedling development (Verma et al. 2017b; Verma and White 2018; Verma et al. 2018). The potential application of the bacterial and fungal endophytes in important food crops has attracted the scientific community and stimulated research to gain a better characterisation and understanding of endophytes and their functional roles in plant development and ecology (Mitter et al. 2013). Rice is a staple crop of nearly half of the world’s total population and one of the most important food crops of the world (Gyaneshwar et al. 2001). A major problem is the burgeoning human population with limited cultivable land; hence, efforts are needed to increase rice productivity. On the other hand, modern agricultural

11

Functional Roles of Seed-Inhabiting Endophytes of Rice

215

practices employed in improving rice production include plant breeding programmes which involve years of breeding, as well as use of chemical fertilisers and pesticides that pose threats to our already suffering environment (Mano and Morisaki 2008). The proper management and application of seed endophytic bacteria in promoting plant growth are gaining importance due to endophyte beneficial effects and their environmentally friendly nature. Studies have reported that certain seed-derived endophytic bacteria play important roles in modulating seedling development and defence against pathogens (Verma et al. 2017a, b; Ma et al. 2011; Glick 2005). Similarly, other experiments conducted on seed endophytes in rice have shown growth promotional activity including nitrogen fixation, hormonal modulation, siderophore formation and phosphate solubilisation (Walitang et al. 2017). Seed endophytic microbes are shown to be important in studies of plant-endophyte interactions (Hardoim et al. 2008). Seed-borne endophytes are better positioned to interact with young developing seedlings, when compared to the bulk of soil bacteria (Rosenblueth and Martínez-Romero 2006). In addition, seed endophytes are transferred vertically and hence remain associated with plants for many generations, thereby establishing a closely adapted and strong association with plants (Johnston-Monje and Raizada 2011). This book chapter provides a comprehensive review of the diversity of seed endophytes inhabiting rice and their possible functional roles in development and growth of rice plants, along with other contributions in various processes such as in providing resistance against biotic and abiotic stresses. This chapter also includes recent studies on functional metagenomics of seeds which tells about the putative functional role of non-cultivable endophytes residing in rice seeds. Future prospects for understanding the basic science of rice seed endophytes as well as their potential applications for sustainable agriculture have also been considered.

11.2

Diversity and Distribution of Rice Seed Endophytes

Research on the presence of endophytic bacteria inside plant tissues originated in the 1870s by Pasteur and group (Hollis 1949), but there was a general tendency that these microorganisms inside plants were pathogenic in nature, causing harmful effects. It was Perotti who firstly reported the incidence of non-pathogenic bacterial isolates inside the root tissues of plants (Perotti 1926). Currently, these endophytic microorganisms are present in all the plant species and have been isolated from many varieties of plants (Lodewyckx et al. 2002). Besides being present in vascular plants (Zhang and Yao 2015), endophytes have been found to colonise marine algae (Mathan 2016), mosses and ferns (Verma et al. 2017a, b; Hoysted et al. 2018). Endophyte communities are affected by host plant species and the environmental conditions under which the host is growing (Rosenblueth and Martínez-Romero 2006). Generally, it is believed that endophytic microbes originate from microbes of the rhizosphere, gaining entrance into the plant through natural openings or wounds or penetrating the plant tissues through enzymes such as cellulases and pectinases (Hallmann et al. 1997). However, the first asymptomatic endophytic fungus of genus

216

G. Pal et al.

Epichloë in grasses was discovered at the end of the nineteenth century in Lolium temulentum (Freeman 1904). Some studies suggest that interactions between fungi and host could result from pathogen-host antagonism (Schulz et al. 1999), while other studies suggest a closely coadapted mutualistic symbiotic association. To obtain information on endophytic bacterial and fungal diversity, culturedependent as well as culture-independent approaches can be employed, but cultureindependent molecular approaches such as 16S rDNA sequencing techniques, denaturing gradient gel electrophoresis (DGGE), etc. are gaining more importance because of the unknown conditions for growth of many endophytic microorganisms (Tholozan et al. 1999). Rice is a gramineous plant and has a deep fibrous root system for drawing for water and nutrient absorption. Various bacteria have been reported from rice plants, including Pantoea from the seeds, Rhizobium and Burkholderia from the roots, Methylobacterium from the shoots, etc. (Mano and Morisaki 2008). Researchers isolated Pantoea ananas from the seeds of Oryza alta, Herbaspirillum seropedicae and Methylobacterium sp. from Oryza meridionalis and Klebsiella oxytoca from Oryza sativa (Elbeltagy et al. 2000). Similarly, Bacillus cereus, Pantoea ananatis, Sphingomonas echinoides and Sphingomonas parapaucimobilis were isolated from Oryza sativa (Okunishi et al. 2005). Various bacterial endophytes such as Pseudomonas fluorescens, Xanthomonas sacchari and Staphylococcus sp. were also identified from milled rice seeds using ribosomal intergenic spacer analysis (RISA). A study was conducted on the diversity of endophytic community of rice seeds of salt-tolerant and salt-sensitive cultivars (Walitang et al. 2017). In this study, it was found that the cultivable seed endophytes were dominated by Proteobacteria notably class Gammaproteobacteria. Extremely identical type strains were isolated that were Flavobacterium sp., Mycobacterium sp. and Xanthomonas sp. from the salt-tolerant as well as salt-sensitive cultivars. The study showed how selective screening of endophytes with physiological characteristics related with tolerance to osmotic stress or ability to detoxify ROS can be done by rice seeds (Walitang et al. 2017). Fungal endophytes such as Alternaria alternata, Epicoccum purpurascens, Fusarium equiseti, Nigrospora oryzae, etc. were isolated from seeds of several different cultivars of rice (Fisher and Petrini 1992). The endophytic bacteria and fungi isolated from seeds of rice plants are listed in Table 11.1.

11.3

Transmission of Seed Endophytes

The transmission of endophytes can take place either from the vegetative parts of the plant to the seed or endosperm via chalaza, funiculus and micropyle, i.e. plant maternal vascular connections, or directly through the ovules, thereby colonising the endosperm and the newly formed embryo (Truyens et al. 2015). The transmission of endophyte directly through the ovules results in vertical transmission of endophytes. The transformation of shoot meristems in which an endophyte grows into reproductive meristems followed by endophytic colonisation is the likely route

11

Functional Roles of Seed-Inhabiting Endophytes of Rice

217

Table 11.1 Various seed endophytic microbes isolated from rice seeds and their functional role Name of bacterial seed endophytes Enterobacter asburiae VWB1

Pantoea dispersa VWB2

Pseudomonas putida VWB3

Pantoea agglomerans CT1

Pantoea agglomerans CT2

Acinetobacter sp. CT5 Curtobacterium citreum CT7 Microbacterium sp. CT8 Pantoea ananatis CT10

Pseudomonas sp. CT11 Paenibacillus sp. CT14 Pantoea sp. CT19 Staphylococcus cohnii CT 21

Role of seed endophytes IAA production Phosphate solubilisation Amylase activity Antifungal activity against Fusarium oxysporum IAA production Phosphate solubilisation Amylase activity Antifungal activity against Fusarium oxysporum Phosphate solubilisation Amylase activity Antifungal activity against Fusarium oxysporum IAA production Nitrogen fixation Phosphate solubilisation Antagonism to Pythium ultimum IAA production Phosphate solubilisation Nitrogen fixation Antagonism to both Curvularia sp. and Fusarium oxysporum IAA production Phosphate solubilisation Nitrogen fixation IAA production Phosphate solubilisation IAA production Phosphate solubilisation Phosphate solubilisation IAA production Nitrogen fixation Antagonism to Curvularia sp., Fusarium oxysporum and Pythium ultimum IAA production Phosphate solubilisation Nitrogen fixation IAA production Phosphate solubilisation Antagonism to Curvularia sp. IAA production Phosphate solubilisation IAA production Phosphate solubilisation

Isolated from rice variety Rex rice seed

Reference Verma et al. (2017a, b)

Rex rice seed

Verma et al. (2017a, b)

Rex rice seed

Verma et al. (2017a, b)

CT6919

Ruiza et al. (2011)

CT6919

Ruiza et al. (2011)

CT6919

Ruiza et al. (2011)

CT6919

Ruiza et al. (2011) Ruiza et al. (2011) Ruiza et al. (2011)

CT6919 CT6919

CT6919

Ruiza et al. (2011)

CT6919

Ruiza et al. (2011)

CT6919

Ruiza et al. (2011) Ruiza et al. (2011)

CT6919

(continued)

218

G. Pal et al.

Table 11.1 (continued) Name of bacterial seed endophytes Sphingomonas sp. CT25 Rhizobium larrymoorei CT26 Microbacterium sp. CT28 Curtobacterium sp. CT30 Microbacterium sp. CT32 Microbacterium sp. CT34 Microbacterium sp. CT39 Kocuria palustris

Role of seed endophytes IAA production Nitrogen fixation Phosphate solubilisation IAA production Phosphate solubilisation IAA production Phosphate solubilisation Antagonism to Pythium ultimum IAA production Phosphate solubilisation Antagonism to Pythium ultimum IAA production Nitrogen fixation Phosphate solubilisation IAA production, Phosphate solubilisation IAA production Phosphate solubilisation Antagonism to Pythium ultimum –

Isolated from rice variety CT6919

CT6919 CT6919

Reference Ruiza et al. (2011) Ruiza et al. (2011) Ruiza et al. (2011)

CT6919

Ruiza et al. (2011)

CT6919

Ruiza et al. (2011)

CT6919

Ruiza et al. (2011) Ruiza et al. (2011)

CT6919

Methylobacterium radiotolerans

–

Methylobacterium fujisawaense

–

Pantoea ananatis

High tolerance to osmotic pressure

Methylobacterium aquaticum Sphingomonas melonis Sphingomonas yabuuchiae Xanthomonas translucens Acidovorax sp.

Amylase activity

Oryza sativa cultivar kinuhikari Oryza sativa cultivar kinuhikari Oryza sativa cultivar kinuhikari Oryza sativa cultivar kinuhikari Oryza sativa

–

Oryza sativa

–

Oryza sativa

Amylase activity

Oryza sativa

–

Oryza sativa

Micrococcus luteus Curtobacterium flaccumfaciens

–

Oryza sativa

–

Oryza sativa

Kaga et al. (2009) Kaga et al. (2009) Kaga et al. (2009) Kaga et al. (2009) Mano and Morisaki (2008) Mano and Morisaki (2008) Mano and Morisaki (2008) Mano and Morisaki (2008) Mano and Morisaki (2008) Mano and Morisaki (2008) Mano and Morisaki (2008) (continued)

11

Functional Roles of Seed-Inhabiting Endophytes of Rice

219

Table 11.1 (continued) Name of bacterial seed endophytes Paenibacillus amylolyticus Bacillus pumilus

Role of seed endophytes –

Isolated from rice variety Oryza sativa

Amylase activity

Oryza sativa

Bacillus subtilis

Amylase activity

Oryza sativa

Sphingomonas echinoides

Catalase activity Oxidase activity

Sphingomonas parapaucimobilis

Oxidase activity

Bacillus cereus

Spore formation Catalase activity Oxidase activity Catalase activity

Oryza sativa cultivar Nipponbare Oryza sativa cultivar Nipponbare Oryza sativa cultivar Nipponbare Oryza sativa cultivar Nipponbare Oryza meridionalis W1627 Oryza alta W0017 Oryza sativa Bu-24

Elbeltagy et al. (2000) Elbeltagy et al. (2000)

Oryza sativa variety Kuruluthuda

Wijesooriya and Deshappriya (2016)

Pantoea ananatis

Herbaspirillum seropedicae B39

–

Pantoea ananas B40 Klebsiella oxytoca B43

Cellulase activity IAA production Pectinase activity Cellulase activity IAA production Improve the growth of rice plants when it’s used with combination of Arthrobotrys

Acremonium sp. (fungi)

Reference Mano and Morisaki (2008) Mano and Morisaki (2008) Mano and Morisaki (2008) Okunishi et al. (2005) Okunishi et al. (2005) Okunishi et al. (2005) Okunishi et al. (2005) Elbeltagy et al. (2000)

of vertical transmission as these will give rise to ovules and finally seed (Pirttilä et al. 2000). Vertical transmission of bacterial endophytes likely occurs in most species of plants. In one study, it was concluded that seeds contributed the major portion of endophytes to mature wheatgrass plants since the same species of endophytes were recovered from seeds and mature tissues (Ringelberg et al. 2012). Similarly, a study was conducted on vertical transmission of seed endophytes in switchgrass plants, and it was found that the same Bacillus sp. and Microbacterium spp. which inhabited the seeds in first year were present in the switchgrass plant in the second year (Gagne-Bourgue et al. 2013). Moreover, another study reported continuity of the endophytic bacterial community of maize from parent to offsprings. Additionally, genetically connected maize hybrids contained similar species and genera of endophytic bacteria (Liu et al. 2012, 2013).

220

G. Pal et al.

Fungal endophytes may be transmitted through varied routes in the case of transmission from one host to another, whereas seed transmission is employed when transmission takes place from one generation to another (Shearin et al. 2018; Vujanovic and Germida 2017). In a study conducted on seed endophytes in forbs, it was proposed that vertical transmission is a widespread phenomenon (Hodgson et al. 2014).

11.4

Functional Role of Rice Seed Endophytes

Seeds contain a diversity of microflora consisting of bacteria and fungi which play various physiological roles, but little work has been done to determine the roles that endophytes play in plant development and ecology. Major functions include plant growth promotion, biocontrol activity, and many other unknown benefits. Some of these functions are described below.

11.4.1 Seed Endophytes as Plant Growth-Promoting Agents Plant growth promotion is one of the major roles that endophytes play in plants. Plant growth promotion activity in plants can be the result of either direct or indirect stimulation of plant growth. Direct stimulation of plant growth involves facilitation of acquisition of essential nutrients or modulation of growth hormones within plants by the bacteria or fungi; indirect growth promotion may involve inhibition of plant pathogens by the endophytes, thereby reducing the level of infection in the plant tissues (Doty et al. 2009; Luo et al. 2012; Sziderics et al. 2007) (Figs. 11.1 and 11.2).

Fig. 11.1 Scheme depicting functional roles, mechanism of action of seed endophytic bacteria during seedling developments and plant protection

11

Functional Roles of Seed-Inhabiting Endophytes of Rice

221

Fig. 11.2 Effect of seed-inhabiting bacteria on seedling development. Where (a) and (d) are control, disinfected with 4% NaOCl and streptomycin (100 μg ml
1), (b) treated with Pantoea dispersa (B2), (c) treated with Bacillus amyloliquefaciens (M4), (e) treated with Pseudomonas sp. and (f) treated with Pantoea dispersa (B2) (From our previous studies; Verma and White 2018; Verma et al. 2017a, b)

11.4.2 Nutrient Acquisition Facilitation The mode of action of many endophytic bacteria involves increasing the availability of nutrients for the plant in the rhizosphere (Glick 1995). The essential nutrients involved in uptake are mainly nitrogen, phosphorus and iron. Enhanced plant growth was observed when rice was inoculated with diazotrophic endophyte Azoarcus sp. strain BH72, with stimulation seen even in Nif
mutants (Hurek et al. 1994). Therefore, it was hypothesised that the observed plant growth might be the result of enhanced nutrient-water uptake associated with the colonisation of Azoarcus and not nitrogen fixation (Hurek et al. 1994). Phosphate solubilisation and siderophore formation are the two major methods by which endophytes facilitate nutrient acquisition by plants.

11.4.2.1 Phosphate Solubilisation Phosphorus is one of the most important minerals limiting the growth of terrestrial plants. Although large reserves of phosphorus are present in soils, most of the phosphorus remains unavailable to plants. The reason behind this unavailability is that most of the phosphorus in soils is present in insoluble forms (Stevenson and Cole 1999). Plants absorb two basic soluble forms of phosphorus, monobasic (H2PO4
) and dibasic (HPO42
) ions (Glass 1989). Secretion of organic acids and

222

G. Pal et al.

phosphatases are commonly utilised by endophytic bacteria for the facilitation of phosphorus solubilisation. A study was conducted to determine the diversity of endophytic bacteria residing inside the seeds of a deepwater rice variety, and mineral phosphate solubilisation activity of the isolates was assessed to judge their growth promotional potentials. Four out of seven isolates showed phosphate solubilisation activity (Verma et al. 2001). Similarly, a diazotrophic bacterium isolated from wild rice (Porteresia coarctata) was tested for its phosphate mineralisation activity and found positive based on clearing zones in the tricalcium phosphate solubilisation test (Loganathan and Nair 2003).

11.4.2.2 Siderophore Formation Iron is an essential element required for the growth of microorganisms; however, most of the iron remains unavailable to microbes since it is present in hard-tosolubilise mineral form in the soil. In order to sequester iron from the environment, many microorganisms secrete low molecular weight, iron-binding compounds known as siderophores. Siderophores have strong affinity to bind with the Fe+3 form of iron. This soluble form of iron is now transported back inside the microbial cell and is utilised for its growth. Acremonium and Fusarium sp. isolated from seeds of a rice variety from Sri Lanka improved growth of rice, and it was hypothesised that siderophore formation and nitrogen scavenging by the endophytic fungal species could be the reasons behind the improved growth of the plant (Wijesooriya and Deshappriya 2016). Siderophores are produced by bacteria as well as fungi, but bacterial siderophores have been found to be more effective than fungal siderophores, and thus they scavenge more effectively than fungi, effectively limiting the growth of fungi. Some plants have the ability to release the iron from iron-siderophore complexes by binding to them and releasing iron for plant growth. In this way, plant growth is boosted in two ways, i.e. by growth suppression of pathogenic fungi and enhanced iron nutrition (Bashan and De-Bashan 2005). A study was conducted on endophytic bacteria producing siderophores in rice in Uruguayan soils, and it was proposed that most of the heterotrophic bacteria were siderophore producing in mature plants. Additionally, in vitro inhibition assays showed that siderophore-producing bacteria of the genus Burkholderia were good antagonists of disease-causing fungi (Loaces et al. 2011). 11.4.2.3 Transference of Nutrients to Plants from Bacteria via the Rhizophagy Cycle Symbiosis The rhizophagy cycle is a process whereby plants may obtain nutrients from bacteria that alternate between a root intracellular endophytic phase and a free-living soil phase (Paungfoo-Lonhienne et al. 2010; Kandel et al. 2017; Prieto et al. 2017; Verma et al. 2018). Bacteria acquire soil nutrients in the free-living soil phase; nutrients are extracted from bacteria oxidatively in the intracellular endophytic phase (White et al. 2018, 2019). We conducted experiments on seed-vectored pseudomonad endophytes from Phragmites australis using Poa annua as surrogate host. We

11

Functional Roles of Seed-Inhabiting Endophytes of Rice

223

found that initially the symbiotic pseudomonads grow on the rhizoplane in the exudate zone behind the root meristem. Bacteria enter root tip meristem cells—locating within the periplasmic space between cell wall and plasma membrane. In the periplasmic spaces of root cells, bacteria convert to wall-less L-forms (White et al. 2018, 2019; Verma and White 2018; Verma et al. 2017a, b, 2018). As root cells mature, bacteria are exposed to reactive oxygen (superoxide) produced by NADPH oxidases (NOX) on the root cell plasma membranes. Reactive oxygen degrades some of the intracellular bacteria—effectively extracting nutrients from them likely in the form of oxidised macro- and micronutrients. Surviving bacteria in root epidermal cells trigger root hair elongation (Verma et al. 2017a, b, 2018), and as hairs elongate bacteria exit at the hair tips, reforming cell walls and rod shapes as they emerge into the rhizosphere where they may obtain additional nutrients. Release of bacteria from elongating root hairs ensures that symbiotic bacteria are deposited into the rhizosphere where additional nutrients may be acquired rather than on the rhizoplane where soil nutrients are scarce. Later attraction of bacteria to the root exudate zone behind the root tip meristem again places bacteria in position to enter root meristem cells. Plants appear to manipulate symbiotic bacteria in the rhizophagy cycle by (1) stimulating bacterial growth around root tip meristems of seedlings by secretion of root exudates around the root tip (Lareen et al. 2016); (2) triggering bacteria to enter into periplasmic spaces in root cells at the root tip meristem by absorbing bacterial fermentation products including butyric acid, causing bacteria to upregulate virulence/endoparasitism genes (White 2017; White et al. 2019; Tramontano and Scanlon 1996; Sun and O’Riordan 2013); (3) subjecting bacteria in periplasmic spaces to superoxide formed on root cell plasma membranes to extract nutrients from bacteria (Verma et al. 2017a, b, 2018); and (4) depositing surviving intracellular bacteria back into the rhizosphere from the tips of elongating root hairs to maximise new nutrient acquisition by bacteria (White et al. 2019). Through the rhizophagy symbiosis, plants appear to ‘farm’ symbiotic microbes. Isotope-tracking experiments where plantlets of Agave tequilana were inoculated with 15N-labelled bacteria demonstrated that nitrogen in bacteria was transferred to the plant likely via rhizophagy symbiosis (Beltran-Garcia et al. 2014). Experiments involving grass seedlings with and without endophytic bacteria grown on 15N-labelled proteins suggest that the rhizophagy cycle could account for 30% of the nutrients absorbed by grass roots (White et al. 2015). Evidence to date suggests that all vascular plants engage in rhizophagy symbiosis to some extent (White et al. 2019). Rhizophagy symbiosis may be a critical means by which plants extract nutrients from symbiotic bacteria.

11.4.2.4 Modulation of Hormonal Levels The mechanism of hormonal modulation is most commonly exploited by endophytic microbes which synthesise several hormones, including auxins, cytokinins and gibberellins. The synthesis of these hormones can be done either singly or in combinations by microbes (Narula et al. 2013). It is believed that phytohormones produced by endophytic microbes have the ability to stimulate plant growth resulting in bigger and more branched roots with greater surface area (Vessey 2003).

224

G. Pal et al.

IAA (indole-3-acetic acid) is a type of auxin generally synthesised by endophytic bacteria and is known to stimulate root initiation, cell division and cell enlargement (Salisbury 1994). Most of the endophytic IAA-producing bacteria are thought to promote root growth and increase root length, thereby increasing the root surface area and enabling the plant to absorb more water and nutrients from soil. Indole acetic acid production by endophytic bacteria was estimated in seeds of deepwater rice varieties, and all seven isolates were found to produce indole acetic acid (Verma et al. 2001). In one study 576 isolates of endophytic bacteria were isolated from different parts of 10 Korean rice cultivars (Ji et al. 2014). From these isolates 12 were identified as diazotrophic, and their growth-promoting activities were assessed by their application to seeds of rice cultivars. It was found that inoculated seeds showed improved plant growth and increased height and dry weight. Ten strains showed higher auxin production activity, and it was proposed that auxin production was one of the possible mechanisms of plant growth promotion (Ji et al. 2014). Similarly, a study was conducted on seed-vectored endophytic bacteria in modulation of development of rice seedlings, and IAA production by three isolates (VWB1, VWB2 and VWB3) was determined. The isolates VWB1 and VWB2 were found to produce IAA, and it was proposed that endophyte-produced IAA may be involved in triggering root hair development (Verma et al. 2017a, b). Cytokinins are also a class of plant hormones known to be involved in cell division promotion, cell enlargement and tissue expansion in specific plant parts. Other groups of phytohormones include gibberellins, abscisic acids and ethylene. Gibberellins are known to be associated with the extension of stem tissues of plants, thereby modifying the plant morphology (Salisbury 1994). Evidence of gibberellin production by endophytic bacteria is rare; however, a study has reported the production of gibberellic acid along with IAA, abscisic acid and cytokinin by a diazotrophic rice endophyte Pantoea agglomerans YS19. The phytohormones were detected by ELISA and finally confirmed by gas chromatography (Feng et al. 2006). An endophytic bacteria RWL isolated from seeds of rice and later identified as Bacillus amyloliquefaciens by sequencing and phylogenetic analysis of 16S rRNA was assessed for its phytohormone production ability. It was reported that the endophytic bacteria secreted various forms of bioactive and inactive gibberellic acids which colonised the roots and promoted rice plant growth (Shahzad et al. 2016).

11.4.2.5 ACC Deaminase Activity In response to pathogenic attack or stress, higher amounts of the hormone ethylene are generally secreted by plants which further stimulates senescence, inhibits plant growth and triggers cell death near the infection site. Hence, lowering the level of ethylene after infection could prove very useful to plants. The bacterial enzyme ACC (1-aminocyclopropane-1-carboxylate) deaminase has been found to have an active role in plant growth promotion and was first characterised by Honma and Shimomura in 1978 (Honma and Shimomura 1978). The enzyme catalyses the conversion of ACC (1-aminocyclopropane-1-carboxylate), the immediate precursor of ethylene synthesis in plants into ammonia and α-ketobutyrate. The seeds or plant roots exude the ACC which is metabolised by the bacteria resulting in stimulation of

11

Functional Roles of Seed-Inhabiting Endophytes of Rice

225

efflux of ACC, thereby decreasing the root ACC concentration as well as root ethylene evolution ultimately increasing root growth (Glick et al. 1998). Several studies have reported that promotion of plant growth by some ACC deaminaseproducing bacteria is done under various stressful conditions like flooding, saline conditions and drought (Holguin and Patten 1999). Isolation of ACC deaminaseproducing bacteria from seeds of non-cultivated plants and then inoculating them onto crop plants could result in improved growth, even in harsh environmental conditions.

11.5

Seed Endophytes as Biocontrol Agents

Different types of biotic stresses such as diseases and pests decrease the agricultural productivity of crop plants. Such pests and diseases are controlled by using pesticides and chemical fertilisers resulting in ill-effects on human health and ecotoxicity. The use of endophytic microflora could solve the toxicity issue and help in improving human health (Boddey et al. 1995). Various seed endophytic fungi and bacteria have been reported to possess anti-pathogenic properties making them key prospects to act as biocontrol agents. In rice seed endophytes, two Bacillus strains were found to show maximum antifungal properties against Rhizoctonia solani, Pythium myriotylum, Gaeumannomyces graminis and Heterobasidion annosum, and it was believed that the antifungal activity was due to the production of volatile antifungal compound ammonia or N-acetyl-β-D-glucosaminidase or both (Mukhopadhyay et al. 1996). A study suggested the presence of antifungal activity in numerous strains of Microbacterium, Pseudomonas, Pantoea, Paenibacillus and Curtobacterium, all isolated from rice seeds and were found to be effective against Curvularia sp., Fusarium oxysporum and Pythium ultimum (Ruiza et al. 2011). Similar study on rice seed endophytes reported in vitro antifungal activity against Rhizoctonia solani and Pyricularia grisea (Cottyn et al. 2001). We also found that presence of seed endophytes protects seedlings from fungal infection during seedling development (Fig. 11.3). Studies have reported that toxin production by endophytic fungi gives them the capacity to deter insects, inducing weight loss and also causing an increase in pest death rates (Azevedo et al. 2000). Several studies suggest that a number of fungi have completely different mode of action and that they render the plant uneatable for various kinds of pests like grasshopper, aphids, etc. (Clay 1989; Carroll 1991). A study was done on two classical rice varieties (Suwandel and Kaluheenati) of Sri Lanka, and it was reported that rice seed-borne fungi Absidia and Cylindrocladium showed high inhibition to pathogen (Magnaporthe grisea), and it was found that these fungi controlled the pathogen by coiling the hyphae around it, forming clamps and loops (Atugala and Deshappriya 2015). The mechanisms behind inhibition of plant pathogens by endophytes are still not very clear, but a number of explanations have been given in this context. Reports suggest that some endophytes inhibit pathogens by direct mechanism involving the secretion of lytic enzymes and hence directly suppressing them by antibiosis. This

226

G. Pal et al.

Fig. 11.3 Effect of seed endophytes Pantoea dispersa on root hair formations and protection from fungal infection. Where (a, b, c) are control and (d, e, f) are treatment with Pantoea dispersa; (a) very few and short root hairs, (b) clean root parenchyma colonise with fungal mycelium (arrows), (c) clean root hairs colonise with fungal mycelium (arrows), (d) very long and dense root hairs (arrows), (e) intracellular bacteria in root parenchyma and no fungal infection (arrows) and (f) internalisation of bacteria in root hairs (from our previous studies; Verma et al. 2017b, 2018)

kind of interaction between fungal endophytes and pathogens can be complex as well as very specific (Arnold et al. 2003). Another mechanism of pathogen inhibition by fungal endophytes involves the production of organic compounds such as alkaloids by the plant, and these alkaloids help in growth suppression of pathogens (Cheplick et al. 1989). Systemic acquired resistance (SAR) mediated by salicylic acid, as well as pathogenesis-related (PR) proteins, and induced systemic resistance (ISR) mediated by jasmonic acid or ethylene are other modes of action by which growth of pathogens may be suppressed in plants by endophytes (Vallad and Goodman 2004).

11.5.1 Synthesis of Allelochemicals Allelochemicals are bioactive compounds, including antibiotics, siderophores, lytic enzymes, etc. that may act defensively against invading competitors, pathogens or pests, while allelopathy refers to the defensive action of allelochemicals (Saraf et al. 2014). These naturally synthesised chemical compounds can be utilised in crop disease management programmes in place of chemical pesticides. As natural pesticides, allelochemicals may solve health-related problems and other soil- and environmental pollution-related issues leading to climate change caused by the use of agrochemicals (Farooq et al. 2011). Allelopathic effects frequently depend on a number of environmental conditions such as presence of water, nutrition, bacterial

11

Functional Roles of Seed-Inhabiting Endophytes of Rice

227

density and soil structure as well as its texture (Barazani and Friedman 2001). Studies suggest that allelopathic rice can have a positive influence on the plant population density as well as soil microbial community (Kong et al. 2008). For allelopathy to be more effective, either improved environmental conditions favouring the biological agent could be provided or the microbe’s allelochemicals may be enhanced/increased through genetic transformation (Hornok 2000). Genetic enhancement of the biocontrol agent can be achieved by various means such as mutation (physical or chemical), development of sexual hybrids, homokaryon production or by genetic manipulations like protoplast fusion, site-directed mutagenesis, recombination, transformation, etc. (Palumbo et al. 2005). Several different methods of allelopathy are described below.

11.5.2 Antibiotic Production Production of antibiotics is one of the most powerful mechanisms utilised by endophytes against the phytopathogens. Antibiotics refer to those naturally synthesised chemical compounds released by microorganisms such as bacteria that inhibit the growth and metabolic activities of other microorganisms, and this phenomenon of suppression of pathogen is known as antibiosis (Fravel 1988). In one study, Pseudomonas fluorescens strain 7–14 isolated from the rice rhizosphere was found to produce antibiotics that were effective against Pyricularia sp., Rhizoctonia solani, Pythium ultimum and Gaeumannomyces graminis var. tritici. The data also showed that Pseudomonas fluorescens strain 7–14 was as effective as the fungicide tricyclazole in controlling several diseases of rice (Chatterjee et al. 1996). The experiments conducted under laboratory conditions have shown production of several antibiotics, as well as their effectiveness against various pathogens, but antibiotic availability under field conditions remains an area of concern. The antibiotic activity of endophytes may be increased as the genes involved in the synthesis of some of the antibiotics are well known, and hence, although theoretical, the suppression of phytopathogens can also be enhanced (Holguin and Patten 1999).

11.5.3 Lytic Enzyme Production Cell wall lysis is one of the potential mechanisms by which endophytic microbes can control growth of pathogens. Several bacteria have the ability to produce various enzymes that can easily hydrolyse chitin, cellulose, hemicellulose and proteins, thereby directly suppressing the growth of pathogenic agents. In a study, Stenotrophomonas maltophilia strain F-81, isolated from the rhizosphere of sugar beet, showed the production of extracellular chitinase and protease enzymes that restricted the growth of the phytopathogenic fungus Pythium ultimum in vitro (Dunne et al. 1997). Similarly, endophytic Bacillus cereus strain 65 was found to produce and excrete the enzyme chitinase that suppressed the growth of Rhizoctonia solani (Pleban et al. 1997). A study was done on endophytic Bacillus sp. isolated from the roots of balloon flower (Platycodon grandiflorus) which showed strong

228

G. Pal et al.

antifungal activity against Rhizoctonia solani, Pythium ultimum and Fusarium oxysporum. The bacterium synthesised iturin A along with cellulase and xylanase (Cho et al. 2003). Most of the studies in this field are focused on rhizospheric bacteria, and hence very little is known about mechanism of cell lysis resulting in suppression of pathogen growth by endophytic microorganisms.

11.5.4 Quorum Sensing Quorum sensing is a process of bacterial cell-cell communication and can be defined as the regulation of gene expression in response to chemical signalling molecules called autoinducers secreted by bacteria with respect to fluctuations in cell density (Miller and Bassler 2001). The accumulation of autoinducers in the environment denotes the increase in cell density of bacteria, and bacteria use this information to monitor changes in their cell numbers and alter gene expression accordingly (Rutherford and Bassler 2012). Quorum sensing controls several important activities of bacteria, including sporulation, antibiotic production, biofilm production, secretion of virulence factors, etc. (Williams and Cámara 2009). A study was done on ToxR regulator of toxoflavin biosynthesis and transport in Burkholderia glumae, which causes rice grain rot and seedling rot in rice, and it was shown that the expression of ToxJ, a transcriptional activator, was controlled by the process of quorum sensing (Kim et al. 2004). Further, the study was extended by a group of scientists that engineered the Burkholderia glumae with an N-acylhomoserine lactonase (aiiA) gene from Bacillus thuringiensis. The results of the study revealed that the introduced gene restricted the production of quorum sensing signals by Burkholderia glumae in vitro and reduced the occurrence of rice seedling rot caused by the pathogenic bacterium Burkholderia glumae in situ (Cho et al. 2007). A recent study also establishes the fact that quorum sensing is an important cell-cell communication process required for effective colonisation and establishment of a beneficial interaction by using a mutant strain of Burkholderia phytofirmans PsJN (Zúñiga et al. 2013).

11.5.5 Induced Systemic Resistance Increasing resistance of plants to disease-causing microbes can help in protecting plants against a wide range of pathogens and for a sustained duration of time. When a plant is exposed to a pathogen (virulent or avirulent) or to metabolites secreted by pathogens, it results in the stimulation of the plant’s natural defence mechanisms resulting in immunisation of plants against bacterial, fungal or viral infections, even before the establishment of pathogenic infection (Bashan and De-Bashan 2005). Induced systemic resistance is a type of generalised resistance already present in plants but is induced or enhanced by plant-associated non-pathogenic bacteria (Van Loon et al. 1998). ISR depends on pathways governed by jasmonic acid or ethylene (Yan et al. 2002). In a study, induced systemic resistance was assessed using two

11

Functional Roles of Seed-Inhabiting Endophytes of Rice

229

Pseudomonas fluorescens strains (PF1 and PF7) against Rhizoctonia solani in rice. It was found that the treatment of rice with Pseudomonas led to the induction of induced systemic resistance against Rhizoctonia solani as a result of increase in chitinase and peroxidase activity. Also, 35 kDa chitinase along with three isozymes of peroxidases (PO3–PO5) was found to be responsible in the induction and establishment of ISR (Nandakumar et al. 2001). Similarly, six fluorescent Pseudomonas strains were used for the induction of induced systemic resistance in rice against Rhizoctonia solani possessing 2,4-diacetlyphloroglucinol (2,4-DAPG) antibiotic genes. The results of the study showed that 2,4-DAPG-positive strains of fluorescent Pseudomonas sp. produced peroxidase, polyphenol oxidase, phenylalanine ammonia lyase and transcinnamic acid that resulted in the induction of induced systemic resistance in rice against the rice sheath blight pathogen Rhizoctonia solani (Reshma et al. 2018). In a study, ISR was also induced in Oryza sativa using Pseudomonas fluorescens WCS374r against the leaf blast causing pathogen Magnaporthe oryzae. This study showed that induction of ISR was dependent on pseudobactin-mediated priming for a salicylic acid-repressible multifaceted defence response (De Vleesschauwer et al. 2008).

11.6

Seed Endophytes for Improving Phytoremediation of Soils

Phytoremediation may be defined as the removal of toxic contaminants from soil through the use of plants (White et al. 2003). Rhizo-degradation is an aspect of phytoremediation that deals with the use of plants to stimulate the microbial community present at the root-soil interface to augment the degradation of toxic contaminants in the soil (Newman and Reynolds 2005). The potential role of seed endophytes in the process of phytoremediation merits study as it is believed that some endophytes contain genetic machinery capable of degrading toxic molecules in the plant rhizospheric region and other contaminated sites (Siciliano et al. 2001). Although endophytes can play an important role in toxin degradation, not all the endophytic microbes have the ability to degrade all toxic compounds as well as have the potential to thrive in the given contaminant site; hence, genetically engineered microbes have been used which can degrade the toxins present and also survive in the contaminated site (Menn et al. 2008). The possible advantage of using endophytic microorganisms in phytoremediation strategies is that these organisms are easier to manipulate and take less time to genetically engineer. In addition to this, the assessment of the efficiency of phytoremediation process can easily be monitored by quantitative gene expression of pollutant catabolic genes within endophytic populations (Newman and Reynolds 2005). At Brookhaven National Laboratory, a genetically engineered plant endophyte was developed using genetic engineering to degrade toluene, a chemical that is highly toxic to plants as well as humans (Barac et al. 2004). Similarly, a transformed Burkholderia cepacia was produced using a conjugation process that was able to reduce toluene levels in the inoculated soils (Shields et al. 1995). However, the use of genetically engineered endophytic organisms requires resolution of social and ethical issues, and field trials must be

230

G. Pal et al.

conducted to determine the actual state of microbial action on the toxins, the survival of the engineered microbe, etc.

11.7

Metagenomic Studies on Rice Endophytes

A fraction of the endophytic microbes in seeds may be cultured from the seedvectored community; therefore, there is a gap in knowledge about activities that these endophytes perform inside the seeds and seedlings (Tsurumaru et al. 2015). In addition to this, the mechanism of interaction between microorganisms is also not clearly understood. The recent advancements in genetic engineering tools such as rapid and inexpensive DNA sequencing have propelled large-scale genomic and metagenomic projects providing insights into the diversity of endophytes in seeds as well as the complexity of the community of endophytes within rice seeds (Brader et al. 2017). Metagenomic analysis relies on culture-independent molecular approaches like 16S rRNA libraries and thus helps in the study of many novel endophytic organisms which were previously impossible to culture in media or were present in a non-viable state (Lucas et al. 2013). A study was conducted in which six independent Gram-negative, facultatively anaerobic, non-spore-forming, nitrogenfixing, rod-shaped isolates were isolated from the endosphere of rice and were characterised using genomic DNA-DNA hybridisation. The results established the existence of two new endophytic species of Enterobacter that were able to enhance plant growth by supplementing nitrogen and phosphorus (Hardoim et al. 2013). In another study, a culture-independent analysis was performed using 16S rDNA amplicons of the bacterial community of rice endosphere that provided important information on bacterial diversity in the endosphere (Bertani et al. 2016).

11.8

Conclusions

The bacterial and fungal species present within, and on the surface of, rice seeds form an important group of microflora. Seed endophytes play important roles in seed germination, seedling development and plant growth. These microbes possess plant growth promotion, biocontrol and phytoremediation properties. Many of the seed endophytes are cultivable, but others have unknown cultivable conditions or are uncultivable; additional metagenomic studies on rice seed endophytes must be done to develop a more basic understanding about the diversity of total endophytic community. The functions of seed-vectored endophytes are still not fully understood. In this respect experiments are needed to develop an understanding of the functions and mechanisms of activity of endophytic microbes. Seed endophytes possess characteristics that can be exploited in various biotechnological applications owing to unique seed environment as well as the transmission to successive generations. Seed endophytes of rice are worthwhile studying for their potential to improve plant growth and development, stress tolerance, disease resistance and bioremediation. The potential for increasing rice productivity to meet the demand

11

Functional Roles of Seed-Inhabiting Endophytes of Rice

231

of the ever-increasing world population makes studies on endophytes an attractive area. The application of seed endophytes in industries, agriculture and environment requires a more detailed study to elucidate the functions of endophytes during seed germination and plant development, the mechanisms through which they control and modulate plant growth and their interactions with other endophytes. Acknowledgement The authors are grateful to the Department of Plant Biology, Rutgers University, NJ, for research facilities. SKV thanks to UGC, India, for providing a Raman Post Doctoral Fellowship No. F 5-11/2016(IC) for the year 2016–2017 to work in the USA and support as grant, Project-UGC-BSR startup-M14-26. The SKV and RNK are also grateful to the Head and Coordinator of CAS and DST-FIST and PURSE of Botany, BHU, Varanasi, for providing facilities and leave to pursue endophyte research. The authors are also thankful for support from the John E. and Christina C. Craighead Foundation, USDA-NIFA Multistate Project W3147, and the New Jersey Agricultural Experiment Station.

References Arnold AE, Maynard Z et al (2000) Are tropical fungal endophytes hyperdiverse? Ecol Lett 3 (4):267–274 Arnold AE, Mejía LC et al (2003) Fungal endophytes limit pathogen damage in a tropical tree. Proc Natl Acad Sci USA 100(26):15649–15654 Atugala DM, Deshappriya N (2015) Effect of endophytic fungi on plant growth and blast disease incidence of two traditional rice varieties. J Natl Sci Found 43(2):173 Azevedo JL, Maccheroni W Jr et al (2000) Endophytic microorganisms: a review on insect control and recent advances on tropical plants. Electron J Biotechnol 3(1):15–16 Barac T, Taghavi S et al (2004) Engineered endophytic bacteria improve phytoremediation of water-soluble, volatile, organic pollutants. Nat Biotechnol 22(5):583 Barazani O, Friedman J (2001) Allelopathic bacteria and their impact on higher plants. Crit Rev Microbiol 27(1):41–55 Barrow JR, Osuna P (2002) Phosphorus solubilization and uptake by dark septate fungi in fourwing saltbush, Atriplex canescens (Pursh) Nutt. J Arid Environ 51(3):449–459 Bashan Y, De-Bashan LE (2005) Plant growth-promoting. In: Encyclopedia of soils in the environment, vol 1. Elsevier, Oxford, pp 103–115 Beltran-Garcia MJ, White JF Jr et al (2014) Nitrogen acquisition in Agave tequilana from degradation of endophytic bacteria. Sci Rep 4:6938 Bertani I, Abbruscato P et al (2016) Rice bacterial endophytes: isolation of a collection, identification of beneficial strains and microbiome analysis. Environ Microbiol Rep 8(3):388–398 Boddey RM, Oliveira OC et al (1995) Biological nitrogen fixation associated with sugar cane and rice: contributions and prospects for improvement. Plant Soil 174(1):195–209 Brader G, Corretto E et al (2017) Metagenomics of plant microbiomes. In: Functional metagenomics: tools and applications. Springer, Cham, pp 179–200 Carroll GC (1991) Fungal associates of woody plants as insect antagonists in leaves and stems. In: Microbial mediation of plant-herbivore interactions. Wiley, New York, pp 253–271 Chatterjee A, Valasubramanian R et al (1996) Isolation of ant mutants of Pseudomonas fluorescens strain Pf7-14 altered in antibiotic production, cloning of antþ DNA, and evaluation of the role of antibiotic production in the control of blast and sheath blight of rice. Biol Control 7 (2):185–195 Cheplick GP, Clay K et al (1989) Interactions between infection by endophytic fungi and nutrient limitation in the grasses Lolium perenne and Festuca arundinacea. New Phytol 111(1):89–97

232

G. Pal et al.

Cho SJ, Lim WJ et al (2003) Endophytic colonization of balloon flower by antifungal strain Bacillus sp. CY22. Biosci Biotechnol Biochem 67(10):2132–2138 Cho HS, Park SY et al (2007) Interference of quorum sensing and virulence of the rice pathogen Burkholderia glumae by an engineered endophytic bacterium. FEMS Microbiol Ecol 60 (1):14–23 Clay K (1989) Clavicipitaceous endophytes of grasses: their potential as biocontrol agents. Mycol Res 92(1):1–12 Compant S, Clément C et al (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(5):669–678 Cottyn B, Regalado E et al (2001) Bacterial populations associated with rice seed in the tropical environment. Phytopathology 91(3):282–292 De Vleesschauwer D, Djavaheri M et al (2008) Pseudomonas fluorescens WCS374r-induced systemic resistance in rice against Magnaporthe oryzae is based on pseudobactin-mediated priming for a salicylic acid-repressible multifaceted defense response. Plant Physiol 148 (4):1996–2012 Doty SL, Oakley B et al (2009) Diazotrophic endophytes of native black cottonwood and willow. Symbiosis 47(1):23–33 Dunne C, Crowley JJ et al (1997) Biological control of Pythium ultimum by Stenotrophomonas maltophilia W81 is mediated by an extracellular proteolytic activity. Microbiology 143 (12):3921–3931 Elbeltagy A, Nishioka K et al (2000) Isolation and characterization of endophytic bacteria from wild and traditionally cultivated rice varieties. Soil Sci Plant Nutr 46(3):617–629 Elbeltagy A, Nishioka K et al (2001) Endophytic colonization and in planta nitrogen fixation by a Herbaspirillum sp. isolated from wild rice species. Appl Environ Microbiol 67(11):5285–5293 Farooq M, Jabran K et al (2011) The role of allelopathy in agricultural pest management. Pest Manag Sci 67:493–506 Feng Y, Shen D et al (2006) Rice endophyte Pantoea agglomerans YS19 promotes host plant growth and affects allocations of host photosynthates. J Appl Microbiol 100(5):938–945 Fisher PJ, Petrini O (1992) Fungal saprobes and pathogens as endophytes of rice (Oryza sativa L.). New Phytol 120(1):137–143 Fravel DR (1988) Role of antibiosis in the biocontrol of plant diseases. Annu Rev Phytopathol 26 (1):75–91 Freeman EM (1904) The seed-fungus of Lolium temulentum L., the darnel. Philos Trans R Soc Lond B 196(214–224):1–27 Gagne-Bourgue F, Aliferis KA et al (2013) Isolation and characterization of indigenous endophytic bacteria associated with leaves of switchgrass (Panicum virgatum L.) cultivars. J Appl Microbiol 114(3):836–853 Glass AD (1989) Plant mineral nutrition. An introduction to current concepts. Jones and Bartlett, Boston Glick BR (1995) The enhancement of plant growth by free-living bacteria. Can J Microbiol 41 (2):109–117 Glick BR (2005) Modulation of plant ethylene levels by the bacterial enzyme ACC deaminase. FEMS Microbiol Lett 251(1):1–7 Glick BR, Penrose DM et al (1998) A model for the lowering of plant ethylene concentrations by plant growth-promoting bacteria. J Theor Biol 190(1):63–68 Gond SK, Bergen MS, Torres MS, White Jr JF (2015) Endophytic bacillus spp. produce antifungal lipopeptides and induce host defence gene expression in maize. Microbiol Res 172:79–87 Gyaneshwar P, James EK et al (2001) Endophytic colonization of rice by a diazotrophic strain of Serratia marcescens. J Bacteriol 183:2634–2645 Hallmann J, Quadt-Hallmann A et al (1997) Bacterial endophytes in agricultural crops. Can J Microbiol 43(10):895–914

11

Functional Roles of Seed-Inhabiting Endophytes of Rice

233

Hardoim PR, van Overbeek LS et al (2008) Properties of bacterial endophytes and their proposed role in plant growth. Trends Microbiol 16(10):463–471 Hardoim PR, Nazir R et al (2013) The new species Enterobacter oryziphilus sp. nov. and Enterobacter oryzendophyticus sp. nov. are key inhabitants of the endosphere of rice. BMC Microbiol 13(1):164 Hodgson S, Cates C et al (2014) Vertical transmission of fungal endophytes is widespread in forbs. Ecol Evol 4(8):1199–1208 Holguin G, Patten CL (1999) Biochemical and genetic mechanisms used by plant growth promoting bacteria. World Scientific, London Hollis J (1949) Location of bacteria in healthy potato tissue. Phytopathology 39(1):9–10 Honma M, Shimomura T (1978) Metabolism of 1-aminocyclopropane-1-carboxylic acid. Agric Biol Chem 42(10):1825–1831 Hornok L (2000) Genetically modified microorganisms in biological control. Növényvédelem 36 (5):229–237 Hoysted GA, Kowal J et al (2018) A mycorrhizal revolution. Curr Opin Plant Biol 44:1–6 Hurek T, Reinhold-Hurek B et al (1994) Root colonization and systemic spreading of Azoarcus sp. strain BH72 in grasses. J Bacteriol 176(7):1913–1923 Irizarry I, White JF (2017) Application of bacteria from non-cultivated plants to promote growth, alter root architecture and alleviate salt stress of cotton. J Appl Microbiol 122(4):1110–1120 Ji SH, Gururani MA et al (2014) Isolation and characterization of plant growth promoting endophytic diazotrophic bacteria from Korean rice cultivars. Microbiol Res 169(1):83–98 Johnston-Monje D, Raizada MN (2011) Conservation and diversity of seed associated endophytes in Zea across boundaries of evolution, ethnography and ecology. PLoS One 6(6):e20396 Johnston-Monje D, Lundberg DS et al (2016) Bacterial populations in juvenile maize rhizospheres originate from both seed and soil. Plant Soil 405(1–2):337–355 Kaga H, Mano H, Tanaka F, Watanabe A, Kaneko S, Morisaki H (2009) Rice seeds as sources of endophytic bacteria. Microbes Environ 24(2):154–162 Kandel SL, Joubert PM et al (2017) Bacterial endophyte colonization and distribution within plants. Microorganisms 5(4):77 Kim J, Kim JG et al (2004) Quorum sensing and the LysR-type transcriptional activator ToxR regulate toxoflavin biosynthesis and transport in Burkholderia glumae. Mol Microbiol 54 (4):921–934 Kong CH, Wang P et al (2008) Impact of allelochemical exuded from allelopathic rice on soil microbial community. Soil Biol Biochem 40(7):1862–1869 Krishnan P, Bhat R et al (2012) Isolation and functional characterization of bacterial endophytes from Carica papaya fruits. J Appl Microbiol 113(2):308–317 Kuldau G, Bacon C (2008) Clavicipitaceous endophytes: their ability to enhance resistance of grasses to multiple stresses. Biol Control 46(1):57–71 Lareen A, Burton F, Schäfer P (2016) Plant root-microbe communication in shaping root microbiomes. Plant Mol Biol 90:575–587 Larran S, Simon MR et al (2016) Endophytes from wheat as biocontrol agents against tan spot disease. Biol Control 92:17–23 Li HY, Wei DQ et al (2012) Endophytes and their role in phytoremediation. Fungal Divers 54 (1):11–18 Liu Y, Zuo S et al (2012) Study on diversity of endophytic bacterial communities in seeds of hybrid maize and their parental lines. Arch Microbiol 194(12):1001–1012 Liu Y, Zuo S et al (2013) Investigation on diversity and population succession dynamics of endophytic bacteria from seeds of maize (Zea mays L., Nongda108) at different growth stages. Ann Microbiol 63(1):71–79 Loaces I, Ferrando L et al (2011) Dynamics, diversity and function of endophytic siderophoreproducing bacteria in rice. Microb Ecol 61(3):606–618 Lodewyckx C, Vangronsveld J et al (2002) Endophytic bacteria and their potential applications. Crit Rev Plant Sci 21(6):583–606

234

G. Pal et al.

Loganathan P, Nair S (2003) Crop-specific endophytic colonization by a novel, salt-tolerant, N2-fixing and phosphate-solubilizing Gluconacetobacter sp. from wild rice. Biotechnol Lett 25(6):497–501 Lopez DC (2015) Ecological roles of two entomopathogenic endophytes: Beauveria bassiana and Purpureocillium lilacinum in cultivated cotton. Texas A&M University Lucas JA, García-Villaraco A et al (2013) Structural and functional study in the rhizosphere of Oryza sativa L. plants growing under biotic and abiotic stress. J Appl Microbiol 115(1):218–235 Luo S, Xu T et al (2012) Endophyte-assisted promotion of biomass production and metal-uptake of energy crop sweet sorghum by plant-growth-promoting endophyte Bacillus sp. SLS18. Appl Microbiol Biotechnol 93(4):1745–1753 Ma Y, Prasad MNV et al (2011) Plant growth promoting rhizobacteria and endophytes accelerate phytoremediation of metalliferous soils. Biotechnol Adv 29(2):248–258 Mano H, Morisaki H (2008) Endophytic bacteria in the rice plant. Microbes Environ 23:109–117 Mathan S (2016) Isolation of endophytic fungi from marine algae and its bioactivity. Int J Res Pharm Biomed Sci 4(1):45–49 Menn FM, Easter JP et al (2008) Genetically engineered microorganisms and bioremediation, 2nd ed. Biotechnology Set, pp 441–463 Miller MB, Bassler BL (2001) Quorum sensing in bacteria. Annu Rev Microbiol 55(1):165–199 Mitter B, Petric A et al (2013) Comparative genome analysis of Burkholderia phytofirmans PsJN reveals a wide spectrum of endophytic lifestyles based on interaction strategies with host plants. Front Plant Sci 4:120 Mukhopadhyay K, Garrison NK et al (1996) Identification and characterization of bacterial endophytes of rice. Mycopathologia 134(3):151–159 Murphy BR, Doohan FM et al (2015) Fungal root endophytes of a wild barley species increase yield in a nutrient-stressed barley cultivar. Symbiosis 65(1):1–7 Nandakumar R, Babu S et al (2001) Induction of systemic resistance in rice against sheath blight disease by Pseudomonas fluorescens. Soil Biol Biochem 33(4–5):603–612 Narula S, Anand RC et al (2013) Beneficial traits of endophytic bacteria from field pea nodules and plant growth promotion of field pea. J Food Legumes 26(3 and 4):73–79 Newman LA, Reynolds CM (2005) Bacteria and phytoremediation: new uses for endophytic bacteria in plants. Trends Biotechnol 23(1):6–8 Okunishi S, Sako K et al (2005) Bacterial flora of endophytes in the maturing seed of cultivated rice (Oryza sativa). Microbes Environ 20(3):168–177 Palumbo JD, Yuen GY et al (2005) Mutagenesis of β-1,3-glucanase genes in Lysobacter enzymogenes strain C3 results in reduced biological control activity toward Bipolaris leaf spot of tall fescue and Pythium damping-off of sugar beet. Phytopathology 95(6):701–707 Paungfoo-Lonhienne C, Rentsch D et al (2010) Turning the table: plants consume microbes as a source of nutrients. PLoS One 5:e11915 Perotti R (1926) On the limits of biological enquiry in soil science. Proc Int Soc Soil Sci 2:146–161 Pirttilä AM, Laukkanen H et al (2000) Detection of intracellular bacteria in the buds of Scotch pine (Pinus sylvestris L.) by in situ hybridization. Appl Environ Microbiol 66(7):3073–3077 Pirttilä AM, Pospiech H et al (2003) Two endophytic fungi in different tissues of Scots pine buds (Pinus sylvestris L.). Microb Ecol 45(1):53–62 Pleban S, Chernin L et al (1997) Chitinolytic activity of an endophytic strain of Bacillus cereus. Lett Appl Microbiol 25(4):284–288 Prieto KR, Echaide-Aquino F et al (2017) Endophytic bacteria and rare earth elements; promising candidates for nutrient use efficiency in plants. In: Plant macronutrient use efficiency: molecular and genomic perspectives in crop plants. Elsevier, pp 285–306 Reshma P, Naik MK et al (2018) Induced systemic resistance by 2,4-diacetylphloroglucinol positive fluorescent Pseudomonas strains against rice sheath blight. Indian J Exp Biol 56 (3):207–212 Ringelberg D, Foley K et al (2012) Bacterial endophyte communities of two wheatgrass varieties following propagation in different growing media. Can J Microbiol 58(1):67–80

11

Functional Roles of Seed-Inhabiting Endophytes of Rice

235

Rojas GJ et al (2016) Infection with a shoot-specific fungal endophyte (Epichloë) alters tall fescue soil microbial communities. Microb Ecol 72(1):197–206 Rosenblueth M, Martínez-Romero E (2006) Bacterial endophytes and their interactions with hosts. Mol Plant-Microbe Interact 19(8):827–837 Ruiza D, Agaras B et al (2011) Characterization and screening of plant probiotic traits of bacteria isolated from rice seeds cultivated in Argentina. J Microbiol 49(6):902–912 Rutherford ST, Bassler BL (2012) Bacterial quorum sensing: its role in virulence and possibilities for its control. Cold Spring Harb Perspect Med 2(11):a012427 Salisbury FB (1994) The role of plant hormones. Plant-environment interactions. Dekker, New York, pp 39–81 Saraf M, Pandya U et al (2014) Role of allelochemicals in plant growth promoting rhizobacteria for biocontrol of phytopathogens. Microbiol Res 169(1):18–29 Schulz B, Römmert AK et al (1999) The endophyte-host interaction: a balanced antagonism? Mycol Res 103(10):1275–1283 Shahzad R, Waqas M et al (2016) Seed-borne endophytic Bacillus amyloliquefaciens RWL-1 produces gibberellins and regulates endogenous phytohormones of Oryza sativa. Plant Physiol Biochem 106:236–243 Shearin ZR, Filipek M et al (2018) Fungal endophytes from seeds of invasive, non-native Phragmites australis and their potential role in germination and seedling growth. Plant Soil 422(1–2):183–194 Shehata HR, Ettinger CL et al (2016) Genes required for the anti-fungal activity of a bacterial endophyte isolated from a corn landrace grown continuously by subsistence farmers since 1000 BC. Front Microbiol 7:1548 Shields MS, Reagin MJ et al (1995) TOM, a new aromatic degradative plasmid from Burkholderia (Pseudomonas) cepacia G4. Appl Environ Microbiol 61(4):1352–1356 Siciliano SD, Fortin N et al (2001) Selection of specific endophytic bacterial genotypes by plants in response to soil contamination. Appl Environ Microbiol 67(6):2469–2475 Stevenson FJ, Cole MA (1999) Cycles of soils: carbon, nitrogen, phosphorus, sulfur, micronutrients. Wiley, New York Sun Y, O’Riordan MX (2013) Regulation of bacterial pathogenesis by intestinal short-chain fatty acids. Adv Appl Microbiol 85:93–118 Sziderics AH, Rasche F et al (2007) Bacterial endophytes contribute to abiotic stress adaptation in pepper plants (Capsicum annuum L.). Can J Microbiol 53(11):1195–1202 Tholozan JL, Cappelier JM et al (1999) Physiological characterization of viable-but-nonculturable Campylobacter jejuni cells. Appl Environ Microbiol 65(3):1110–1116 Tramontano WA, Scanlon C (1996) Cell cycle inhibition by butyrate in legume root meristems. Phytochemistry 41:85–88 Truyens S, Weyens N et al (2015) Bacterial seed endophytes: genera, vertical transmission and interaction with plants. Environ Microbiol 7(1):40–50 Tsurumaru H, Okubo T et al (2015) Metagenomic analysis of the bacterial community associated with the taproot of sugar beet. Microbes Environ 30(1):63–69 Vallad GE, Goodman RM (2004) Systemic acquired resistance and induced systemic resistance in conventional agriculture. J Trop Crop Sci 44(6):1920–1934 Van Loon LC, Bakker PAHM et al (1998) Systemic resistance induced by rhizosphere bacteria. Annu Rev Phytopathol 36(1):453–483 Verma SC, Ladha JK et al (2001) Evaluation of plant growth promoting and colonization ability of endophytic diazotrophs from deep water rice. J Biotechnol 91(2–3):127–141 Verma SK, Gond SK et al (2017a) Fungal endophytes representing diverse habitats and their role in plant protection. In: Developments in fungal biology and applied mycology. Springer, Singapore, pp 135–157 Verma SK, Kingsley K et al (2017b) Seed vectored endophytic bacteria modulate development of rice seedlings. J Appl Microbiol 122(6):1680–1691 Verma SK, White JF (2018) Indigenous endophytic seed bacteria promote seedling development and defend against fungal disease in browntop millet (L.). J Appl Microbiol 124(3):764–778

236

G. Pal et al.

Verma SK, Kingsley K et al (2018) Bacterial endophytes from rice cut grass (Leersia oryzoides L.) increase growth, promote root gravitropic response, stimulate root hair formation, and protect rice seedlings from disease. Plant Soil 422(1–2):223–238 Vessey JK (2003) Plant growth promoting rhizobacteria as biofertilizers. Plant Soil 255(2):571–586 Vujanovic V, Germida JJ (2017) Seed endosymbiosis: a vital relationship in providing prenatal care to plants. Can J Plant Sci 97(6):972–981 Walitang DI, Kim K et al (2017) Characterizing endophytic competence and plant growth promotion of bacterial endophytes inhabiting the seed endosphere of Rice. BMC Microbiol 17(1):209 White JF (2017) Syntrophic imbalance and the etiology of bacterial endoparasitism diseases. Med Hypotheses 107:14–15 White JC, Wang X et al (2003) Subspecies-level variation in the phytoextraction of weathered p, p0 -DDE by Cucurbita pepo. Environ Sci Technol 37(19):4368–4373 White JF, Chen Q et al (2015) Collaboration between grass seedlings and rhizobacteria to scavenge organic nitrogen in soils. AoB Plants 7 White J F, Kingsley K L et al (2018) Reactive oxygen defense against cellular endoparasites and the origin of eukaryotes. Transformative paleobotany: papers to commemorate the life and legacy of Thomas N. Taylor. Elsevier, Amsterdam White JF, Torres MS et al (2019) Evidence for widespread microbivory of endophytic bacteria in roots of vascular plants through oxidative degradation in root cell periplasmic spaces. In: PGPR amelioration in sustainable agriculture: food security and environmental management. Elsevier, London Wijesooriya WADK, Deshappriya N (2016) An inoculum of endophytic fungi for improved growth of a traditional rice variety in Sri Lanka. Trop Plant Res 3(3):470–480 Williams P, Cámara M (2009) Quorum sensing and environmental adaptation in Pseudomonas aeruginosa: a tale of regulatory networks and multifunctional signal molecules. Curr Opin Microbiol 12(2):182–191 Wilson D (1995) Endophyte: the evolution of a term, and clarification of its use and definition. Oikos 73:274–276 Yan Z, Reddy MS et al (2002) Induced systemic protection against tomato late blight elicited by plant growth-promoting rhizobacteria. Phytopathology 92(12):1329–1333 Yumlembam RA, Borkar SG (2014) Assessment of antibacterial properties of medicinal plants having bacterial leaf endophytes against plant pathogenic Xanthomonads. Indian Phytopathol 67(4):353–357 Zhang T, Yao YF (2015) Endophytic fungal communities associated with vascular plants in the high arctic zone are highly diverse and host-plant specific. PLoS One 10(6):e0130051 Zúñiga A, Poupin MJ et al (2013) Quorum sensing and indole-3-acetic acid degradation play a role in colonization and plant growth promotion of Arabidopsis thaliana by Burkholderia phytofirmans PsJN. Mol Plant-Microbe Interact 26(5):546–553

Mechanism of Interaction of Endophytic Microbes with Plants

12

Neethu Sahadevan, E. K. Radhakrishnan, and Jyothis Mathew

Contents 12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2 Spermosphere . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2.1 Zone of Spermosphere . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2.2 Seed Exudates and the Time Duration of Its Release . . . . . . . . . . . . . . . . . . . . . . . . . 12.2.3 Exudate Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3 Microbiology of the Spermosphere . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3.1 Dynamics of Seed-Associated Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.4 Mechanism of Interaction of Seed Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.4.1 Plant Growth Promotion by Phytohormone Synthesis . . . . . . . . . . . . . . . . . . . . . . . . 12.4.2 Phosphorus Solubilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.4.3 ACC Deaminase Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.4.4 Antagonistic Properties by the Production of Lytic Enzymes . . . . . . . . . . . . . . . . 12.5 Role of Seed Endophytes in Plant Growth Promotion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.6 Seed Endophytes as Biocontrol Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.7 Characterization of Seed Microbial Communities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.8 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

238 239 239 240 241 242 243 245 246 246 247 248 248 249 250 252 252

Abstract

Microbial association with seeds as endophyte may play significant role in its germination and seedling establishment. However, the microbial communities associated with seeds and its diversity are still unexplored due to the difficulties in the characterization of spermosphere and its temporary duration and also due to the complexity of interactions involved. The rapidly changing spermosphere is a microbiologically rich area where interactions between seed and associated microbial communities take place. Recent technological advances are giving us significant promises to study the characteristics of seed exudates, microbial communities, and their mechanisms of interactions with seed and soil. In this N. Sahadevan · E. K. Radhakrishnan · J. Mathew (*) School of BioSciences, Mahatma Gandhi University, Kottayam, Kerala, India # Springer Nature Switzerland AG 2019 S. K. Verma, J. F. White, Jr (eds.), Seed Endophytes, https://doi.org/10.1007/978-3-030-10504-4_12

237

238

N. Sahadevan et al.

chapter, we focused on the current understanding concerning the spermosphere and endophytic microbes that have been isolated from seeds of different plant species. Endophytic colonization route and mode of transmission to the seeds and mechanisms of interaction are also considered under the purview of this chapter. In addition, examples of recent techniques used for the characterization of seed microbiome are also given. Keywords

Spermosphere · Microbial community · Vertical transmission · Mechanisms · Characterization

12.1

Introduction

Seed forms an exceptional stage of plant development as it can persist for decades in a state of dormancy with the potential to arose rapidly and develop into a new plant under suitable conditions (Nelson 2004). Hence, seed germination is an important step in plant growth and development. The seed germination process starts with imbibition, the uptake of water, and is completed when the radicle tip is visible. Imbibition causes swelling of the seed as the cellular components get rehydrated. The swelling mechanically ruptures the seed coat and facilitates the radicle to come out in the form of primary root. Imbibition of water leads to enhanced respiration to restore the metabolic activities of the seeds. Initially, their respiration may be anaerobic, but soon it becomes aerobic with the entry of oxygen into the seeds. The third phase is characterized by a resumption of water uptake, endosperm rupture, and elongation of the embryo axes resulting in radicle protrusion and development of seedlings which marks the end of germination. During imbibition, the size and shape of seed increase, and a temporary structural disruption occurs leading to the rapid and immediate leakage of low-molecular-weight metabolites to the surrounding medium (Schiltz et al. 2015) which can have a significant role in designing the associated microbial communities (Nelson 2004). Just like plants, the seeds also have associated with a plethora of microorganisms as their biological partner. These associations can have a role in seed dormancy and also in its germination or development in the soil. The germinating seed and associated soil also serve as a rich habitat for diverse microorganisms, and this zone is known as “spermosphere.” The main energy source for seed-associated microorganisms is the carbon released by germinating seeds in the spermosphere. The associations between seeds and microorganisms involve specific interactions, as in the case of nitrogen fixation by root-associated organisms, or may be nonspecific and casual. These associations further determine their contact with plants, soil microorganisms, and pathogens as beneficial or harmful. With the understanding on plant microbiome, microorganisms closely associated with plants can be considered to have a significant influence on host metabolic processes and growth (BacilioJiménez et al. 2001).

12

Mechanism of Interaction of Endophytic Microbes with Plants

239

Microbial association with plants as endophyte has been reported from diverse plants and tissues. But the information on the same is minimal from seeds. Endophytic microbial communities are mainly bacteria and fungi, which asymptomatically inhabit the plant without causing any visible harm. Baker and Smith (1966) were the first to suggest the role of seeds as a carrier of promising endophytes. Spermosphere is crucial for the future development of rhizosphere also. Some of the associated microorganisms can have long-lasting effect on plant growth and development. Hence these plant growth-promoting bacteria can have commercial promises as a green alternative to chemical fertilizers. Microbial associations have been investigated in detail from several plants like wheat, maize, oat, peas, barley, canola, potatoes, soy, tomatoes, lentils, and cucumber. However, only very few studies have focused on the interaction between seeds and its associated microorganisms (Avis et al. 2008; Babalola 2010; Bhattacharyya and Jha 2012). Many of the seed-associated microorganisms can expect to have the array of mechanisms reported for other plant-associated bacteria. These involve nitrogen fixation, production of siderophores, solubilization of nutrients such as phosphorus and iron, production of phytohormones such as IAA and cytokinins, and ACC deaminase production for the suppression of ethylene (Sabu et al. 2017b). In addition, they can also have inhibitory effect to phytopathogenic microorganisms through the competition for space and nutrients, by the production of hydrolytic enzymes and antibiosis and induction of plant defense mechanisms (Ahemad and Kibret 2014). Hence the objective of this chapter is to examine the present knowledge about the environment in the immediate proximity to the germinating seeds, the spermosphere, its microbial dynamics, possible colonization routes of the endophytes, mechanism of interactions, seed endophyte functions, and characterization of seed microbial communities.

12.2

Spermosphere

The spermosphere can be defined as the region of soil directly under the influence of seeds and is an important interface between plant and microorganisms. Spermosphere has only a temporary existence during the germination. The soil environment surrounding the seeds after the emergence of radicle is defined as rhizosphere (Schiltz et al. 2015).

12.2.1 Zone of Spermosphere The heterogeneous environment in the soil contributed by several biotic and abiotic factors may impose several stress effects on microorganisms. These can determine the physiology, distribution, and survival of microbial communities. The diverse bioactive compounds of seed origin which are being released into the soil can also affect the physical and chemical properties of spermosphere and thereby influence the soil microorganisms. The density of bacteria is considered to be higher toward

240

N. Sahadevan et al.

Fig. 12.1 Diagrammatic representation of the spermosphere showing microbial seed interaction. During germination, seed passively exudes a variety of compounds (double arrow) into the environment. The composition and quality of these exudates are highly variable and contribute to creating specific microenvironments in the vicinity of seed. Specifically adapted microorganisms (filled rectangle) colonize this zone and set up specific interactions with seeds

proximity to the future radicle emerging sites. At the same time, seed-associated microorganisms also release diverse diffusible compounds into the soil which spread radially at millimeter distance from the plant (Schiltz et al. 2015) (Fig. 12.1). Many previous studies showed the area of spermosphere to be variable, depending upon the type of seeds, microorganisms, and the environmental conditions. Higher soil moisture has been suggested to improve the diffusion of seed exudates through the soil and thereby extend the area of spermosphere (Short and Lacy 1974). The zone of seed influence is not uniformly distributed in the soil around the seeds, and it varies according to moisture, soil texture, and temperature (Roberts et al. 2009; Short 1976; Short and Lacy 1974).

12.2.2 Seed Exudates and the Time Duration of Its Release The compounds released by the seeds are not continuously released following imbibition. Previous studies revealed three distinct phases of seed exudation; the

12

Mechanism of Interaction of Endophytic Microbes with Plants

241

first phase is associated with the initiation of seed imbibitions followed by a second phase a few hours later (Simon and Harun 1972). The first two phases occur as a result of rupture of seed membranes due to rapid water uptake in the first phase of seed germination. When the exudation ceases, the integrity of the membrane is restored. The third and last phase of exudation happens when the radicle emerges through the seed coat at the end of germination. Corn and cucumber seeds have been demonstrated to release significant levels of fatty acids and sugars within 15 min after the initiation of imbibition, but there was no exudation peak within 6 h after imbibition (Windstam and Nelson 2008). Here the temperature was also found to affect the pattern of exudation. The majority of carbohydrates were exuded during the first 18 h of incubation at 22 or 30  C and persisted for about 48 h at 10  C. The nutrient distribution in the spermosphere may also vary over time due to the growth and maturation of root and associated microbial communities. Thus, the spermosphere is a temporally heterogeneous zone on both micro- and macroscales in terms of individual nutrients and their availability and microbial community (Roberts and Kobayashi 2011).

12.2.3 Exudate Composition When seeds germinate, a variety of compounds are passively released into the spermosphere. The composition of these exudates varies with plant species and with factors like soil type, moisture, available nutrients in the soil, temperature, and the presence of microorganisms. Little research has been done on the spermosphere when compared with the analogous rhizosphere around roots. Exudates released from the germinating seeds are usually the by-products of seed metabolism, and they generally consist of carbohydrates, amino acids, sterols, flavonoids, and salts (Vancura and Hanzlikova 1972; Rovira 1969). Volatile compounds such as alcohols, aldehydes, ethylene, CO2, and volatile carboxylic acids are also released from germinating seeds (Schiltz et al. 2015). Moreover, sugars like sucrose, fructose, glucose, and maltose are also released from the germinating seeds, but the ratio of these sugars released greatly depends upon the developmental phase of seeds (Lugtenberg et al. 1999). The molecules released during the germination can have an effect on the immediate biotic and abiotic environment. Organic acids exuded into the spermosphere change the surrounding soil pH and enhance the solubility of inorganic phosphorus compounds. Furthermore, these organic acids can also make metal-chelating complexes which can increase iron solubility in iron-limiting conditions and reduce aluminum toxicity (Jones 1998). It has also been known that other micronutrients like zinc and copper may also be solubilized by the organic acid. Seed exudates may attract the soil microbes and determine their ability to adhere and grow competitively in the seed vicinity. Interestingly, seed exudates released from soybeans were found to induce the chemotaxis, cell division, and biofilm formation of Bacillus amyloliquefaciens BNM 339, but the same was not observed with root exudates. Here differences between root and seed exudates were demonstrated to induce differential responses in bacteria (Yaryura et al. 2008).

242

12.3

N. Sahadevan et al.

Microbiology of the Spermosphere

The ecological diversity of seed-colonizing microorganisms in the spermosphere, especially those colonizing the seeds immediately following sowing, has rarely been studied. Moreover, the concept of seeds as an important source of endophytic microorganisms has been called controversial until the recent past. But now, numerous studies have explained that seeds harbor diverse microbial communities not only within the embryo but also on their surfaces. The microbial communities that colonize the seeds during the early stage of germination were found to be soil microorganisms. This was explained in a study of microbial colonization of seeds germinating in Pythium suppressive and nonsuppressive leaf composites (Mckellar and Nelson 2003). The main factor which certainly affects the quantitative levels of indigenous bacterial populations that colonize the spermosphere and endophytic association with seeds is the genotype of seeds. Seeds may also select specific endophytic communities since the microbe which proliferates in the spermosphere appears to be different from rhizosphere colonizers. The nature and properties of germinating seed-associated microbial communities can have a direct impact on plant growth promotion, nitrogen fixation, and biological disease control (Sabu et al. 2017a). Moreover, indigenous microbial communities associated with seeds can have a long-term effect on plant health and seedling establishment. However, indigenous microbial communities associated with spermosphere are still poorly unexplored (Nelson 2004). The origin of microbial communities in the spermosphere can be from many sources. The seed could acquire microbiome prior to the extension of primary root and is facilitated by a passive encounter between the microorganisms conveyed by the soil and the germinating seed (Buyer et al. 1999; Green et al. 2006; Ofek et al. 2011). Here, the composition of microbial communities associated with the seed is exclusively influenced on the medium. According to Buyer et al. (1999), even the seeds of different plant species with varying compositions of seed exudates were found to share highly similar spermosphere microbial communities. At the same time, spermosphere-colonizing bacteria can also be host dependent. Here, the microbial metabolic activities are based on the composition of host exudates released during germination (Roberts et al. 2009), and hence microbial communities of the spermosphere in these cases are entirely dependent upon the seeds and their exudates. Kageyama and Nelson (2003) showed the germination of sporangia of Pythium sporangium to be higher in the presence of plants such as corn, carrot, lettuce, pea, radish, and wheat than in the presence of cucumber, cotton, tomato, and sunflower seed exudates. This was also in relation to the findings of Simon et al. (2001), where the population densities of microbial communities associated with tomato seeds were observed to be influenced by the genotype of the seeds. Furthermore, the findings of Liu et al. (2012) demonstrated significant differences in the number and species of endophytic bacteria among the seeds of four offspring hybrid maize when compared with their parental lines. Bacteria and fungi are the main microbial communities associated with the seeds. The colonization of microorganisms in the seed occurs within a few hours after

12

Mechanism of Interaction of Endophytic Microbes with Plants

243

sowing. The microbial strains are mainly from the initial potting mix or populations present in and on the seeds (Buyer et al. 1999; Green et al. 2006; Nelson 2004; Ofek et al. 2011). The evolution of microbial communities in the spermosphere indicates specific design of microbial strains during seed to root conversion because microorganisms in the spermosphere appear to differ from those present in the rhizosphere (Singh et al. 2009). Microbial strains on or adjacent to seeds constitute the first inoculum of the spermosphere. Then, the compounds released by the germinating seeds and the pedoclimatic conditions determine the structure of microbiome in the spermosphere which can be similar or different from the initial microbial communities. Apart from plant pathogenic species, the identities of seed-colonizing microbial species have generally not been determined. Pythium and Fusarium species were reported to be the most frequently occurred spermosphere fungi recovered from turnip seeds germinated for 72 h in the soil. High-frequency occurrence of the oomycetes Thraustotheca and Achlya was also reported. Rhizoctonia solani, Penicillium species, Gliocladium, Trichoderma, Cylindrocarpon, Cephalosporium, Cunninghamella, Helicocephalum, and Mucor were also recovered at low frequency. These were also reported from the spermosphere around the onion, tomato, cabbage, mustard, bean, and melon. Among the bacteria, species of Acinetobacter, Bacillus, Pantoea, Burkholderia, and Pseudomonas were reported to colonize barley seeds during the early stage of germination, whereas cottonseeds were identified to be colonized by species of Enterobacter, Xanthomonas, Microbacterium, Paracoccus, Micrococcus, Curtobacterium, Paenibacillus, and Agrobacterium.

12.3.1 Dynamics of Seed-Associated Endophytes 12.3.1.1 Colonization of Seeds by Endophytes Majority of the microbial communities that colonize the seeds as endophytes are common soil microbial strains (Jasim et al. 2018). Plant roots are exposed to soil microbial reservoir during its growth and development, which facilitate the entry of selected microbial strains into the plants for further consideration as endophyte in the seed. Several bacterial traits play an important role in plant colonization and endophytic association (Chithra et al. 2017). The most important mechanism of soil bacteria which is considered to determine its endophytic potential is the chemotaxis-induced motility (Bacilio-jim et al. 2003). Because of the absence of flagella-driven chemotaxis, several pilA or cheA mutants of Pseudomonas strains showed reduced competitive potential for root colonization. Subsequent to colonization further steps involve penetration at root tip or active penetration using cell wall degrading pectinase and cellulase. The major determinants of plant-endophyte interactions have been suggested to be the bacterial protein secretion system for the uptake of plant-synthesized nutrients, transcriptional regulators for metabolic adaptation and quorum sensing, secretion and delivery systems involved in the switching of free-living microbe to an endophytic lifestyle, and detoxification mechanisms to manage oxidative stress within the host (Sessitsch et al. 2012; Ali et al. 2014). Also,

244

N. Sahadevan et al.

endophytic microorganisms seem to be better protected from abiotic stresses when compared with rhizosphere strains. Once inside the plant, some endophytes are able to spread systemically and finally reach the fruits, flowers, and seeds. Further, some endophytes use the root xylem vessels for flagella-mediated movement (James et al. 2002), and others use nutrient-rich intracellular spaces facilitated by cell wall degrading enzymes. Microorganisms associated with the anthosphere, phyllosphere, caulosphere, and carposphere can also colonize the plant reproductive organs (James et al. 2002) and thereby form endophyte of seeds.

12.3.1.2 Vertical Transmission Soil microorganisms can reach the seeds in different ways. Microorganisms can get transmitted from different parts of plants to the seed endosperm through vascular connections from the maternal plant via the funiculus and chalaza and also through micropyle. Puente et al. (2009) described the transfer of bacteria from mature fruits to seeds and explained the bacterial presence in the exocarp, mesocarp, and endocarp of the embryonic site of cactus seeds. Moreover, endophytes conveyed through gametes can colonize the resulting embryo and endosperm. Vertical transmission through shoot meristems has also been suggested to result in their presence in ovules and seeds (Pirttila et al. 2000). Another possible route is through the shoot apical meristem, which consists of undifferentiated cells that give rise to postembryonic aerial organs. Tissues developing from this to seeds may contain bacteria residing in the meristem. This route ensures transfer of endophytes from mother plant to the seedling. Inside the seeds, most microbial strains are associated with seed husk, seed cortex, and seed coat (Puente et al. 2009), while some endophytes have also been reported from endosperm and embryonic tissues (Cankar et al. 2005). Vertical transmission of endophytic microbes has also been reported in several plant species. Ringelberg et al. (2012) isolated the same genera of endophytes from wheat grass seeds and also from mature plant tissues and suggested seeds to be the major contributor of endophytes for the mature plant. Mukhopadhyay et al. (1996) found the same Enterobacter species in seeds of consecutive rice generations. Johnston-Monje and Raizada (2011) demonstrated the maize to have a core microbiota as the same species conserved across boundaries of evolution and human selection, following the cross-continental migration. Then, Liu et al. (2012) observed endophytic bacterial transmission from parent to offspring in maize seeds, and even the seed endophytic population of genetically similar maize hybrids possessed the same pattern of endophytic microbiota. Studies of Croes et al. (2013) explained the endophytic communities of plants in the lead-contaminated and non-contaminated field and revealed a correlation between endophytic communities of roots. Here the plants on the two fields contained similar obligate endophytes possibly derived from a common seed endophytic community. As suggested by Truyens et al. (2013), the presence of identical 16S rRNA gene sequences among seeds from different genotypes, between seed and seedlings, and between seeds of consecutive generations cannot corroborate vertical transmission until strain-level information is available. As vertical transmission allows a plant with an established

12

Mechanism of Interaction of Endophytic Microbes with Plants

245

endophytic community to pass the bacteria to their offspring, the mechanism involved in the same needs detailed study (Ferreira et al. 2008). Bacteria have been identified both inside and on the surface of pollen of different plant species, and hence endophytic entry through male gametes is also possible. As pollen grains are exposed to the environment, it could be colonized by bacteria from the atmosphere or via pollinators or by other animals. Vertical transmission of microbial communities to the seed could be achieved if the bacteria present in the pollen originate from within the plant. Reports of Madmony (2005) described the isolation of endophytic bacteria Enterobacter cloacae from the surface sterilized pollens of the Turkish pine (Pinus brutia), Mediterranean pines Aleppo pine (Pinus halepensis), and stone pine (Pinus pinea). The authors also isolated the same bacterial species from fertilized P. brutia ovules which indicate the vertical transmission of Enterobacter species in pines via pollens. Truyens et al. (2013) also described the isolation of endophytic bacteria associated with two wind-pollinated and two insect-pollinated species of plant Betula pendula (birch), Secale cereale (rye), Brassica napus (rape), and Colchicum autumnale (autumn crocus). The results showed abundant bacterial distribution (106–109 cultivable bacteria per gram of pollen) on the outer surface as single cells, as clusters, or as thin biofilms. Bacterial communities from insect-pollinated species were more similar to each other than to bacterial communities from wind-pollinated species, suggesting an influence of pollinators on pollen bacterial community composition.

12.4

Mechanism of Interaction of Seed Endophytes

Till now, only limited research is carried out to understand the role and potential applications of seed endophytes. The available studies mainly focused on the use of seed endophytes as plant growth-promoting and biocontrol agents. Generally, the useful functions of seed endophytes can be comparable to endophytes isolated from other tissues. The mechanisms used by the soil bacteria to aid plant growth are reasonably well described (Gamalero 2011; Glick and Glick 2012). Plant growth-promoting bacteria affect the plant growth directly by facilitating the acquisition of resources from the environment including phosphorous, nitrogen, and iron and modulating plant growth by providing plant hormones such as auxin, cytokinin, and ethylene (Jishma et al. 2017; Basheer et al. 2018). Indirect growth promotion by them involves prevention of the damage to plants caused by pathogenic fungi, bacteria, and nematodes. This also involved the production of antibiotics, cell wall degrading enzymes, lowering ethylene levels, decreasing the amount of iron available to pathogens, induced systemic resistance, and the synthesis of pathogen-inhibiting volatile compounds (Glick and Glick 2012).

246

N. Sahadevan et al.

12.4.1 Plant Growth Promotion by Phytohormone Synthesis The phytohormones auxins, cytokinins, gibberellins, ethylene, and abscisic acid (ABA) all play key roles in the regulation of plant growth and development. The levels of endogenous phytohormones are often insufficient for optimal growth under diverse environmental conditions. Most endophytic bacteria have got the ability to alter phytohormone levels to interfere plant’s hormonal balance (Jasim et al. 2015a, 2016). Indole acetic acid (IAA) is the most common and well-studied phytohormone of the auxin class. IAA influences plant growth and development and has an impact on plant cell division, extension, and differentiation; increases the rate of xylem and root development; stimulates seed and tuber germination; controls processes of vegetative growth; mediates responses to light, gravity, and fluorescence; initiates lateral and adventitious root formation; and affects photosynthesis, biosynthesis of various metabolites, pigment formation, and resistance to stressful conditions. IAA can be generated in bacteria through different biosynthetic pathways. Idris et al. (2007) showed the ability of gram-positive bacterium Bacillus amyloliquefaciens to produce and secrete significant amount of IAA with the positive effect on the growth of Lemna minor. Patten and Glick (2002) also demonstrated the direct positive effect of IAA produced by Pseudomonas putida through the indole pyruvic acid pathway, on root development. Roots from canola seeds treated with the wild-type P. putida were longer than that of seeds treated with an IAA-deficient mutant. The phytohormone ethylene affects plant growth and development in different ways including promotion of root initiation, leaf abscission, fruit ripening, flower wilting, inhibiting root elongation, stimulating seed germination, activating the synthesis of other plant hormones, and responding to both biotic and abiotic stresses (Jasim et al. 2015b). Ethylene synthesized in response to various stresses may lead to senescence, chlorosis, and abscission which inhibit plant growth and survival. Mayak et al. (2004) showed the ability of bacteria such as Burkholderia caryophylli, Pseudomonas spp., and Achromobacter piechaudii to lower the endogenous ethylene level in plants is by the production of degradative enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase. The effects of ACC deaminase-producing bacteria on plants include increased root growth and improved tolerance to salt and water stress (Mayak et al. 2004).

12.4.2 Phosphorus Solubilization Unlike the case of nitrogen, phosphorus (P) is a major growth-limiting nutrient. Root development, flowering, and seed formation, stalk and stem strength, crop maturity and production, crop quality, N-fixation in legumes, and resistance to plant diseases are the characteristics associated with phosphorus nutrition. Several studies clearly demonstrated the beneficial and positive effects of soil microbes on growth and yield of different crops through P solubilization. In fact, soil microbial community plays a pivotal role in soil phosphorus dynamics and making the phosphate available to plants (Jasim et al. 2014a, b). These microbiomes release organic acids

12

Mechanism of Interaction of Endophytic Microbes with Plants

247

that dissolve the available phosphate minerals in the soil and chelate cationic partners of the phosphorous ions to release phosphate into the solutions. Many studies proved the capability of soil bacteria to transform soil P to the forms available to plant (Jimtha John et al. 2017). These bacteria serve as a sink for P, in the presence of labile carbon, by rapidly immobilizing it even in low P soils. Subsequently, PSB becomes a source of P to plants upon its release from their cell (Khan et al. 2014). The mechanism of phosphate solubilization is solely a microbial process which consists of proton extrusion and organic acid production. Many soil microbes have the mineralization and solubilization potential for organic and inorganic phosphorus in the soil. Phosphorus-solubilizing ability is determined by the release of organic acids, chelation of the cation bound to phosphate through their carboxyl and hydroxyl groups, and then conversion to soluble forms (Gamalero 2011; Glick and Glick 2012; Khan et al. 2014).

12.4.3 ACC Deaminase Production One mechanism by which plant growth-promoting bacteria enhance the plant growth is through the production of ACC deaminase. Ethylene is a gaseous plant hormone involved in both plant development and stress response. It mediates a myriad of plant processes including germination, root elongation, and plant responses to both biotic and abiotic stress. Common stress factors include ultraviolet light, drought, flooding, temperature extremes, pathogens, high salt concentrations, and exposure to chemical contaminants such as metals and organics. Many plant growth-promoting bacteria enhance plant growth by lowering ethylene concentrations through a mechanism that involves the bacterial enzyme 1-aminocyclopropane 1-carboxylic acid (ACC) deaminase (Glick 2014). The proposed role of ACC deaminase in alleviating the ethylene-mediated growth inhibition was described by Glick (2014). ACC deaminase binds to the seed or root surface of a developing plant. Tryptophan and other small molecules in the seed or root exudates can stimulate the synthesis and secretion of bacterial indole acetic acid. Some of this IAA can be taken up by the plant, and in combination with the endogenous plant IAA, it initiates several plant responses including cell proliferation, cell elongation, and synthesis of ACC synthase. This enzyme converts S-adenosylmethionine to ACC, the immediate precursor of ethylene in the biosynthetic pathway. Some of this ACC is exuded by roots or seeds and is subsequently taken up by the plant growth-promoting bacteria. Within the bacterial cell, ACC is converted to ammonia and α-ketobutyrate via the action of ACC deaminase. The lower level of ACC in plants finally leads to the reduction in the amount of ethylene produced, thereby alleviating ethylene-mediated growth inhibition (Jasim et al. 2013, 2014b).

248

N. Sahadevan et al.

12.4.4 Antagonistic Properties by the Production of Lytic Enzymes Diverse microorganisms secrete and excrete other biochemicals that can interfere with pathogen growth or activities. These include enzymes like chitinase, protease, cellulase, lipase, and HCN production (Rohini et al. 2018; Sabu et al. 2018). Chitinases are enzymes which degrade the chitin directly into low-molecular-weight products. Chitinase enzyme has a significant role in pharmaceutical and chemical industries due to their ability to convert chitin containing raw materials. Moreover, chitinase may be applied as insecticides and fungicides to control pests and fungal pathogens of plants, respectively. Hydrogen Cyanide (HCN) Production Many soil bacteria are capable of producing HCN, a dreadful chemical with toxic properties and metabolized to a lesser degree into other compounds. Even if cyanide acts as a general inhibitor of metabolism, it is synthesized, metabolized, and excreted by many plant growth-promoting bacteria. The host plant is usually not affected by the inoculation of cyanide-producing microbial strains. Hence they are widely used for biological control (Jayakumar et al. 2018). HCN first inhibits the electron transport chain and energy supply to the cell leading to the death of organisms. It is known to inhibit the action of enzyme cytochrome oxidase and inhibits proper functioning of other enzymes. Voisard et al. (1989) showed production of HCN by certain strains of fluorescent Pseudomonas as a suppression mechanism against soilborne pathogens. The suppression of black root rot of tobacco and take-all of wheat by P. fluorescens strain CHAO has been considered to be connected to the production of HCN. In another experiment, Weststeijn (1990) showed ability of HCN producing P. fluorescens to inhibit the mycelial growth of Pythium in vitro.

12.5

Role of Seed Endophytes in Plant Growth Promotion

Díaz Herrera et al. (2016) isolated six endophytic bacteria from wheat seeds and studied its plant growth-promoting and biocontrol abilities. The strains from Paenibacillus genus and Pantoea genus produced IAA and siderophores and solubilized phosphate. These isolates also inhibited Fusarium graminearum growth on dual culture and in a bioassay using barley and wheat kernels. Paenibacillus was found to have outstanding ability to form biofilm on inert surface and displayed greater biocontrol of F. graminearum. Inoculating plants with bacteria isolated from seeds has been suggested to increase plant growth, especially in harsh or suboptimal environmental conditions. Johnston-Monje and Raizada (2011) used a potato bioassay to test the plant growthpromoting traits of endophytes, Burkholderia and Hafnia strains, isolated from maize. Puente et al. (2009) showed the seed endophytes isolated from cactus to have an enhancement effect on the growth of cactus seedlings in extreme conditions without any fertilization. These seed endophytes were able to solubilize inorganic nutrients such as phosphorus, from pulverized rock by the release of organic acids

12

Mechanism of Interaction of Endophytic Microbes with Plants

249

and possessed the capacity to fix nitrogen. Gagne-Bourgue et al. (2013) demonstrated enhanced growth of switchgrass seedlings due to inoculation with seed-borne endophytic Bacillus or Microbacterium strains. Both the endophytic strains produced IAA, cytokinins, and volatiles such as butanediol and acetoin, but Bacillus was also able to solubilize phosphorus. Then Xu et al. (2014) showed the property of a seed-borne Bacillus species to enhance the shoot and root growth of tomato plants due to the production of ACC deaminase and nitrogen fixation. Many other studies also assumed the seed endophytes to have beneficial plant growthpromoting traits (López-López et al. 2010; Cankar et al. 2005). Plant growth-promoting properties of endophytic microorganisms associated with seeds in harsh environmental conditions have been exploited in practical applications such as phytoremediation. In the studies of Mastretta et al. (2009), when tobacco was inoculated with its seed endophytes, biomass production was found to increase under cadmium exposure. Moreover, seed endophytes of the grass Agrostis capillaris were found to be capable of producing ACC deaminase, siderophores, IAA, and acetoin and were positive for phosphate solubilization also. These were found to have the promises for phytostabilization and phytoextraction of Agrostis capillaris in Cd-contaminated areas as it showed cadmium tolerance also (Truyens et al. 2013). In case of nonexposed plants, inoculation resulted in a significantly improved plant growth. After inoculation of cadmium-exposed plants, an increased cadmium uptake was seen without affecting the plant growth. Observation by Truyens et al. (2013) showed seed endophytic bacteria isolated from Arabidopsis thaliana, to have the ability to produce IAA, ACC deaminase, siderophores, and organic acids.

12.6

Seed Endophytes as Biocontrol Agents

Certain endophytes isolated from seeds were also found to possess antifungal activities. Bacillus and Microbacterium strains isolated from seeds of switchgrass inhibited the mycelial growth of fungal pathogens by producing several toxins such as surfactants and the lipo-peptides iturin and mycobacillin (Gagne-Bourgue et al. 2013). Among the endophytes isolated from rice seeds, Enterobacter strains showed highest antifungal activities against fungal pathogens Rhizoctonia solani, Pythium myriotylum, Heterobasidium annosum, and Gaeumannomyces graminis. The Enterobacter strains were producers of volatile antifungal compounds, probably ammonia and chitinolytic enzyme N-acetyl-β-D-glucosaminidase (Mukhopadhyay et al. 1996). Cottyn et al. (2001) have also demonstrated antifungal activity among rice seed endophytes Cellulomonas, Bacillus species, Pantoea, Enterobacter, Stenotrophomonas, Xanthomonas, Paenibacillus, and Acinetobacter against Rhizoctonia solani and Pyricularia grisea. In other studies (Ruiza et al. 2011; Sessitsch et al. 2012), several strains of Pantoea, Microbacterium, Paenibacillus, Pseudomonas, and Curtobacterium isolated from rice seeds were found to have antifungal activities against Curvularia species, Fusarium oxysporum, and Pythium ultimum. These rice seed endophytes produced antifungal compounds, quorumsensing molecules, hydrolytic enzymes, and siderophores.

250

12.7

N. Sahadevan et al.

Characterization of Seed Microbial Communities

The spermosphere can also be characterized based on the microbiota present in and around the germinating seeds (Buyer et al. 1999; Green et al. 2006; Ofek et al. 2011). But due to the divergence of microbial populations and their interactions, characterization and functionalization of different microbial communities in the spermosphere are rather complex. To simplify the experimental model to understand the mechanism of these complex interactions, seeds could be sterilized and then inoculated with some specific bacterial strains in a sterilized or controlled inert medium. By the strong influence of genotype, species, and stage of development of seeds and by the pedoclimatic conditions on the structure, form, and working of the spermosphere, it is essential to study the interactions between the seed endophytes in the spermosphere under controlled experimental conditions to have explicable and reproducible results. For example, the influence of host seeds on the metabolic activity of Enterobacter cloacae during the colonization of pea and cucumber seeds was studied in the controlled culture medium. In addition, Simon et al. (2001) tested the influence of host seeds on the growth of Bacillus and Pseudomonas strains by using tomato lines from a recombinant inbred line. Till now, no specific method has been used to study the diversity of microbiota in the spermosphere. Even though the same methods as those used for analyzing the rhizosphere or the bulk soil are used, there is a limitation with short time duration (70% suppression of test pathogens in antagonistic dual culture assays. The endophyte strain Trichoderma harzianum TharDOB-31 showed in vitro mycelia growth inhibition against P. aphanidermatum (76.0%) and R. solani (76.9%) significantly, whereas the antagonistic potential of strains T. harzianum TharDOB-31 is followed by T. asperellum TaspDOB-19 > 70% against P. aphanidermatum and R. solani. Anisha et al. (2018) reported antagonistic property of processed methanolic extract of Rhizopycnis vagum ZM6 and endophytic isolates of ginger against the strains like Colletotrichum falcatum, Fusarium oxysporum, Sclerotium rolfsii, Phytophthora infestans, Corynespora cassiicola, Rhizoctonia solani, and Pythium myriotylum by the method of agar well diffusion and observed significant inhibition of all these pathogenic strains. Endophytic microbes have been recently used as a novel source of bioactive compounds (Singh et al. 2017a) and being broadly used in the nutraceutical or pharmaceutical industries (Strobel and Daisy 2003). The endophytic isolates of herbal plants gained high attention in the metabolite discovery (Theantana et al. 2009). Rhizome is a storehouse for the large number of phenolic compounds, terpenoids,

19

Rhizome Endophytes: Roles and Applications in Sustainable Agriculture

415

and flavonoids, which supports large number of endophytic microorganisms. Most of the chemotherapeutics have antifungal and antibacterial properties (Rohini et al. 2018). In case of rhizome, Krishnapura et al. (2016) isolated 50 endophytes (14 bacteria, 22 actinomycetes, and 14 fungi) from the 5 medicinal plants, Alpinia galanga, Curcuma amada, Curcuma longa, Hedychium coronarium, and Zingiber officinale, of Zingiberaceae family, and 31 out of 50 strains evidenced positive for L-asparaginase production. L-Asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) hydrolyzes amino acid L-asparagine into aspartic acid and ammonia. This enzyme has attained appreciable importance, owing to its use in the treatment of acute lymphoblastic leukemia. Lee et al. (2017) isolated dark septate endophytic fungal strain from the rhizome of Phragmites communis (a halophyte). Halophytes are known to overcome salt stress via associations with their endophytes. After extraction with ethyl acetate, the strain synthesizes two glycosylated dialkylresorcinol and anthraquinone derivative compounds, which showed significant nitric oxide reduction activity in lipopolysaccharide-stimulated microglia BV-2 cells. Another important property of endophytic strain is used as a valuable resource for the bioactive compound production.

19.7

Concluding Remarks

Currently increasing world population and changing climatic conditions are the two major global problems for humans. Both the problems are somewhat associated with each other. An increasing population needs extra food from the present limited resources which creates extra pressure to the farmers. To overcome the problems of foods, farmers use huge amount of chemical fertilizers and pesticides, which adversely affect the climatic conditions, ecology of plant, and soil as well as health of human beings. In this context, like plant growth-promoting bacteria, endophytic bacterial and fungal strains appear as a suitable alternative to chemical fertilizers and pesticides. Endophytic microbes had been isolated from a range of agricultural plants and broadly used as plant and soil inoculants to enhance the plant production or manage the phytopathogens; besides these endophytes also play an essential role in maintaining plant physiology. The diversity of endophytic populations depends upon the host and microbial genotype and biotic and abiotic environmental factors. Bacillus and Pseudomonas are the most common bacterial group identified as frequently occurring in agricultural crops; in most of the plant rhizome, these two groups are also commonly found. Currently many endophytic strains are also used in abiotic stress management, including drought and salinity. In this context, there is need to explore some hidden endophytic strains that may be used as biofertilizers and biocontrols, in biotic or abiotic stress management, and also as sources of novel bioactive compounds. Acknowledgment Authors thank the University Grants Commission and CSIR, New Delhi, for fellowship in the form of JRF and SRF.

416

A. Gupta et al.

References Aggarwal BB, Shishodia S (2004) Suppression of the nuclear factor-kappa B activation pathway by spice-derived phytochemicals: reasoning for seasoning. Ann N Y Acad Sci 1030:434–441 Alibrandi P, Cardinale M, Rahman MM, Strati F, Ciná P, de Viana ML, Giamminola EM, Gallo G, Schnell S, De Filippo C (2018) The seed endosphere of Anadenanthera colubrina is inhabited by a complex microbiota, including Methylobacterium spp. and Staphylococcus spp. with potential plant-growth promoting activities. Plant Soil 422(1–2):81–99 Alkotaini B, Anuar N, Kadhum AAH, Sani AAA (2013) Detection of secreted antimicrobial peptides isolated from cell-free culture supernatant of Paenibacillus alvei AN5. J Ind Microbiol Biotechnol 40(6):571–579 Alström S (2001) Characteristics of bacteria from oilseed rape in relation to their biocontrol activity against Verticillium dahliae. J Phytopathol 149(2):57–64 Amalraj A, Pius A, Gopi S, Gopi S (2017) Biological activities of curcuminoids, other biomolecules from turmeric and their derivatives – a review. J Tradit Complement Med 7(2):205–233 Anandaraj B, Vellaichamy A, Kachman M, Selvamanikandan A, Pegu S, Murugan V (2009) Co-production of two new peptide antibiotics by a bacterial isolate Paenibacillus alvei NP75. Biochem Biophys Res Commun 379(2):179–185 Anisha C, Mathew J, Radhakrishnan E (2013) Plant growth promoting properties of endophytic Klebsiella sp. isolated from Curcuma longa. IJBPAS 2(3):593–601 Anisha C, Jishma P, Bilzamol VS, Radhakrishnan EK (2018) Effect of ginger endophyte Rhizopycnis vagum on rhizome bud formation and protection from phytopathogens. Biocat Agric Biotechnol 14:116–119 Antoun H (2012) Beneficial microorganisms for the sustainable use of phosphates in agriculture. Procedia Eng 46:62–67 Arora D, Kumar S, Singh D, Jindal N, Mahajan NK (2013) Isolation, characterization and antibiogram pattern of Salmonella from poultry in parts of Haryana. India Adv Anim Vet Sci 1(5):161–163 Aswathy AJ, Jasim B, Jyothis M, Radhakrishnan E (2013) Identification of two strains of Paenibacillus sp. as indole 3 acetic acid-producing rhizome-associated endophytic bacteria from Curcuma longa. 3 Biotech 3(3):219–224 Balogh B, Jones J, Iriarte F, Momol M (2010) Phage therapy for plant disease control. Curr Pharm Biotechnol 11(1):48–57 Barik BP, Tayung K, Jagadev PN, Dutta SK (2010) Phylogenetic placement of an endophytic fungus Fusarium oxysporum isolated from Acorus calamus rhizomes with antimicrobial activity. EJBS 2(1):8–16 Benhamou N, Chet I (1996) Parasitism of sclerotia of Sclerotium rolfsii by Trichoderma harzianum: ultrastructural and cytochemical aspects of the interaction. Phytopathology 86 (4):405 Benhamou N, Kloepper JW, Quadt-Hallman A, Tuzun S (1996) Induction of defense-related ultrastructural modifications in pea root tissues inoculated with endophytic bacteria. Plant Physiol 112(3):919–929 Boiero L, Perrig D, Masciarelli O, Penna C, Cassán F, Luna V (2007) Phytohormone production by three strains of Bradyrhizobium japonicum and possible physiological and technological implications. Appl Microbiol Biotechnol 74(4):874–880 Boominathan U, Sivakumaar P (2012) A liquid chromatography method for the determination of curcumin in PGPR inoculated Curcuma longa. L. plant. Int J Pharm Sci Res 3(11):4438 Bulgarelli D, Schlaeppi K, Spaepen S, van Themaat EVL, Schulze-Lefert P (2013) Structure and functions of the bacterial microbiota of plants. Annu Rev Plant Biol 64(1):807–838 Bustanussalam B, Rachman F, Septiana E, Lekatompessy SJR, Widowati T, Sukiman HI, Simanjuntak P (2015) Screening for endophytic fungi from turmeric plant (Curcuma longa L.) of Sukabumi and Cibinong with potency as antioxidant compounds producer. Pak J Biol Sci 18(1):42–45

19

Rhizome Endophytes: Roles and Applications in Sustainable Agriculture

417

Chanway CP (1996) Endophytes: they’re not just fungi! Can J Bot 74(3):321–322 Chen T, Chen Z, Ma GH, Du BH, Shen B, Ding YQ, Xu K (2014) Diversity and potential application of endophytic bacteria in ginger. Genet Mol Res 13(3):4918–4931 Chevrot R, Didelot S, van den Bossche L, Tambadou F, Caradec T, Marchand P, Izquierdo E, Sopéna V, Caillon J, Barthélémy C, van Schepdael A, Hoogmartens J, Rosenfeld E (2013) A novel depsipeptide produced by Paenibacillus alvei 32 isolated from a cystic fibrosis patient. Probiotics Antimicrob Proteins 5(1):18–25 Choudhary DK, Sharma KP, Gaur RK (2011) Biotechnological perspectives of microbes in agroecosystems. Biotechnol Lett 33(10):1905–1910 Compant S, Mitter B, Colli-Mull JG, Gangl H, Sessitsch A (2011) Endophytes of grapevine flowers, berries, and seeds: identification of cultivable bacteria, comparison with other plant parts, and visualization of niches of colonization. Microb Ecol 62(1):188–197 de Almeida Lopes KB, Carpentieri-Pipolo V, Oro TH, Stefani Pagliosa E, Degrassi G (2016) Culturable endophytic bacterial communities associated with field-grown soybean. J Appl Microbiol 120(3):740–755 Dias ACF, Costa FEC, Andreote FD, Lacava PT, Teixeira MA, Assumpção LC, Araújo WL, Azevedo JL, Melo IS (2009) Isolation of micropropagated strawberry endophytic bacteria and assessment of their potential for plant growth promotion. World J Microbiol Biotechnol 25(2):189–195 Dong Z, Canny MJ, McCully ME, Roboredo MR, Cabadilla CF, Ortega E, Rodes R (1994) A nitrogen-fixing endophyte of sugarcane stems (a new role for the apoplast). Plant Physiol 105 (4):1139–1147 Dutta SC, Neog B (2016) Accumulation of secondary metabolites in response to antioxidant activity of turmeric rhizomes co-inoculated with native arbuscular mycorrhizal fungi and plant growth promoting rhizobacteria. Sci Hortic 204:179–184 Frampton RA, Pitman AR, Fineran PC (2012) Advances in bacteriophage-mediated control of plant pathogens. Int J Microbiol 2012(6079):1–11 Frommel MI, Nowak J, Lazarovits G (1991) Growth enhancement and developmental modifications of in vitro grown potato (Solanum tuberosum spp. tuberosum) as affected by a nonfluorescent Pseudomonas sp. Plant Physiol 96(3):928–936 Gaiero JR, McCall CA, Thompson KA, Day NJ, Best AS, Dunfield KE (2013) Inside the root microbiome: bacterial root endophytes and plant growth promotion. Am J Bot 100(9):1738–1750 Gantar M, Kerby NW, Rowell P (1991) Colonization of wheat (Triticum vulgare L.) by N2-fixing cyanobacteria: II. An ultrastructural study. New Phytol 118(3):485–492 García-Fraile P, Menéndez E, Rivas R (2015) Role of bacterial biofertilizers in agriculture and forestry. AIMS Bioeng 2(3):183–205 Gizmawy I, Kigel J, Koller D, Ofir M (1985) Initiation, orientation and early development of primary rhizomes in Sorghum halepense (L.) Pers. Ann Bot 55(3):343–350 Glick BR (2014) Bacteria with ACC deaminase can promote plant growth and help to feed the world. Microbiol Res 169(1):30–39 Glick BR, Patten CL, Holguin G, Penrose DM (1999) Biochemical and genetic mechanisms used by plant growth promoting bacteria. Imperial College Press, London Goswami D, Vaghela H, Parmar S, Dhandhukia P, Thakker JN (2013) Plant growth promoting potentials of Pseudomonas spp. strain OG isolated from marine water. J Plant Interact 8(4):281–290 Hawksworth DL (2001) The magnitude of fungal diversity: the 1.5 million species estimate revisited. Mycol Res 105(12):1422–1432 Hider RC, Kong X (2010) Chemistry and biology of siderophores. Nat Prod Rep 27(5):637 Hinton DM, Bacon CW (1995) Enterobacter cloacae is an endophytic symbiont of corn. Mycopathologia 129(2):117–125 Hu F, Wang D, Zhao X, Zhang T, Sun H, Zhu L, Zhang F, Li L, Li Q, Tao D, Fu B, Li Z (2011) Identification of rhizome-specific genes by genome-wide differential expression analysis in Oryza longistaminata. BMC Plant Biol 11:18

418

A. Gupta et al.

Hurek T, Reinhold-Hurek B, van Montagu M, Kellenberger E (1994) Root colonization and systemic spreading of Azoarcus sp. strain BH72 in grasses. J Bacteriol 176(7):1913–1923 Jalgaonwala RE, Mahajan RT (2014) Production of anticancer enzyme asparaginase from endophytic Eurotium sp. isolated from rhizomes of Curcuma longa. Eur J Exp Biol 4(3):36–43 James EK, Reis VM, Olivares FL, Baldani JI, Döbereiner J (1994) Infection of sugar cane by the nitrogen-fixing bacterium Acetobacter diazotrophicus. J Exp Bot 45(6):757–766 Jang CS, Kamps TL, Skinner DN, Schulze SR, Vencill WK, Paterson AH (2006) Functional classification, genomic organization, putatively cis-acting regulatory elements, and relationship to quantitative trait loci, of sorghum genes with rhizome-enriched expression. Plant Physiol 142(3):1148–1159 Jasim B, Jimtha CJ, Jyothis M, Radhakrishnan EK (2013) Plant growth promoting potential of endophytic bacteria isolated from Piper nigrum. Plant Growth Regul 71(1):1–11 Jasim B, Anisha C, Rohini S, Kurian JM, Jyothis M, Radhakrishnan EK (2014a) Phenazine carboxylic acid production and rhizome protective effect of endophytic Pseudomonas aeruginosa isolated from Zingiber officinale. World J Microbiol Biotechnol 30(5):1649–1654 Jasim B, Joseph AA, John CJ, Mathew J, Radhakrishnan EK (2014b) Isolation and characterization of plant growth promoting endophytic bacteria from the rhizome of Zingiber officinale. 3 Biotech 4(2):197–204 Kauppinen M, Saikkonen K, Helander M, Pirttilä AM, Wäli PR (2016) Epichloë grass endophytes in sustainable agriculture. Nat Plants 2:15224 Khammas KM, Kaiser P (1991) Characterization of a pectinolytic activity in Azospirillum irakense. Plant Soil 137(1):75–79 Kloepper JW, Beauchamp CJ (1992) A review of issues related to measuring colonization of plant roots by bacteria. Can J Microbiol 38(12):1219–1232 Knolhoff AM, Zheng J, McFarland MA, Luo Y, Callahan JH, Brown EW, Croley TR (2015) Identification and structural characterization of naturally-occurring broad-spectrum cyclic antibiotics isolated from Paenibacillus. J Am Soc Mass Spectrom 26(10):1768–1779 Koo HJ, McDowell ET, Ma X, Greer KA, Kapteyn J, Xie Z, Descour A, Kim H, Yu Y, Kudrna D, Wing RA, Soderlund CA, Gang DR (2013) Ginger and turmeric expressed sequence tags identify signature genes for rhizome identity and development and the biosynthesis of curcuminoids, gingerols and terpenoids. BMC Plant Biol 13(1):27 Krishnapura PR, Belur PD, Subramanya S (2016) A critical review on properties and applications of microbial l-asparaginases. Crit Rev Microbiol 42(5):720–737 Kumar A, Singh R, Giri DD, Singh PK, Pandey KD (2014) Effect of Azotobacter chroococcum CL13 inoculation on growth and curcumin content of turmeric (Curcuma longa L.). Int J Curr Microbiol App Sci 3(9):275–283 Kumar V, Kumar A, Pandey KD, Roy BK (2015a) Isolation and characterization of bacterial endophytes from the roots of Cassia tora L. Ann Microbiol 65:1391–1139 Kumar A, Vandana S, Yadav A, Giri DD, Singh PK, Pandey KD (2015b) Rhizosphere and their role in plant–microbe interaction. In: Chaudhary KK, Dhar DW (eds) Microbes in soil and their agricultural prospects. Nova Science, New York, pp 83–97 Kumar A, Vandana SR, Singh M, Pandey KD (2015c) Plant growth promoting rhizobacteria (PGPR): a promising approach for disease management. In: Singh JS, Singh DP (eds) Microbes and environmental management. Studium Press, New Delhi, pp 195–209 Kumar A, Singh R, Yadav A, Giri DD, Singh PK, Pandey KD (2016a) Isolation and characterization of bacterial endophytes of Curcuma longa L. 3 Biotech 6(1):60 Kumar A, Singh V, Singh M, Singh PP, Singh SK, Singh PK, Pandey KD (2016b) Isolation of plant growth promoting rhizobacteria and their impact on growth and curcumin content in Curcuma longa L. Biocatal Agric Biotechnol 8:1–7 Kumar A, Verma H, Singh VK, Singh PP, Singh SK, Ansari WA, Yadav A, Singh PK, Pandey KD (2017) Role of Pseudomonas sp. in sustainable agriculture and disease management. In: Meena V, Mishra P, Bisht J, Pattanayak A (eds) Agriculturally important microbes for sustainable agriculture. Springer, Singapore, pp 195–215

19

Rhizome Endophytes: Roles and Applications in Sustainable Agriculture

419

Kumar A, Singh VK, Tripathi V, Singh PP, Singh AK (2018) Plant growth-promoting rhizobacteria (PGPR): perspective in agriculture under biotic and abiotic stress. In: Crop improvement through microbial biotechnology, pp 333–342. https://doi.org/10.1016/B978-0-444-63987-5.00016-5 Lahlali R, Hijri M (2010) Screening, identification and evaluation of potential biocontrol fungal endophytes against Rhizoctonia solani AG3 on potato plants. FEMS Microbiol Lett 311(2):152–159 Lee C, Kim S, Li W, Bang S, Lee H, Lee H-J, Noh E-Y, Park J-E, Bang WY, Shim SH (2017) Bioactive secondary metabolites produced by an endophytic fungus Gaeumannomyces sp. JS0464 from a maritime halophyte Phragmites communis. J Antibiot 70(6):737–742 Levanony H, Bashan Y, Romano B, Klein E (1989) Ultrastructural localization and identification of Azospirillum brasilense Cd on and within wheat root by immuno-gold labeling. Plant Soil 117(2):207–218 Ling L, Lei L, Feng L, He N, Ding L (2014) Isolation and identification of endophytic bacterium TG116 from Typhonium giganteum and its antimicrobial characteristics. J Northwest Univ Nat Sci 5:019 Liu Y-G, Mitsukawa N, Oosumi T, Whittier RF (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 8(3):457–463 Liu K-W, Li Z-L, Pu S-B, Xu D-R, Zhou H-H, Shen W-B (2014) Chemical constituents of the rhizome of Typhonium giganteum. Chem Nat Compd 29:168 Lodewyckx C, Vangronsveld J, Porteous F, Moore ERB, Taghavi S, Mezgeay M, van der Lelie D (2002) Endophytic bacteria and their potential applications. Crit Rev Plant Sci 21(6):583–606 Mahaffee W, Kloepper J (1997) Bacterial communities of the rhizosphere and endorhiza associated with field-grown cucumber plants inoculated with a plant growth-promoting rhizobacterium or its genetically modified derivative. Can J Microbiol 43(4):344–353 Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES science gateway for inference of large phylogenetic trees. In: Gateway computing environments workshop (GCE) IEEE, pp 1–8 Mukerjee A, Vishwanatha JK (2009) Formulation, characterization and evaluation of curcuminloaded PLGA nanospheres for cancer therapy. Anticancer Res 29(10):3867–3875 Niemhom N, Chutrakul C, Suriyachadkun C, Thawai C (2016) Asanoa endophytica sp. nov., an endophytic actinomycete isolated from the rhizome of Boesenbergia rotunda. Int J Syst Evol Microbiol 66(3):1377–1382 Nongalleima K, Dey A, Deb L, Singh C, Thongam B, Devi HS, Devi SI (2013) Endophytic fungus isolated from Zingiber zerumbet (L.) Sm. inhibits free radicals and cyclooxygenase activity. Int J PharmTech Res 5(2):301–307 Ohshiro M, Kuroyanagi M, Ueno A (1990) Structures of sesquiterpenes from Curcuma longa. Phytochemistry 29(7):2201–2205 Oteino N, Lally RD, Kiwanuka S, Lloyd A, Ryan D, Germaine KJ, Dowling DN (2015) Plant growth promotion induced by phosphate solubilizing endophytic Pseudomonas isolates. Front Microbiol 6:111 Panahi Y, Saadat A, Beiraghdar F, Hosseini Nouzari SM, Jalalian HR, Sahebkar A (2014) Antioxidant effects of bioavailability-enhanced curcuminoids in patients with solid tumors: a randomized double-blind placebo-controlled trial. J Funct Foods 6:615–622 Patriquin DG, Döbereiner J (1978) Light microscopy observations of tetrazolium-reducing bacteria in the endorhizosphere of maize and other grasses in Brazil. Can J Microbiol 24(6):734–742 Patriquin DG, Döbereiner J, Jain DK (1983) Sites and processes of association between diazotrophs and grasses. Can J Microbiol 29(8):900–915 Quadt-Hallmann A, Kloepper J (1996) Immunological detection and localization of the cotton endophyte Enterobacter asburiae JM22 in different plant species. Can J Microbiol 42(11):1144–1154 Quadt-Hallmann A, Kloepper JW, Benhamou N (1997) Bacterial endophytes in cotton: mechanisms of entering the plant. Can J Microbiol 43(6):577–582 Rao CV, Rivenson A, Simi B, Reddy BS (1995) Chemoprevention of colon carcinogenesis by dietary curcumin, a naturally occurring plant phenolic compound. Cancer Res 55(2):259–266

420

A. Gupta et al.

Reinhold-Hurek B, Hurek T (1998) Interactions of gramineous plants with Azoarcus spp. and other diazotrophs: identification, localization, and perspectives to study their function. Crit Rev Plant Sci 17(1):29–54 Rohini S, Aswani R, Kannan M, Sylas VP, Radhakrishnan EK (2018) Culturable endophytic bacteria of ginger rhizome and their remarkable multi-trait plant growth-promoting features. Curr Microbiol 75(4):505–511 Rosenblueth M, Martínez-Romero E (2006) Bacterial endophytes and their interactions with hosts. Mol Plant Microbe Interact 19(8):827–837 Rout ME, Chrzanowski TH, Westlie TK, DeLuca TH, Callaway RM, Holben WE (2013) Bacterial endophytes enhance competition by invasive plants. Am J Bot 100(9):1726–1737 Ruppel S, Hecht-Buchholz C, Remus R, Ortmann U, Schmelzer R (1992) Settlement of the diazotrophic, phytoeffective bacterial strain Pantoea agglomerans on and within winter wheat: an investigation using ELISA and transmission electron microscopy. Plant Soil 145(2):261–273 Sabu R, Aswani R, Prabhakaran P, Krishnakumar B, Radhakrishnan EK (2018) Differential modulation of endophytic microbiome of ginger in the presence of beneficial organisms, pathogens and both as identified by DGGE analysis. Curr Microbiol 26(3):555 Saini R, Dudeja SS, Giri R, Kumar V (2015) Isolation, characterization, and evaluation of bacterial root and nodule endophytes from chickpea cultivated in Northern India. J Basic Microbiol 55(1):74–81 Sauvêtre A, Schröder P (2015) Uptake of carbamazepine by rhizomes and endophytic bacteria of Phragmites australis. Front Plant Sci 6(148):232 Seo WT, Lim WJ, Kim EJ, Yun HD, Lee YH, Cho KM (2010) Endophytic bacterial diversity in the young radish and their antimicrobial activity against pathogens. J Korean Soc Appl Biol Chem 53(4):493–503 Shishido M, Massicotte HB, Chanway CP (1996) Effect of plant growth promoting Bacillus strains on pine and spruce seedling growth and mycorrhizal infection. Ann Bot 77(5):433–442 Shubin L, Juan H, RenChao Z, ShiRu X, YuanXiao J (2014) Fungal endophytes of Alpinia officinarum rhizomes: insights on diversity and variation across growth years, growth sites, and the inner active chemical concentration. PLoS One 9(12):e115289 Singh D, Rathod V, Ninganagouda S, Herimath J, Kulkarni P (2013) Biosynthesis of silver nanoparticle by endophytic fungi Penicillium sp. isolated from Curcuma longa (turmeric) and its antibacterial activity against pathogenic gram negative bacteria. J Pharm Res 7(5):448–453 Singh M, Kumar A, Singh R, Pandey KD (2017a) Endophytic bacteria: a new source of bioactive compounds. 3 Biotech 7:315 Singh VK, Singh AK, Kumar A (2017b) Disease management of tomato through PGPB: current trends and future perspective. 3 Biotech 7(4):255 Singh R, Pandey DK, Kumar A, Singh M (2017c) PGPR isolates from the rhizosphere of vegetable crop Momordica charantia: characterization and application as biofertilizer. Int J Curr Microbiol App Sci 6(3):1789–1802 Spaepen S, Vanderleyden J (2011) Auxin and plant-microbe interactions. Cold Spring Harb Perspect Biol 3(4):a001438–a001438 Srimal R (1997) Turmeric: a brief review of medicinal properties. Fitoterapia 68:483–493 Strobel G, Daisy B (2003) Bioprospecting for microbial endophytes and their natural products. Microbiol Mol Biol Rev 67(4):491–502 Sturz AV, Christie BR, Matheson BG, Nowak J (1997) Biodiversity of endophytic bacteria which colonize red clover nodules, roots, stems and foliage and their influence on host growth. Biol Fertil Soils 25(1):13–19 Sturz AV, Christie BR, Matheson BG, Arsenault WJ, Buchanan NA (1999) Endophytic bacterial communities in the periderm of potato tubers and their potential to improve resistance to soilborne plant pathogens. Plant Pathol 48(3):360–369 Sulistiyani TR, Lisdiyanti P (2016) Diversity of endophytic bacteria associated with (Curcuma heyneana) and their potency for nitrogen fixation. Widyariset 2(2):106

19

Rhizome Endophytes: Roles and Applications in Sustainable Agriculture

421

Sulistiyani S, Ardyati T, Winarsih S (2016) Antimicrobial and antioxidant activity of endophyte bacteria associated with Curcuma longa rhizome. J Exp Life Sci 6(1):45–51 Suryadevara N, Ponmurugan P (2012) Response of turmeric to plant growth promoting rhizobacteria (PGPR) inoculation under different levels of nitrogen. Int J Biol Technol 3(1):39–44 Theantana T, Hyde KD, Lumyong S (2009) Asparaginase production by endophytic fungi from Thai medicinal plants: cytoxicity properties. Int J Integr Biol 7:1–8 Vejan P, Abdullah R, Khadiran T, Ismail S, Nasrulhaq Boyce A (2016) Role of plant growth promoting rhizobacteria in agricultural sustainability – a review. Molecules 21(5):573 Vinayarani G, Prakash HS (2018) Fungal endophytes of turmeric (Curcuma longa L.) and their biocontrol potential against pathogens Pythium aphanidermatum and Rhizoctonia solani. World J Microbiol Biotechnol 34(3):49 Vu T, Sikora R, Hauschild R (2006) Fusarium oxysporum endophytes induced systemic resistance against Radopholus similis on banana. Nematology 8(6):847–852 Wang H, Jiang X, Mu H, Liang X, Guan H (2007) Structure and protective effect of exopolysaccharide from P. agglomerans strain KFS-9 against UV radiation. Microbiol Res 162(2):124–129 Wiehe W, Hecht-Buchholz C, Hoflich G (1994) Electron microscopic investigations on root colonization of Lupinus albus and Pisum sativum with two association plant growth promoting rhizobacteria, Pseudomonas fluorescens and Rhizobium leguminosarum bv. trifolii. Symbiosis (Philadelphia, PA) (USA) Xu L, Zhou L, Zhao J, Li J, Li X, Wang J (2008) Fungal endophytes from Dioscorea zingiberensis rhizomes and their antibacterial activity. Lett Appl Microbiol 46(1):68–72 Zandi P, Basu SK (2016) Role of plant growth-promoting rhizobacteria (PGPR) as biofertilizers in stabilizing agricultural ecosystems. In: Nandwani D (ed) Organic farming for sustainable agriculture. Springer, pp 71–87 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 L, Deng Z, Yang W, Cao Y, Wang E, Wei G (2010) 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

Agriculturally Important Biosynthetic Features of Endophytic Microorganisms

20

S. Sreejith, R. Aswani, and E. K. Radhakrishnan

Contents 20.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2 Types and Colonization of Endophytic Microbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.3 Endophytes Isolated from Various Parts of the Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.3.1 Root Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.3.2 Leaf and Stem Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.3.3 Flower and Fruit Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.3.4 Seed Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.4 Agricultural Promises of Endophytic Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.5 Plant Growth-Promoting Features of Endophytic Microorganisms . . . . . . . . . . . . . . . . . . . . 20.6 Endophytic Microorganisms as Biocontrol Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.6.1 Antimicrobial Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.6.2 Production of Hydrogen Cyanide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.6.3 Production of Lytic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

424 424 425 428 428 429 429 431 432 434 435 437 438 438 438

Abstract

Endophytic microorganisms mainly include bacteria and fungi that colonize the plant internally without causing any adverse effect. Due to the mutualistic association with microorganisms accommodated internally, plants are benefited significantly in growth and resistance against various pathogens. The multiple plant-beneficial functions of endophytes like plant growth promotion, biocontrol and alleviation of abiotic stress are mediated through the production of diverse biomolecules. Hence there are immense possibilities to explore endophytes for various agricultural applications to substitute the use of agrochemicals. Endophytology with concepts of holobiome (plant and endophytes) and hologenome (genome of plant and S. Sreejith · R. Aswani · E. K. Radhakrishnan (*) School of Biosciences, Mahatma Gandhi University, Kottayam, India e-mail: [email protected] # Springer Nature Switzerland AG 2019 S. K. Verma, J. F. White, Jr (eds.), Seed Endophytes, https://doi.org/10.1007/978-3-030-10504-4_20

423

424

S. Sreejith et al.

endophytes) is gaining acceptance in recent years, both in basic and applied sciences. The current chapter describes biosynthetic features of endophytes especially those from seed endophytes, which are least investigated. Keywords

Endophytic microorganisms · Plant-microbe interactions · Plant growth promotion · Bioactive metabolites

20.1

Introduction

Agricultural yield and the quality of products are highly challenged by diverse biotic and abiotic stress conditions and the methods used to manage the same. Agrochemical-based approach has shown to cause severe environmental toxicity and thereby diverse health problems. Hence, the exploitation of natural methods and mechanisms is considered to have immense promises to meet the global food security challenge and also to reduce the toxic input to agricultural field. Due to the same, plant microbiome has been identified as the ultimate resource for sustainable agricultural practices (Jain and Pundir 2017). Because of this, the plant-beneficial mechanisms of endophytic microorganisms have recently gained much attraction (Pimentel et al. 2011; Singh and Dubey 2015). The biosynthetic features of endophytes are very influential to the growth and disease resistance of host plants. In addition, these microbial communities also contribute to the induced systemic resistance which enable the plant to significantly resist most of the devastating diseases (Hubbard et al. 2014). The term endophyte has originated from the Greek words endon (within) and phyton (plant) which means inside the plant (De Bary 1866). This term broadly describes microorganisms which live inside the plants without any obvious harmful effects to the host (Hallmann et al. 1997). The direct interaction with the plants makes the endophyte a highly valuable biotool for plant growth and biocontrol. Even though endophytic microorganisms have been characterized from various parts of plants, only limited reports are there on its mode of transmission. The endophytic microorganisms associated with the seeds and vegetatively propagated plant materials can expect to have remarkable significance as these are being transmitted from plant to plant vertically. Hence the current chapter describes the biosynthetic features of endophytic microorganisms and recent understanding on endophytes associated with seeds and vegetatively propagated plant materials.

20.2

Types and Colonization of Endophytic Microbes

Various types of microorganisms have been identified to be colonized within the plant tissues as endophytes which include bacteria and fungi. Bacterial members from different phyla like actinobacteria, proteobacteria and firmicutes (Golinska et al. 2015) are reported to have association with plants as endophytes, and these include Achromobacter, Acinetobacter, Agrobacterium, Bacillus, Brevibacterium, Serratia,

20

Agriculturally Important Biosynthetic Features of Endophytic Microorganisms

425

Microbacterium, Pseudomonas, Xanthomonas, etc. (Sun et al. 2013). Even though many endophytes have been reported from various plants, strains of Bacillus, Burkholderia, Enterobacter, Pseudomonas and Serratia are found to be most effective against various fungal pathogens (Kloepper and Ryu 2006; Larran et al. 2016). Among these, Bacillus and Pseudomonas are also reported as the most common genera with biocontrol and plant growth-promoting features (Jasim et al. 2014a, 2016a). Endophytic actinomycetes are also remarkable for the presence of various chemical compounds with both antibacterial and antifungal properties (Sabu et al. 2017b). Mycoplasma species are also reported to have endophytic association with red algae such as Bryopsis pennata and B. hypnoides (Hollants et al. 2011). Streptomyces is one of the dominant genera commonly isolated as endophytic actinomycetes (Zhao et al. 2011; Golinska et al. 2015). Fungal endophytes mainly include clavicipitaceous and non-clavicipitaceous endophytes (Bhardwaj and Agrawal 2014; Sushanto Gouda et al. 2016; Rajamanikyam et al. 2017). Loading of microorganisms within the plant as endophyte can be the result of either vertical or horizontal transmission. In vertical transmission, the endophytes are being transferred to seeds via vascular connections (Liu et al. 2013; Malfanova et al. 2013; Truyens et al. 2015). Subsequent to seed germination, these endophytic populations are considered to increase its number and get distributed to parts such as root and shoot (Fig. 20.1). While in horizontal transmission, the endophytes will be recruited mainly from soil, and this requires a stage of rhizoplane interaction before it is transported to intercellular spaces in the root (Chi et al. 2005). Host plant and the tissue types are the major factors determining the diversity and population of associated endophytic microbial communities. Hence seeds are remarkably significant to its microbiome as it can have candidate microbial partners with determining effect on plant competitiveness and growth (Hu et al. 2011). The same is applicable to plant parts used in vegetative propagation (Fig. 20.2). A large number of endophytic bacteria and fungi have been reported from ginger rhizome with broadspectrum plant-beneficial features due to its continuous interaction with soil (Jasim et al. 2014b; Anisha and Radhakrishnan 2017; Anisha et al. 2018b; Rohini et al. 2018). Some of these microflora can likely to get transmitted by vertical transmission. Also the endophytic microorganisms which reside within the seeds are considered to have the superior potential to provide support to the plant to manage adverse environmental conditions than those isolated from other plant tissues (Mano et al. 2006; Compant et al. 2011). They can also possess the cell motility and phytase activity to migrate freely inside the plant and get loaded within the seeds before they harden.

20.3

Endophytes Isolated from Various Parts of the Plants

Endophytic microbes live inside the plant tissues without inducing any disease symptoms (Brader et al. 2017). As all plants are likely to have endophytic association in its various parts, these microbes can have great agricultural promises (Smith et al. 2008). These organisms have been reported from parts of the plant like roots, leaves, stems, fruits, seeds and flowers (Wearn et al. 2012; Saikkonen et al. 2004;

Germination of seedlings

Di s t ri pa but rts ion of of ge en rm do p i n at hy ed te se s to e dl va in rio gs u s

of al ers hyte s p s Di dop eed en ed s d loa

Endophytes transfer to seeds via vascular connection, Gamates or through reproductive meristem

Vertical transmission of endophytes

Fig. 20.1 Vertical transmission of endophyte and associated mechanisms

ble ura ns vo Fa ditio n co

Induction of plant

defence

Direct mechanisms Phytohormone production ACC deaminase, Nutrient solubilization Plant growth and development

Indirect mechanisms Antimicrobial compounds, Hydrolytic enzymes

Functioning of endophytic micrororganisms with in plants as its second genome Mechanisms involved

Further recruitment of endophytes from soil

Pl a en ma nt do na gr ph ge ow yt me th ic nt an m a d ic ss st ro is re or te ss ga d ni by sm s

426 S. Sreejith et al.

5,7-dimethaxy-4-pmethoxylphenylcou marin,5,7dimethoxy-4phenylcoumarin Phenol 2,4-bis(1,1dimethylethyl and phenazine gene

(Taechowisan et al. 2005)

Streptomyces aureofaciens

Sabu et al. 2017b)

Rohini et al. 2018)

ACC deaminase, nitrogen fixation, phosphate solubilization, IAA

Serratia sp.

Antifungal activity

(Onja, 2016)

Pyrrolnitrin gene, ACC deaminase, Phosphate & zinc solubilization, ammonia production, IAA (Sabu et al. 2017b)

Decursin

Streptomyces chrysomallus

Rohini et al. 2018)

ACC deaminase, nitrogen fixation, IAA, phosphate solubilization

Burkholderia vietnaminesis,Bu rkholderia sp., Burkholderia cenocepacia

(Taechowisan et al. 2013)

Streptomyces sp.

Endophytic Actinomycetes

Rohini et al. 2018)

Nocardiopsis

(Jasim et al. 2014b)

IAA, ACC deaminase and siderophore

Phosphate solubilization, nirogen fixation, IAA

Enterobacter asburiae,

Enterobacter cloacae,

Ginting et al. 2013)

Bacillus sp., Bacillus altitudinis, Alcaligenes faecalis, Pantoea agglomerans

Endophytic Bacteria

(Anisha et al. 2018b)

Antifungal activity

(Anisha and Radhakrishnan 2017)

Acremonium macroclavatum,Belt raniella,

Tyrosol prduction

Fungal sp.

Agriculturally Important Biosynthetic Features of Endophytic Microorganisms

Fig. 20.2 Endophytes characterized from ginger rhizome

Rhizome of Zingiber officinale Rosc.

(Jasim et al. 2014a)

phenazine 1carboxylic acid

Pseudomonas sp.

(Anisha et al. 2018a)

(Anisha and Radhakrishnan 2015)

Antifungal activity, enhancemnet of germination and bud formation

Rhizopycnis vagum

Enterobacter hormaechei

Production of Danthrone

Gliotoxin production

Pseudomonas aeruginosa

Paraconiothyrium sp.

Acremonium sp. ,

Endophytic Fungi

20 427

428

S. Sreejith et al.

Shahzad et al. 2016; Brader et al. 2017). Among the endophytes distributed in various parts of plant, those which are being accommodated in the seeds can have significant role in seedling growth and establishment. So an overview of endophytes from various parts of plants can provide insight into endophytic reservoir of plants. Importantly some of these might have originated from seeds or some can further be transmitted through seeds.

20.3.1 Root Endophytes Root hairs offer a common port of entry for endophytes from soil reserve. This is well studied in the case of nodulating bacteria (Hardoim et al. 2008; Prieto et al. 2011). In the case of Rhizobia-mediated nodulation in legumes, the colonization happens inside the root hairs, before the formation of infection threads (Garg and Geetanjali 2007), and is mediated by chemotaxis and nod factors. The attached endophytes may directly enter into plant by secreting cell wall-degrading enzymes or through the phenomenon called rhizophagy (White et al. 2014; Paungfoo-Lonhienne et al. 2010). This facilitates active recruitment of microbes into plant from soil (Paungfoo-Lonhienne et al. 2013). Further endophytic distribution and transport within the host plants are mediated by microbial factors like lipopolysaccharides, flagella, pili and twitching motility (Bohm et al. 2007). The secretion of cell walldegrading enzymes can have role in both bacterial penetration (Lodewyckx et al. 2002) and spreading within the plant (Fouts et al. 2008). Insight into endophytic association as a universal phenomenon in the root has been identified by its isolation from diverse roots. The isolation procedure generally involves surface sterilization followed by plating the root samples directly or by maceration and subsequent plating onto various culture media. Regarding the function of root endophytes, it follows the mechanisms similar to mycorrhiza and is mainly related to enhancement of plant growth and nutrient uptake (Newsham 2011). These root endophytes can also provide direct antagonism to plant pathogens or may induce plant resistance systemically (Jallow et al. 2004).

20.3.2 Leaf and Stem Endophytes Systemic spread of endophytes within the plant is considered to facilitate its distribution and functional assistance in the stem and leaf tissues (Hardoim et al. 2008). In leaves, bacterial endophytes have been observed to be present at the intercellular spaces of mesophyll and xylem tissues and substomatal areas. Its presence in xylem and substomatal chambers of grapevine plants has been proved by green fluorescent protein (GFP) labelling and glucuronidase (GUS) staining. Interestingly, bacterial cells leaving through the stomatal aperture were also observed in grapevine leaves by in-depth observation (Compant et al. 2005b). The endophytes from maize plants were demonstrated to be more in the lower stem than in the stem closer to the shoot apex (Fisher et al. 1992). Diverse endophytic fungal isolates have also been

20

Agriculturally Important Biosynthetic Features of Endophytic Microorganisms

429

characterized from leaf and sapwood of Hevea brasiliensis, and this described its diversity within the plant parts (Gazis and Chaverri 2010).

20.3.3 Flower and Fruit Endophytes Endophytic colonization has also been reported from flowers and fruits but at a very low density under natural conditions (Hallmann 2001). Compant et al. (2011) have reported the presence of Gammaproteobacteria and Firmicutes inside the epidermis layer of flowers and xylem of ovaries and are suggestive of the presence of unique niches for endophytic colonization within flowers. At the same time, Bacillus has also been identified as the predominant species in papaya fruit along with Kocuria, Acinetobacter and Enterobacter species (Shi et al. 2010; Krishnan et al. 2012). Jasim et al. (2015) have also identified the presence of fruit-associated bacteria from Elettaria cardamomum with multiple plant-beneficial properties especially ACC deaminase production.

20.3.4 Seed Endophytes Seeds are biologically remarkable as it exists in an inactive state till the favourable and suitable conditions to develop into a new plant (Lopez-Lopez et al. 2010; Cope-Selby et al. 2017; Shade et al. 2017; Geisen et al. 2017). The presence of various types of endophytic microorganisms in seeds has been reported from a variety of plants including monocotyledons, dicotyledons and herbaceous plants (Shen et al. 2014; Truyens et al. 2015; Parsa et al. 2016). The microbiome of seed forms immediate source of microbial assistance for seedling establishment and is especially important for invasive plant species, where identifying suitable microbiome partner is highly challenging for the establishment of seedlings (Newcombe et al. 2009; Hodgson et al. 2014; Klaedtke et al. 2015). Here, the seed-borne bacterial and fungal endophytes can have significant impact on seed germination (Chee-Sanford et al. 2006; Shearin et al. 2017). Seed endophytes are mainly mobilized through the vascular connections or through the transgenerational transfer via vertical transmission (Kaga et al. 2009; Ruiz et al. 2011; Hodgson et al. 2014). Several studies have reported the plant-beneficial features of both seed-borne endophytic bacteria and fungi (Table 20.1). They have been reported to have direct or indirect mechanisms to improve plant growth and development and also to enhance plant tolerance to biotic and abiotic stresses (Santoyo et al. 2016; Shahzad et al. 2017; Khan et al. 2016). However the mechanisms behind the preservation of beneficial organisms inside the seed are least studied. Detailed mechanistic insight into seed endophyte will enable the development of tailor-made plant probiotic formulations for agricultural applications.

430

S. Sreejith et al.

Table 20.1 Endophytic microorganisms isolated from seeds Sl. No. 1.

2. 3.

Seed Bacteria Cucumis sativus, Cucumis melo, Citrullus lanatus and Cucurbita sp. Chenopodium quinoa Cannabis sativa L.

4.

Leersia oryzoides

5.

Lolium perenne

6.

Oryza sativa

7.

Phaseolus vulgaris Phragmites australis

8.

Endophytes present

Plant beneficial traits

References

Bacillus, Paenibacillus, Enterobacteriaceae, Staphylococcus, Microbacterium Bacillus sp.

Antifungal activity

Khalaf and Raizada (2018)

Plant development and stress tolerance Siderophores, phosphorus solubilization IAA (auxin) production, phosphate solubilization, root hair formation and growth Plant growth promotion Indole-3-acetic acid, phosphate solubilization, siderophore production

Pitzschke (2018) Scott et al. (2018)

Pseudomonas, Pantoea and Bacillus Pantoea sp., Pseudomonas sp., Microbacterium sp., Paenibacillus sp., Chryseobacterium sp. Epichloë festucae Herbaspirillum, Microbacterium, Kosakonia, Pseudomonas, Paenibacillus, Ochrobactrum sp. Flavobacterium sp. Aureobasidium pullulans Pseudomonas spp., Pantoea sp.

9.

Triticum aestivum

Paenibacillus sp. Pantoea sp., Bacillus sp.

10.

Lycopersicum esculentum

Bacillus subtilis

11.

Nicotiana tabacum

Enterobacter sp., Pseudomonas sp., Stenotrophomonas sp.

12.

Fungi Raphanus sativus

Aspergillus oryzae

Biocontrol Phosphate solubilization, antifungal property IAA production, siderophore production, phosphate solubilization, antifungal activity IAA production, ACC deaminase, antifungal activity Plant growth promotion

Plant growth promotion and protection against herbivores

Verma et al. (2018a)

Liu et al. (2017) Walitang et al. (2017), Hardoim et al. (2012)

Parsa et al. (2016) White et al. (2017), Verma et al. (2018b) Herrera et al. (2016)

Xu et al. (2014) Mastretta et al. (2009)

Sun et al. (2018)

(continued)

20

Agriculturally Important Biosynthetic Features of Endophytic Microorganisms

431

Table 20.1 (continued) Sl. No. 13.

Seed Cannabis sativa L.

14.

Phragmites australis

15.

Dendrobium friedericksianum

16.

Dactylis glomerata

20.4

Endophytes present Aureobasidium, Alternaria and Cochliobolus Alternaria sp., Phoma sp., Penicillium corylophilum Fusarium sp., Beauveria sp., Tulasnella violea, T. violea, Epulorhiza sp., Trichosporiella multisporum Epichloë typhina

Plant beneficial traits Siderophores, phosphate solubilization Enhance seed germination and seedling growth Growth promotion

References Scott et al. (2018)

Improved host plant growth and photosynthesis

Rozpadek et al. (2015)

Shearin et al. (2017) Khamchatra et al. (2016)

Agricultural Promises of Endophytic Microorganisms

The excessive use of agrochemicals for the enhanced crop yield has caused serious environmental and health problems. Therefore, there is a growing demand for safer methods to avoid or minimize the use of chemicals. Here comes the importance of exploitation of endophytic microorganisms, which have the natural plant growthpromoting and antagonistic mechanisms executed through diverse biosynthetic processes (Santoyo et al. 2016; Shahzad et al. 2017). Their direct mechanisms to support plant growth and development mainly involve solubilization of soil supplements, nitrogen fixation, phytohormone synthesis (Tayung et al. 2012; Maehara et al. 2016; Shahzad et al. 2016) and the lowering effect of ethylene levels by synthesizing the ACC deaminase (1-amino-cyclopropane-1-carboxylate) (Coutinho et al. 2014; Pandya et al. 2015; Saini et al. 2015; Khamchatra et al. 2016). The indirect mechanisms involve the production of secondary metabolites which strengthen the plant resistance towards various pathogens (Maehara et al. 2016; Shahzad et al. 2016). These general mechanisms can expect to function effectively in seed endophytes. As plant-associated bacteria have been explored for agricultural applications, seed endophytes can also be considered to have promising applications. Many studies have suggested the potential application of Pseudomonas spp. in agriculture as plant growth-promoting and biocontrol agents (Govindarajan et al. 2008; Mattos et al. 2008; Paungfoo-Lonhienne et al. 2014; Bernabeu et al. 2015). In addition to this, various endophytic bacterial species of Azospirillum, Azoarcus, Enterobacter, Serratia and Stenotrophomonas have also been reported to enhance plant growth by utilizing the mechanisms like nitrogen fixation, phytohormone production, siderophore production and ACC deaminase synthesis (Krause et al. 2006; Taghavi et al. 2009; Wisniewski-Dyé et al. 2011; Kwak et al. 2012). Plant growthpromoting activities of endophytic Bacillus spp. and Penicillium spp. have also been

432

S. Sreejith et al.

reported to be due to the synthesis of IAA and by phosphate solubilization (Hassan 2017). The biocontrol effect of endophytes is also considered to promote plant growth indirectly. The antagonistic analysis of endophytic bacterial genera Aureobacterium, Bacillus, Paenibacillus, Phyllobacterium, Pseudomonas and Burkholderia has also revealed its inhibition towards pathogens like Fusarium oxysporum, Rhizoctonia solani, Sclerotium rolfsii, Verticillium dahliae and many other fungi (Chen et al. 1995; Rybakova et al. 2016). Although several endophytic microorganisms have been reported as candidates with potential for biocontrol, Bacillus-based products are mainly marketed as microbial pesticides, fungicides and fertilizers. Because of their biological safety, they are being commonly used in agriculture. The sporulation capacity of Bacillus also provides high resistance, ubiquity in diverse habitats and stability in the formulated products (Ongena and Jacques 2008; Tanaka et al. 2015; Mnif et al. 2016). In a previous study, Herrera et al. (2016) have demonstrated the isolation of Paenibacillus sp., Pantoea sp. and Bacillus sp., with plant growth promotion and resistance to F. graminearum from wheat seeds. This is an indication of the presence of Bacillus sp. in seed as endophyte and its agricultural promises. The biocontrol basis of endophyte mainly involves synthesis of antibiotics, lytic enzymes and induction of induced systemic resistance mechanisms in plants (Compant et al. 2005a; Gagne-Bourgue et al. 2013; Pageni et al. 2014; Sicuia et al. 2015). Hence, the seed endophytes can be considered as a reservoir of microbial candidates with highly specialized plant-beneficial functions.

20.5

Plant Growth-Promoting Features of Endophytic Microorganisms

Irrespective of their source of isolation, endophytic organisms are demonstrated to have a range of plant-beneficial mechanisms. These microorganisms influence the growth and development of host plant by increasing the nutrient uptake and modulating phytohormone levels (Dovana et al. 2015; Santoyo et al. 2016). Several plant-associated microorganisms like Pantoea agglomerans and Azoarcus sp. have been reported to fix atmospheric nitrogen and thereby converting it into plant utilizable form (Verma et al. 2001; Hurek et al. 2002). Phosphate solubilization ability of microorganism is one of the major mechanisms for plant growth promotion as it is required for the development of stem and root, flowering and also for the formation of seed and fruit. Some of the endophytic microorganisms could convert the insoluble phosphate compounds into available forms for plant through a series of reactions like acidification, chelation and exchange reaction (Chung et al. 2005). Oteino et al. (2015) have suggested the phosphate-solubilizing ability of endophytic Pseudomonas spp. and its growth enhancement effect on Pisum sativum. Microbially produced siderophore also supports the plant for the absorption of Fe3+ from its environment. This mechanism is useful in agriculture as a defence against phytopathogens also as the beneficial organisms limit the nutrients to pathogens especially fungal pathogens. Endophytic microbiome of seed can have organisms which are highly specialized for these mechanisms. Hence they play key role in seedling establishment and plant growth.

20

Agriculturally Important Biosynthetic Features of Endophytic Microorganisms

433

Endophytic production of phytohormones also indicates their key role in modulation of plant metabolism and growth. One of the well-known and widely distributed phytohormone biosyntheses in endophyte is indole-3-acetic acid (IAA) production. This phytohormone is involved in root initiation, plant cell division, extension and differentiation. IAA also increases the plant growth and development by stimulating the seed and tuber germination, increasing the rate of xylem functioning, controlling the processes of vegetative growth, initiating lateral and adventitious root formation, regulating biosynthesis of various metabolites, affecting photosynthesis and providing resistance to stressful conditions. Based on the wide spectrum of activity of IAA in plants, this seems to be a major feature of endophytes of seeds. Microbial production of IAA occurs through several pathways, and these are indole-3-acetonitrile (IAN) pathway, indole-3-acetamide (IAM) pathway, tryptamine pathway, indole-3acetaldoxime pathway and the indole-3-pyruvate (IPyA) pathway (Mano and Nemoto 2012). The indole-3-pyruvic acid pathway (IPyA pathway) function for IAA production in the presence of exogenous tryptophan. The first step in this pathway is the conversion of tryptophan into indole-3-pyruvic acid by an aminotransferase enzyme. Then it forms indole-3-acetaldehyde (IAAld) via the decarboxylation reaction catalysed by an enzyme indole-3-pyruvate decarboxylase (IPDC), and this IAAld is then oxidized to form IAA. The produced IAA will then be utilized to stimulate plant growth. IAA-producing endophytes from Curcuma longa and Piper nigrum have also demonstrated to stimulate plant growth (Aswathy et al. 2013; Jasim et al. 2014b). Endophytic fungi like Fusarium tricinctum and Alternaria alternata isolated from Solanum nigrum were also reported to enhance plant growth through the synthesis of IAA (Khan et al. 2015). The presence of multiple biosynthetic pathways for IAA production in microorganisms itself is indicative of their functional suitability as second genome of plants. To further execute its regulatory effect on plant, endophytic microorganisms have also developed mechanisms to quantitatively regulate plant hormones. In the case of ethylene, it is endogenously produced in plants and is also known as stress hormone due to its association with stress conditions like drought, salinity, waterlogging and heavy metals. The biosynthesis of ethylene from methionine takes place in three steps. First, ATP and water bind to methionine to form S-adenosyl methionine (SAM). It is further converted into ACC with the help of an enzyme 1-aminocyclopropane-1-carboxylic acid synthase (ACC-synthase). Finally ACC is enzymatically converted to ethylene. However, the increased level of ethylene adversely affects the overall plant growth and development resulting in reduced crop yield (Saleem et al. 2007). Microbial regulation of ethylene production is mediated through the production of ACC deaminase. It is a sulfhydryl enzyme with a molecular weight of 35–42 kDa. It utilizes pyridoxal 5-phosphate as an essential cofactor and several amino acids such as D-serine and D-cysteine as substrates. ACC deaminase utilizes the ACC from plant root and converts it into ammonia and α-ketobutyrate through the deamination and fragmentation of cyclopropane ring. The decreased ACC level lowers ethylene production and minimizes plant stress. Hence inoculation of plants with ACC deaminase-producing bacteria can have the promises to protect plants from stress

434

S. Sreejith et al.

conditions. The ACC deaminase activity has been reported for many bacterial genera like Acinetobacter, Achromobacter, Agrobacterium, Alcaligenes, Azospirillum, Bacillus, Burkholderia, Enterobacter, Pseudomonas, Ralstonia, Serratia and Rhizobium and also in fungi like Penicillium citrinum (Jia et al. 2000). As ethylene may have significant impact on fruit development and ripening, the adverse effects of its increased concentration in plants could be controlled by the endophytes with ACC deaminase production properties. Hence by producing ACC deaminase, endophytic organisms regulate plant physiology significantly.

20.6

Endophytic Microorganisms as Biocontrol Agents

Endophytic microorganisms produce diverse range of bioactive metabolites with potential applications in agriculture, medicine and food industries (Strobel and Daisy 2003; Jalgaonwala et al. 2011). Even though these were synthesized to provide a primary plant-beneficial function, they have enormous applications and are also considered as the least explored drug resource. The close biological association and genetic exchange between endophytes and their host plant are likely to favour the production of a greater number and types of biological molecules. Recent microbiome research has revealed the tremendous biosynthetic potential of bacterial and fungal endophytes (Godstime et al. 2014; Wu et al. 2015). This has also been considered to be directly associated with the evolutionary adaptations in endophyte to protect the plant from pathogens, insects and grazing animals (Strobel and Daisy 2003). In a recent study, Shahzad et al. (2017) have suggested the organic acids produced by seed-borne endophytic Bacillus amyloliquefaciens to have inhibitory effect to Fusarium oxysporum and also with the ability to induce induced systemic resistance in tomato plants. This is explanatory to the potential of seed endophyte to protect plant multimechanistically. Several studies have demonstrated the role of endophytes to control many of the plant diseases through the production of various secondary metabolites (Ryan et al. 2008; Compant et al. 2010). The camptothecin-producing endophytic fungi from Nothapodytes foetida have also reported to have antifungal properties (Puri et al. 2005). The antifungal and anti-oomycete metabolite ambuic acid from endophytic fungus Pestalotiopsis microspora has been demonstrated to have activity against several Fusarium species and Pythium ultimum (Li et al. 2001). Gliotoxin- and danthron-producing endophytic Acremonium sp. and Paraconiothyrium sp. from Zingiber officinale Rosc. also exhibited promising antifungal activity against soft rot pathogen Pythium myriotylum (Anisha and Radhakrishnan 2015; Anisha et al. 2018a). In addition to these, various other biomolecules such as hypericin, podophyllotoxin, tyrosol and vinblastine have also been reported from endophytic fungi which can have pharmaceutical and agricultural applications (Joseph and Priya 2011; Anisha and Radhakrishnan 2017; Zhao et al. 2011). Endophytic Pseudomonas aeruginosa isolated from Zingiber officinale was also reported to produce antifungal compounds phenazine-1-carboxylic acid with protective effect to rhizome from soft rot diseases caused by Pythium myriotylum (Jasim et al. 2014a; Sabu et al. 2017a). Seed

20

Agriculturally Important Biosynthetic Features of Endophytic Microorganisms

435

endophytic Bacillus and Pseudomonas have also been reported to have antagonistic effects against F. oxysporum f. sp. lycopersici (Fol.), the causative agent of tomato wilt (Sundaramoorthy and Balabaska 2013). Volatile antifungal compounds with biocontrol activity have also been reported from endophytic Enterobacter strains obtained from rice (Mukhopadhyay et al. 1996). Therefore the identification and characterization of a large number of diverse seed endophytes can expect to accelerate development of bioformulations to manage agricultural challenges by minimizing the use of agrochemicals. The following spectrum of biomolecules from endophytes is indicative of its agricultural potential as a living, natural and harmless bioagrochemical resource.

20.6.1 Antimicrobial Peptides Antimicrobial peptides (peptide antibiotics) are natural products present in various taxa as a part of their innate immune system against pathogen attack. These low molecular weight products were first discovered from the culture supernatant of the soil bacteria Bacillus brevis (Dubos 1939). The positively charged amino acids and the hydrophobic or hydrophilic moieties of antimicrobial peptides facilitate their binding to the negatively charged lipopolysaccharide of Gram-negative or to the lipoteichoic acid of Gram-positive bacteria. In the case of fungi, these peptides bind with the negatively charged phosphatidylinositol or to the cell wall (Makovitzki et al. 2006; Vincent and Bedon 2013). Finally, the pathogens will be killed by (1) membrane depolarization, (2) cell wall degradation, (3) modification of lipid composition of membrane bilayer or (4) micelle formation leading to cell leakage. These peptides have been evolved in diverse ways among microorganisms especially among plantassociated microorganisms. One interesting group of this is the lipopeptides synthesized by non-ribosomal peptide synthetases (Gond et al. 2015; Deng et al. 2017; Torel et al. 2018) which act against diverse phytopathogens. The mode of action of these lipopeptides involves the inhibition of synthesis of cell wall components such as β-D glucan or chitin or through the disruption of membrane barriers by forming pores leading to cell lysis. The lipopeptides like fengycin, surfactin and iturin are shown to have the promises to fight against plant pathogens (Romero et al. 2007; Ongena and Jacques 2008; Roongsawang et al. 2010; Romano et al. 2011; Jasim et al. 2016b; Dimkic et al. 2017). These families share a cyclic β-amino or β-hydroxy fatty acid linked to a lipid tail (Tapi et al. 2010; Jemil et al. 2017). Biological activities may differ from one compound to another depending on the type of amino acid residues, the cyclization of the peptide and the length and branching of the fatty acid chain (Ongena and Jacques 2008; Frikha-Gargouri et al. 2017). Several bacterial endophytes including those from seeds have reported to have antifungal activity mediated through surfactin, iturin and mycobacillin (GagneBourgue et al. 2013).

20.6.1.1 Surfactin Surfactin is a cyclic lipopeptide antibiotic produced by most of the endophytic of Bacillus species and was initially reported in 1968 by Arima et al. (1968). Surfactin

436

S. Sreejith et al.

works against the pathogens by lowering the surface tension of phospholipid bilayer. Also, surfactin directly binds to the lipid bilayer of the target organism and causes depolarization of membrane. Though surfactin is mainly reported to act against fungal pathogens, it can have haemolytic, antibacterial, antiviral and antimycoplasma properties. This lipopeptide is composed of a hydrophilic peptide portion and a hydrophobic fatty acid part. The peptide portion of surfactin is composed of seven amino acids (Glu-Leu-Leu-Val-Asp-Leu-Leu) in a LLLDLLDL chiral series which are linked with a cyclic lactone bond to a β-hydroxy fatty acid. The synthesis of surfactin is mediated by non-ribosomal peptide synthetase. Among various Bacillus spp., Bacillus subtilis has been reported as a major endophyte with surfactin biosynthetic features along with iturin and other lipopeptides as coproducts. The several variants of surfactin include lichenysin and pumilacidin which were produced by other Bacillus species.

20.6.1.2 Iturin Antibiotics of iturin family are basically cyclic lipopeptides and include iturin A, mycosubtilin, bacillomycin, etc. and are one of the most commonly studied compounds for their antifungal activities (Moyne et al. 2004). It consists of a cyclic peptide composed of 7 amino acid residues and a hydrophobic tail with 11–12 carbon atoms. Due to this structure, the compound has a strong amphiphilic character and thus has high probability to act towards the target cell membrane (Aranda et al. 2005). It has strong antibiotic activity against a wide range of pathogens which makes the compound a promising candidate for biological control (Hsieh et al. 2008). Bacillomycin D of iturin family has been demonstrated to act against various plant pathogenic fungi such as Fusarium graminearum (Gu et al. 2017). 20.6.1.3 Fengycin Fengycin or plipastatin is the third family of lipopeptides produced by Bacillus subtilis. The structure of fengycin is characterized by the decapeptides linked to a fatty acid chain of varying lengths from C-14 to C-17 (Meena and Kanwar 2015). The production of fengycin was primarily reported by Ulrike Koch (1986) from Bacillus subtilis strain F-29-3 with antifungal activity against filamentous fungi. Fengycin consists of two main components differing by one amino acid sequence. Fengycin A is composed of I D-Ala, 1 L-Ile, 1 L-Pro, 1 D-allo-Thr, 3 L-Glx, 1 D-Tyr, 1 L-Tyr and I D-Orn, whereas in fengycin B the D-Ala is replaced by D-Val. Wang et al. (2015) have reported Bacillus subtilis NCD-2 to be an excellent biocontrol agent for tomato grey mould and cotton soil-borne diseases due to the production of fengycin lipopeptides. 20.6.1.4 Cryptocandin and Cryptocin Cryptocandin is another lipopeptide isolated from the fungus Cryptosporiopsis quercina associated with hardwood species in Europe. It is active against a number of plant pathogenic fungi, including Sclerotinia sclerotiorum and Botrytis cinerea (Strobel et al. 1999), and is related to antimycotic echinocandins and the pneumocandins. Cryptocin produced by C. quercina also demonstrated to have potent activity against Pyricularia oryzae, Fusarium oxysporum, Geotrichum

20

Agriculturally Important Biosynthetic Features of Endophytic Microorganisms

437

candidum, Rhizoctonia solani, S. sclerotiorum, P. ultimum, Phytophthora cinnamon and P. citrophthora (Li et al. 2000).

20.6.1.5 Ecomycins These lipopeptides were identified from the plant-associated Pseudomonas viridiflava present on or within the leaf tissues of Lactuca sativa and many grass species (Miller et al. 1998). The three types of ecomycin lipopeptide compounds (A, B and C) have been demonstrated to have diverse bioactivity. 20.6.1.6 Fusaricidins and LI-F Lipopeptides Fusaricidins and the closely related LI-F lipopeptides from Paenibacillus polymyxa are found to have inhibitory activity against plant pathogens including Rhodotorula aurantiaca, F. oxysporum, B. cinerea, Cladosporium cucumerinum, Pythium aphanidermatum and P. syringae (Debois et al. 2013). 20.6.1.7 Phenazines Phenazines are nitrogen-containing heterocyclic metabolites produced by Gramnegative and Gram-positive bacteria like Pseudomonas spp., Streptomyces, Brevibacterium and Xanthomonas and the archaeal genus Methanosarcina. There are different derivatives of phenazine antibiotics. The major group is phenazine-1carboxylic acid followed by 2-hydroxy phenazine-1-carboxylic acid and the minor phenazine group 2-hydroxy phenazine. The production of phenazine antibiotics occurs via the shikimic acid pathway by utilizing nitrogen from glutamine and joining two chorismic acid molecules to form the basic phenazine structure. The addition of different functional groups can determine its solubility and thereby biological function. The small modifications in the functional groups generate the pigments of Pseudomonas which range from deep red to lemon yellow and to bright blue. The antimicrobial mechanism of phenazine compounds involves inhibition of DNA replication and uncoupling the electron transport, inhibiting energy production and disrupting membrane function.

20.6.2 Production of Hydrogen Cyanide Hydrogen cyanide (HCN) is a highly diffusible metabolite which works against phytopathogens. The killing effect of HCN involves inhibition of terminal cytochrome C oxidase and other metalloenzymes. HCN synthase, the membrane-associated enzyme, synthesizes HCN and is mainly present in Proteobacteria especially in Pseudomonas species. HCN is also produced by fungi and several cyanobacteria but has less importance. HCN synthase is highly sensitive to oxygen and in Pseudomonas and Chromobacterium; glycine protects the enzyme from oxygen toxicity. In Pseudomonas, threonine is converted to glycine for subsequent production of HCN. During the process of HCN production, CO2 is formed as by-product and imino acetic acid as an intermediate metabolite.

438

S. Sreejith et al.

20.6.3 Production of Lytic Enzymes Several enzymes are also produced by plant-associated microorganisms. Chitinases degrade chitin present in cell wall of pathogens to water-soluble chitooligosaccharides. This has wide physiological activities in bacteria, fungi, plants, archaea and animals (Das et al. 2018; Krolicka et al. 2018). Plants utilize this enzyme for the protection from fungal pathogens (Zarei et al. 2011). Its cell wall lytic ability exposes the cellular material of microbes and thereby prevents multiplication of pathogens (Zarei et al. 2011; Hong et al. 2017). However, the enzymatic potential of seed endophytes is least studied. Due to their functional association with seeds, enzymatic features of seed endophytes can have both industrial and agricultural applications. All the plantbeneficial features of endophytes as mentioned in this chapter indicate the immense biosynthetic promises of seed endophytes which in coming days can be exploited for agricultural applications.

20.7

Conclusion

Endophytic microorganisms have attracted considerable attention due to their plant growth-promoting and disease resistance properties by which they protect plants from biotic and abiotic stresses. Biosynthetically promising endophytic microbiome can be vertically transmitted through seeds to the next generation. By analysing the biosynthetic potential of already characterized endophytes, many biochemical opportunities with agricultural applications can be expected from seed endophytes. The role of seeds as a carrier of plant-beneficial microorganisms is just beginning to be explored.

References Anisha C, Radhakrishnan EK (2015) Gliotoxin-producing endophytic Acremonium sp. from Zingiber officinale found antagonistic to soft rot pathogen Pythium myriotylum. Appl Biochem Biotechnol 175(7):3458–3467 Anisha C, Radhakrishnan EK (2017) Metabolite analysis of endophytic fungi from cultivars of Zingiber officinale Rosc identifies myriad of bioactive compounds including tyrosol. 3 Biotech 7(2):146 Anisha C, Sachidanandan P, Radhakrishnan EK (2018a) Endophytic Paraconiothyrium sp. from Zingiber officinale Rosc. displays broad-spectrum antimicrobial activity by production of danthron. Curr Microbiol 75(3):343–352 Anisha C, Jishma P, Sasi Bilzamol V, Radhakrishnan EK (2018b) Effect of ginger endophyte Rhizopycnis vagum on rhizome bud formation and protection from phytopathogens. Biocatal Agric Biotechnol 14:116–119 Aranda FJ, Teruel JA, Ortiz A (2005) Further aspects on the hemolytic activity of the antibiotic lipopeptide iturin A. Biochim Biophys Acta Biomembr 1713(1):51–56 Arima K, Kakinuma A, Tamura G (1968) Surfactin, a crystalline peptide lipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation. Biochem Biophys Res Commun 31(3):488–494 Aswathy AJ, Jasim B, Jyothis M, Radhakrishnan EK (2013) Identification of two strains of Paenibacillus sp. as indole 3 acetic acid-producing rhizome-associated endophytic bacteria from Curcuma longa. 3 Biotech 3(3):219–224

20

Agriculturally Important Biosynthetic Features of Endophytic Microorganisms

439

Bernabeu PR, Pistorio M, Torres-tejerizo G, Santos PEL, Galar ML, Boiardi JL et al (2015) Colonization and plant growth-promotion of tomato by Burkholderia tropica. Sci Hortic (Amsterdam) 191:113–120. https://doi.org/10.1016/j.scienta.2015.05.014 Bhardwaj A, Agrawal P (2014) A review fungal endophytes: as a store house of bioactive compound. World J Pharm Pharm Sci 3:228–237 Bohm 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 Brader G, Compant S, Vescio K, Mitter B, Trognitz F, Ma L-J et al (2017) Ecology and genomic insights into plant-pathogenic and plant-non pathogenic endophytes. Annu Rev Phytopathol 55:61–83. https://doi.org/10.1146/annurev-phyto-080516-035641 Chee-Sanford JC, Williams MM, Davis AS, Sims GK (2006) Do microorganisms influence seedbank dynamics. Weed Sci 54:575–587. https://doi.org/10.1614/WS-05-055R.1 Chen C, Bauske EM, Musson G, Rodriguezkabana R, Kloepper JW (1995) Biological control of fusarium wilt on cotton by use of endophytic bacteria. Biol Control 5:83–91. https://doi.org/10. 1006/bcon.1995.1009 Chi F, Shen S, Cheng H, Jing Y, Yanni YG, Dazzo FB (2005) Ascending migration of endophytic rhizobia, from roots to leaves, inside rice plants and assessment of benefits to rice growth. Physiology 71(11):7271–7278. https://doi.org/10.1128/AEM.71.11.7271 Chung H, Park M, Madhaiyan M, Seshadri S, Song J, Cho H, Sa T (2005) Isolation and characterization of phosphate solubilizing bacteria from the rhizosphere of crop plants of Korea. Soil Biol Biochem 37(10):1970–1974 Compant S, Duffy B, Nowak J, Clément C, Barka EA (2005a) Use of plant growth-promoting bacteria for biocontrol of plant diseases: principles, mechanisms of action, and future prospects. Appl Environ Microbiol 71(9):4951–4959 Compant S, Reiter B, Nowak J, Sessitsch A, Clément C, Barka EA (2005b) Endophytic colonization of Vitis vinifera L. by plant growth-promoting bacterium Burkholderia sp. strain PsJN. Appl Environ Microbiol 71:1685–1693 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 Compant S, Mitter B, Colli-Mull JG, Gangl H, Sessitsch A (2011) Endophytes of grapevine flowers, berries and seeds: identification of cultivable bacteria, comparison with other plant parts, and visualization of niches of colonization. Microb Ecol 62:188–197 Cope-Selby N, Cookson A, Squance M, Donnison I, Flavell R, Farrar K (2017) Endophytic bacteria in Miscanthus seed: implications for germination, vertical inheritance of endophytes, plant evolution and breeding. GCB Bioeng 9:57–77. https://doi.org/10.1111/gcbb.12364 Coutinho BG, Licastro D, Mendonça-Previato L, Camara M, Venturi V (2014) Plant-influenced gene expression in the rice endophyte Burkholderia kururiensis M130. Mol Plant-Microbe Interact 28:10–21. https://doi.org/10.1094/MPMI-07-14-0225-R Das S, Dey P, Roy D et al (2018) N-acetyl-D-glucosamine production by a chitinase of marine fungal origin : a case study of potential industrial significance for valorization of waste chitins. Appl Biochem Biotechnol 187(1):407–423 De Bary A (1866) Morphologie und Physiologie Pilze, Flechten, und myxomyceten. In: Hofmeister’s handbook of physiological botany, vol 2. Verlag Von Wilhelm Engelmann, Leipzig Debois D, Ongena M, Cawoy H, De Pauw E (2013) MALDI-FTICR MS imaging as a powerful tool to identify Paenibacillus antibiotics involved in the inhibition of plant pathogens. J Am Soc Mass Spectrom 24(8):1202–1213 Deng Q, Wang W, Sun L, Wang Y, Liao J, Xu D, Liu Y, Ye R, Gooneratne R (2017) A sensitive method for simultaneous quantitative determination of surfactin and iturin by LC-MS/MS. Anal Bioanal Chem 409(1):179–191

440

S. Sreejith et al.

Dimkić I, Stanković S, Nišavić M, Petković M, Ristivojević P, Fira D, Berić T (2017) The profile and antimicrobial activity of Bacillus lipopeptide extracts of five potential biocontrol strains. Front Microbiol 8:925 Dovana F, Mucciarelli M, Mascarello M, Fusconi A (2015) In vitro morphogenesis of Arabidopsis to search for novel endophytic fungi modulating plant growth. PLoS One 10(12):e0143353 Dubos RJ (1939) Studies on a bactericidal agent extracted from a soil Bacillus: I. Preparation of the agent. Its activity in vitro. J Exp Med 70(1):1 Fisher PJ, Petrini O, Lappin-Scott HM (1992) The distribution of some fungal and bacteria endophytes in maize (Zea mays L.). New Phytol 122:299–305 Fouts DE, Tyler HL, DeBoy RT, Daugherty S, Ren Q, Badger JH, Durkin AS, Huot H, Shrivastava S, Kothari S, Dodson RJ, Mohamoud Y, Khouri H, Roesch LFW, Krogfelt KA, Struve C, Triplett EW, Methé BA (2008) Complete genome sequence of the N2-fixing broad host range endophyte Klebsiella pneumoniae 342 and virulence predictions verified in mice. PLoS Genet 4(7):1–18 Frikha-Gargouri O, Ben Abdallah D, Ghorbel I, Charfeddine I, Jlaiel L, Triki MA, Tounsi S (2017) Lipopeptides from a novel Bacillus methylotrophicus 39b strain suppress agrobacterium crown gall tumours on tomato plants. Pest Manag Sci 73(3):568–574 Gagne-Bourgue F, Aliferis KA, Seguin P, Rani M, Samson R, Jabaji S (2013) Isolation and characterization of indigenous endophytic bacteria associated with leaves of switchgrass (Panicum virgatum L.) cultivars. J Appl Microbiol 114:836–853. https://doi.org/10.1111/jam.12088 Garg N, Geetanjali (2007) Symbiotic nitrogen fixation in legume nodules: process and signalling. A review. Agron Sustain Dev 27:59–68 Gazis R, Chaverri P (2010) Diversity of fungal endophytes in leaves and stems of wild rubber trees (Hevea brasiliensis) in Peru. Fungal Ecol 3:240–254. https://doi.org/10.1016/j.funeco.2009.12.001 Geisen S, Kostenko O, Cnossen MC, ten Hooven FC, Vres B, van der Putten WH (2017) Seed and root endophytic fungi in a range expanding and a related plant species. Front Microbiol 8:1645 Ginting RCB, Sukarno N, Widyastuti U et al (2013) Diversity of endophytic fungi from red ginger (Zingiber officinale Rosc.) plant and their inhibitory effect to Fusarium oxysporum plant pathogenic fungi. HAYATI J Biosci 20:127–137. https://doi.org/10.4308/hjb.20.3.127 Godstime OC, Enwa FO, Augustina JO, Christopher EO (2014) Mechanisms of antimicrobial actions of phytochemicals against enteric pathogens–a review. J Pharm Chem Biol Sci 2:77–85 Golinska P, Wypij M, Agarkar G, Rathod D, Dahm H, Rai M (2015) Endophytic actinobacteria of medicinal plants: diversity and bioactivity. Antonie Van Leeuwenhoek 108:267–289. https:// doi.org/10.1007/s10482-015-0502-7 Gond SK, Bergen MS, Torres MS, White JF Jr (2015) Endophytic Bacillus spp. produce antifungal lipopeptides and induce host defence gene expression in maize. Microbiol Res 172:79–87 Gouda S, Das G, Sen SK, Shin H-S, Patra JK (2016) Endophytes: a treasure house of bioactive compounds of medicinal importance. Front Microbiol 7:1538 Govindarajan M, Balandreau J, Kwon S-W, Weon H-Y, Lakshminarasimhan C (2008) Effects of the inoculation of Burkholderia vietnamensis and related endophytic diazotrophic bacteria on grain yield of rice. Microb Ecol 55:21–37. https://doi.org/10.1007/s00248-007-9247-9 Gu Q, Yang Y, Yuan Q, Shi G, Wu L, Lou Z, Huo R, Wu H, Borriss R, Gao X (2017) Bacillomycin D produced by Bacillus amyloliquefaciens is involved in the antagonistic interaction with the plant pathogenic fungus Fusarium graminearum. Appl Environ Microbiol 83:AEM-01075 Hallmann J (2001) Plant interactions with endophytic bacteria. In: Jeger MJ, Spence NJ (eds) Biotic interactions in plant–pathogen associations. CABI, Wallingford, pp 87–119 Hallmann J, Quadt-Hallmann A, Mahaffee WF, Kloepper JW (1997) Bacterial endophytes in agricultural crops. Can J Microbiol 43(10):895–914. https://doi.org/10.1139/m97-131 Hardoim PR, van Overbeek LS, Elsa v (2008) Properties of bacterial endophytes and their proposed role in plant growth. Trends Microbiol 16:463–471 Hardoim PR, Hardoim CC, Van Overbeek LS, Van Elsas JD (2012) Dynamics of seed-borne rice endophytes on early plant growth stages. PLoS One 7(2):e30438

20

Agriculturally Important Biosynthetic Features of Endophytic Microorganisms

441

Hassan SE-D (2017) Plant growth-promoting activities for bacterial and fungal endophytes isolated from medicinal plant of Teucrium polium L. J Adv Res 8(6):687–695. https://doi.org/10.1016/j. jare.2017.09.001 Herrera SD, Grossi C, Zawoznik M, Groppa MD (2016) Wheat seeds harbour bacterial endophytes with potential as plant growth promoters and biocontrol agents of Fusarium graminearum. Microbiol Res 186–187:37–43. https://doi.org/10.1016/j.micres.2016.03.002 Hodgson S, de Cates C, Hodgson J, Morley NJ, Sutton BC, Gange AC (2014) Vertical transmission of fungal endophytes is widespread in forbs. Ecol Evol 4:1199–1208. https://doi.org/10.1002/ece3.953 Hollants J, Leroux O, Leliaert F, Decleyre H, De Clerck O, Willems A (2011) Who is in there? Exploration of endophytic bacteria with in the siphonous green seaweed Bryopsis (Bryopsidales, Chlorophyta). PLoS One 6:e26458. https://doi.org/10.1371/journal.pone.0026458 Hong SH, Song YS, Seo DJ, Kim KY, Jung WJ (2017) Antifungal activity and expression patterns of extracellular chitinase and β-1, 3-glucanase in Wickerhamomyces anomalus EG2 treated with chitin and glucan. Microb Pathog 110:159–164 Hsieh FC, Lin TC, Meng M, Kao SS (2008) Comparing methods for identifying Bacillus strains capable of producing the antifungal lipopeptide iturin A. Curr Microbiol 56(1):1–5 Hu X, Roberts DP, Maul JE et al (2011) Formulations of the endophytic bacterium Bacillus subtilis Tu-100 suppress Sclerotinia sclerotiorum on oilseed rape and improve plant vigor in field trials conducted at separate locations. Can J Microbiol 57:539–546 Hubbard M, Germida JJ, Vujanovic V (2014) Fungal endophytes enhance wheat heat and drought tolerance in terms of grain yield and second-generation seed viability. J Appl Microbiol 116:109–122 Hurek T, Handley LL, Reinhold-Hurek B et al (2002) Azoarcus grass endophytes contribute fixed nitrogen to the plant in an unculturable state. Mol Plant-Microbe Interact 15(3):233–242 Jain P, Pundir RK (2017) Potential role of endophytes in sustainable agriculture-recent developments and future prospects. In: Maheshwari D (ed) Endophytes: biology and biotechnology. Sustainable development and biodiversity, vol 15. Springer, Cham Jalgaonwala RE, Mohite BV, Mahajan RT (2011) Natural products from plant associated endophytic fungi. J Microbiol Biotechnol Res 1:21–32 Jallow MFA, Dugassa-Gobena D, Vidal S (2004) Indirect interaction between an unspecialised endophytic fungus and a polyphagous moth. Basic Appl Ecol 5:183–191 Jasim B, Anisha C, Rohini S, Kurian JM, Jyothis M, Radhakrishnan EK (2014a) Phenazine carboxylic acid production and rhizome protective effect of endophytic Pseudomonas aeruginosa isolated from Zingiber officinale. World J Microbiol Biotechnol 30(5):1649–1654 Jasim B, Joseph AA, John CJ, Mathew J, Radhakrishnan EK (2014b) Isolation and characterization of plant growth promoting endophytic bacteria from the rhizome of Zingiber officinale. 3 Biotech 4(2):197–204 Jasim B, Anish MC, Shimil V, Jyothis M, Radhakrishnan EK (2015) Studies on plant growth promoting properties of fruit-associated bacteria from Elettaria cardamomum and molecular analysis of ACC deaminase gene. Appl Biochem Biotechnol 177(1):175–189. https://doi.org/ 10.1007/s12010-015-1736-6 Jasim B, Mathew J, Radhakrishnan EK (2016a) Identification of a novel endophytic Bacillus sp. from Capsicum annuum with highly efficient and broad spectrum plant probiotic effect. J Appl Microbiol 121(4):1079–1094. https://doi.org/10.1111/jam.13214 Jasim B, Sreelakshmi KS, Mathew J, Radhakrishnan EK (2016b) Surfactin, iturin, and fengycin biosynthesis by endophytic Bacillus sp. from Bacopa monnieri. Microb Ecol 72(1):106–119 Jemil N, Manresa A, Rabanal F, Ayed HB, Hmidet N, Nasri M (2017) Structural characterization and identification of cyclic lipopeptides produced by Bacillus methylotrophicus DCS1 strain. J Chromatogr B 1060:374–386 Jia Y, McAdams SA, Bryan GT, Hershey HP, Valent B (2000) Direct interaction of resistance gene and a virulence gene products confers rice blast resistance. EMBO J 19(15):4004–4014 Joseph B, Priya M (2011) Review on nutritional, medicinal and pharmacological properties of guava (Psidium guajava Linn.). Int J Pharm Biol Sci 2(1):53–69

442

S. Sreejith et al.

Kaga H, Mano H, Tanaka F, Watanabe A, Kaneko S, Morisaki H (2009) Rice seeds as sources of endophytic bacteria. Microbes Environ 24:154–162 Khalaf EM, Raizada MN (2018) Bacterial seed endophytes of domesticated cucurbits antagonize fungal and oomycete pathogens including powdery mildew. Front Microbiol 9(FEB):1–18. https://doi.org/10.3389/fmicb.2018.00042. Khamchatra N, Dixon K, Chayamarit K, Apisitwanich S, Tantiwiwat S (2016) Using in situ seed baiting technique to isolate and identify endophytic and mycorrhizal fungi from seeds of a threatened epiphytic orchid, Dendrobium friedericksianum Rchb.f. (Orchidaceae). Agric Nat Resour 50:8–13. https://doi.org/10.1016/j.anres.2016.01.002 Khan AR, Ullah I, Waqas M, Shahzad R, Hong SJ, Park GS, Jung BK, Lee IJ, Shin JH (2015) Plant growth-promoting potential of endophytic fungi isolated from Solanum nigrum leaves. World J Microbiol Biotechnol 31(9):1461–1466 Khan Z, Rho H, Firrincieli A, Hung SH, Luna V, Masciarelli O, Kim SH, Doty SL (2016) Growth enhancement and drought tolerance of hybrid poplar upon inoculation with endophyte consortia. Curr Plant Biol 6:38–47 Klaedtke S, Jacques M-A, Raggi L et al (2015) Terroir is a key driver of seed-associated microbial assemblages. Environ Microbiol 18(6):1792–1804 Kloepper JW, Ryu C-M (2006) Bacterial endophytes as elicitors of induced systemic resistance. In: Schulz B, Boyle C, Sieber TN (eds) Microbial root endophytes. Springer, Berlin, pp 33–52 Krause A, Ramakumar A, Bartels D, Battistoni F, Bekel T, Boch J, Böhm M, Friedrich F, Hurek T, Krause L, Linke B (2006) Complete genome of the mutualistic, N2-fixing grass endophyte Azoarcus sp. strain BH72. Nat Biotechnol 24(11):1 Krishnan P, Bhat R, Kush A, Ravikumar P (2012) Isolation and functional characterization of bacterial endophytes from Carica papaya fruits. J Appl Microbiol 113(2):308–317 Krolicka M, Hinz SW, Koetsier MJ, Joosten R, Eggink G, van den Broek LA, Boeriu CG (2018) Chitinase Chi1 from Myceliophthora thermophila C1, a thermostable enzyme for chitin and chitosan depolymerization. J Agric Food Chem 66(7):1658–1669 Kwak JY, Song SY, Kim H, Jeong SG, Kang BK, Kim SK, Kwon CH, Lee DSY, Park SH, Kim JF (2012) Complete genome sequence of the endophytic bacterium Burkholderia sp. strain KJ006. J Bacteriol 194:4432–4433 Larran S, Simon MR, Moreno MV, Siurana MPS, Perell A (2016) Endophytes from wheat as biocontrol agents against tan spot disease. Biol Control 92:17–23 Li JY, Strobel G, Harper J, Lobkovsky E, Clardy J (2000) Cryptocin, a potent tetramic acid antimycotic from the endophytic fungus Cryptosporiopsis cf. quercina. Org Lett 2(6):767–770 Li JY, Harper JK, Grant DM, Tombe BO, Bashyal B, Hess WM, Strobel GA (2001) Ambuic acid, a highly functionalized cyclohexenone with antifungal activity from Pestalotiopsis spp. and Monochaetia sp. Phytochemistry 56(5):463–468 Liu Y, Zuo S, Zou Y, Wang J, Song W (2013) Investigation on diversity and population succession dynamics of endophytic bacteria from seeds of maize (Zea mays L., Nongda108) at different growth stages. Ann Microbiol 63:71–79. https://doi.org/10.1007/s13213-012-0446-3 Liu J, Nagabhyru P, Schardl CL (2017) Epichloë festucae endophytic growth in florets, seeds, and seedlings of perennial ryegrass (Lolium perenne). Mycologia 109(5):691–700. https://doi.org/ 10.1080/00275514.2017.1400305 Lodewyckx C, Vangronsveld J, Porteous F, Moore ERB, Taghavi S, Mezgeay M, Van Der Lelie D (2002) Endophytic bacteria and their potential applications. Crit Rev Plant Sci 21:583–606 López-López A, Rogel MA, Ormeño-Orrillo E, Martínez-Romero J, Martínez-Romero E (2010) Phaseolus vulgaris seed-borne endophytic community with novel bacterial species such as Rhizobium endophyticum sp. nov. Syst Appl Microbiol 33:322–327. https://doi.org/10.1016/j. syapm.2010.07.005 Maehara S, Agusta A, Kitamura C, Ohashi K, Shibuya H (2016) Composition of the endophytic filamentous fungi associated with Cinchona ledgeriana seeds and production of cinchona alkaloids. J Nat Med 70:271–275. https://doi.org/10.1007/s11418-015-0954-0

20

Agriculturally Important Biosynthetic Features of Endophytic Microorganisms

443

Makovitzki A, Avrahami D, Shai Y (2006) Ultrashort antibacterial and antifungal lipopeptides. Proc Natl Acad Sci 103(43):15997–16002 Malfanova N, Lugtenberg BJJ, Berg G (2013) Bacterial endophytes: who and where, and what are they doing there. In: de Bruijn FJ (ed) Molecular microbial ecology of the rhizosphere. WileyBlackwell, Hoboken, NJ, pp 391–403 Mano Y, Nemoto K (2012) The pathway of auxin biosynthesis in plants. J Exp Bot 63(8):2853–2872 Mano H, Tanaka F, Watanabe A, Kaga H, Okunishi S, Morisaki H (2006) Culturable surface and endophytic bacterial flora of the maturing seeds of rice plants (Oryza sativa) cultivated in a paddy field. Microbes Environ 21:86–100. https://doi.org/10.1264/jsme2.21.86 Mastretta C, Taghavi S, van der Lelie D, Mengoni A, Galardi F, Gonnelli C et al (2009) Endophytic bacteria from seeds of Nicotiana Tabacum can reduce cadmium phytotoxicity. Int J Phytoremed 11:251–267. https://doi.org/10.1080/15226510802432678 Mattos KA, Pádua VLM, Romeiro A, Hallack LF, Neves BC, Ulisses TMU et al (2008) Endophytic colonization of rice (Oryza sativa L.) by the diazotrophic bacterium Burkholderia kururiensis and its ability to enhance plant growth. An Acad Bras Cienc 80:477–493. https://doi.org/10. 1590/S0001-37652008000300009 Meena KR, Kanwar SS (2015) Lipopeptides as the antifungal and antibacterial agents: applications in food safety and therapeutics. Biomed Res Int 2015:1 Miller JT, Dong F, Jackson SA, Song J, Jiang J (1998) Retrotransposon-related DNA sequences in the centromeres of grass chromosomes. Genetics 150(4):1615–1623 Mnif I, Grau-Campistany A, Coronel-León J, Hammami I, Triki MA, Manresa A, Ghribi D (2016) Purification and identification of Bacillus subtilis SPB1 lipopeptide biosurfactant exhibiting antifungal activity against Rhizoctonia bataticola and Rhizoctonia solani. Environ Sci Pollut Res 23(7):6690–6699 Moyne AL, Cleveland TE, Tuzun S (2004) Molecular characterization and analysis of the operon encoding the antifungal lipopeptide bacillomycin D. FEMS Microbiol Lett 234(1):43–49 Mukhopadhyay K, Garrison NK, Hinton DM, Bacon CW, Khush GS, Peck HD, Datta N (1996) Identification and characterization of bacterial endophytes of rice. Mycopathologia 134 (3):151–159 Newcombe G, Shipunov A, Eigenbrode S, Raghavendra AK, Ding H, Anderson CL, Schwarzländer M (2009) Endophytes influence protection and growth of an invasive plant. Commun Integr Biol 2 (1):29–31 Newsham KK (2011) A meta-analysis of plant responses to dark septate root endophytes. New Phytol 190:783–793 Ongena M, Jacques P (2008) Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends Microbiol 16:115–125. https://doi.org/10.1016/j.tim.2007.12.009 Onja Andriambeloson H (2016) Biological potentials of ginger associated Streptomyces compared with ginger essential oil. Am J Life Sci 4:152 Oteino N, Lally RD, Kiwanuka S, Lloyd A, Ryan D, Germaine KJ, Dowling DN (2015) Plant growth promotion induced by phosphate solubilizing endophytic Pseudomonas isolates. Front Microbiol 6:745 Pageni BB, Lupwayi NZ, Akter Z, Larney FJ, Kawchuk LM, Gan Y (2014) Plant growth-promoting and phytopathogen-antagonistic properties of bacterial endophytes from potato (Solanum tuberosum L.) cropping systems. Can J Plant Sci 94(5):835–844 Pandya M, Rajput M, Rajkumar S (2015) Exploring plant growth promoting potential of non rhizobial root nodules endophytes of Vigna radiata. Microbiology 84:80–89. https://doi.org/10. 1134/S0026261715010105 Parsa S, García-Lemos AM, Castillo K et al (2016) Fungal endophytes in germinated seeds of the common bean, Phaseolus vulgaris. Fungal Biol 120:783–790. https://doi.org/10.1016/j.funbio. 2016.01.017 Paungfoo-Lonhienne C, Rentsch D, Robatzek S, Webb RI, Sagulenko E, Näsholm T, Schmidt S, Lonhienne TGA (2010) Turning the table: plants consume microbes as a source of nutrients. PLoS One 5:e11915

444

S. Sreejith et al.

Paungfoo-Lonhienne C, Schmidt S, Webb RI (2013) Interactions, P.M. Rhizophagy—a new dimension of plant—microbe interactions. In: de Bruijn FJ (ed) Molecular microbial ecology of the rhizosphere, vol 2. Wiley-Blackwell, Hoboken, NJ, pp 1201–1207 Paungfoo-Lonhienne C, Lonhienne TGA, Yeoh YK, Webb RI, Lakshmanan P, Chan CX et al (2014) A new species of Burkholderia isolated from sugarcane roots promotes plant growth. Microb Biotechnol 7:142–154. https://doi.org/10.1111/1751-7915.12105 Pimentel MR, Molina G, Dionisio AP, Maróstica MR, Pastore GM (2011) Use of endophytes to obtain bioactive compounds and their application in biotransformation process. Bio Technol Res Int 576286:2011. https://doi.org/10.4061/2011/576286 Pitzschke A (2018) Molecular dynamics in germinating, endophyte-colonized quinoa seeds. Plant Soil 422(1–2):135–154. https://doi.org/10.1007/s11104-017-3184-2. Prieto P, Schilirò E, Maldonado-González MM, Valderrama R, Barroso-Albarracín JB, MercadoBlanco J (2011) Root hairs play a key role in the endophytic colonization of olive roots by Pseudomonas spp. with biocontrol activity. Microb Ecol 62:435–445 Puri SC, Verma V, Amna T, Qazi GN, Spiteller M (2005) An endophytic fungus from Nothapodytes foetida that produces camptothecin. J Nat Prod 68(12):1717–1719 Rajamanikyam M, Vadlapudi V, Amanchy R, Upadhyayula SM (2017) Endophytic fungi as novel resources of natural therapeutics. Braz Arch Biol Technol 60:e17160542 Rohini S, Aswani R, Kannan M et al (2018) Culturable endophytic bacteria of ginger rhizome and their remarkable multi-trait plant growth-promoting features. Curr Microbiol 75:505. https://doi. org/10.1007/s00284-017-1410-z Romano A, Vitullo D, Di Pietro A, Lima G, Lanzotti V (2011) Antifungal lipopeptides from Bacillus amyloliquefaciens strain BO7. J Nat Prod 74(2):145–151 Romero D, de Vicente A, Rakotoaly RH, Dufour SE, Veening JW, Arrebola E, Cazorla FM, Kuipers OP, Paquot M, Pérez-García A (2007) The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca. Mol Plant-Microbe Interact 20(4):430–440 Roongsawang N, Washio K, Morikawa M (2010) Diversity of nonribosomal peptide synthetases involved in the biosynthesis of lipopeptide biosurfactants. Int J Mol Sci 12(1):141–172 Rozpadek P, Wezowicz K, Nosek M, Wazny R, Tokarz K, Lembicz M et al (2015) The fungal endophyte Epichloë typhina improves photosynthesis efficiency of its host orchard grass (Dactylis glomerata). Planta 242:1025–1035. https://doi.org/10.1007/s00425-015-2337-x Ruiz D, Agaras B, Werra P, Wall LG, Valverde C (2011) Characterization and screening of plant probiotic traits of bacteria isolated from rice seeds cultivated in Argentina. J Microbiol 49:902–912 Ryan RP, Germaine K, Franks A, Ryan DJ, Dowling DN (2008) Bacterial endophytes: recent developments and applications. FEMS Microbiol Lett 278:1–9 Rybakova D, Cernava T, Köberl M, Liebminger S, Etemadi M, Berg G (2016) Endophytes-assisted biocontrol: novel insights in ecology and the mode of action of Paenibacillus. Plant Soil 405:125–140. https://doi.org/10.1007/s11104-015-2526-1 Sabu R, Aswani R, Jishma P, Jasim B, Mathew J, Radhakrishnan EK (2017a) Plant growth promoting endophytic Serratia sp. ZoB14 protecting ginger from fungal pathogens. Proc Natl Acad Sci India Sect B Biol Sci 7:1–8 Sabu R, Soumya KR, Radhakrishnan EK (2017b) Endophytic Nocardiopsis sp. from Zingiber officinale with both antiphytopathogenic mechanisms and antibiofilm activity against clinical isolates. 3 Biotech 7(115):115. https://doi.org/10.1007/s13205-017-0735-4 Saikkonen K, Wäli P, Helander M, Faeth SH (2004) Evolution of endophyte–plant symbioses. Trends Plant Sci 9:275–280. https://doi.org/10.1016/j.tplants.2004.04.005 Saini R, Dudeja SS, Giri R, Kumar V (2015) Isolation, characterization, and evaluation of bacterial root and nodule endophytes from chickpea cultivated in northern India. J Basic Microbiol 55:74–81. https://doi.org/10.1002/jobm.201300173

20

Agriculturally Important Biosynthetic Features of Endophytic Microorganisms

445

Saleem M, Arshad M, Hussain S, Bhatti AS (2007) Perspective of plant growth promoting rhizobacteria (PGPR) containing ACC deaminase in stress agriculture. J Ind Microbiol Biotechnol 34(10):635–648 Santoyo G, Moreno-Hagelsieb G, del Carmen Orozco-Mosqueda M, Glick BR (2016) Plant growth-promoting bacterial endophytes. Microbiol Res 183:92–99 Scott MMR, Samsatly J, Charron J-B, Jabaji S (2018) Endophytes of industrial hemp (Cannabis sativa L.) cultivars: identification of culturable bacteria and fungi in leaves, petioles, and seeds. Can J Microbiol 64:1–17. https://doi.org/10.1139/cjm-2018-0108 Shade A, Jacques M-A, Barret M (2017) Ecological patterns of seed microbiome diversity, transmission, and assembly. Curr Opin Microbiol 37:15–22. https://doi.org/10.1016/j.mib.2017.03.010 Shahzad R, Waqas M, Khan AL, Asaf S, Khan MA, Kang S-M, Yun B-W, Lee I-J (2016) Seedborne endophytic Bacillus amyloliquefaciens RWL-1 produces gibberellins and regulates endogenous phytohormones of Oryza sativa. Plant Physiol Biochem 106:236–243. https://doi. org/10.1016/j.plaphy.2016.05.006 Shahzad R, Khan AL, Bilal S, Asaf S, Lee I-J (2017) Plant growth-promoting endophytic bacteria versus pathogenic infections: an example of Bacillus amyloliquefaciens RWL-1 and Fusarium oxysporum f. sp. lycopersici in tomato. Peer J 5:e3107. https://doi.org/10.7717/peerj.3107 Shearin ZRC, Filipek M, Desai R, Bickford WA, Kowalski KP, Clay K (2017) Fungal endophytes from seeds of invasive, non-native Phragmites australis and their potential role in germination and seedling growth. Plant Soil 2017:1–12. https://doi.org/10.1007/s11104-017-3241-x Shen XY, Cheng YL, Cai CJ, Fan L, Gao J, Hou CL (2014) Diversity and antimicrobial activity of culturable endophytic fungi isolated from moso bamboo seeds. PLoS One 9(4):e95838 Shi J, Liu A, Li X et al (2010) Identification of endophytic bacterial strain MGP1 selected from papaya and its biocontrol effects on pathogens infecting harvested papaya fruit. J Sci Food Agric 90:227–232 Sicuia OA, Grosu I, Constantinescu F, Voaides C, Cornea CP (2015) Enzymatic and genetic variability in Bacillus spp. strains with plant beneficial qualities. AgroLife Sci J 4:124–131 Singh R, Dubey AK (2015) Endophytic actinomycetes as emerging source for therapeutic compounds. Indo Global J Pharm Sci 5:106–116 Smith SA, Tank DC, Boulanger L-A, Bascom-Slack CA, Eisenman K, Kingery D et al (2008) Bioactive endophytes warrant intensified exploration and conservation. PLoS One 3:e3052. https://doi.org/10.1371/journal.pone.0003052 Strobel G, Daisy B (2003) Bioprospecting for microbial endophytes and their natural products. Microbiol Mol Biol Rev 67(4):491–502 Strobel GA, Miller RV, Martinez-Miller C, Condron MM, Teplow DB, Hess WM (1999) Cryptocandin, a potent antimycotic from the endophytic fungus Cryptosporiopsis cf. quercina. Microbiology 145(8):1919–1926 Sun H, He Y, Xiao Q, Ye R, Tian Y (2013) Isolation, characterization, and antimicrobial activity of endophytic bacteria from Polygonum cuspidatum. Afr J Microbiol Res 7:1496–1504. https:// doi.org/10.5897/AJMR12.899 Sun B-T, Senyo Akutse K, Xia X-F et al (2018) Endophytic effects of Aspergillus oryzae on radish (Raphanus sativus) and its herbivore, Plutella xylostella. Planta 1(3):705. https://doi.org/10. 1007/s00425-018-2928-4 Sundaramoorthy S, Balabaska P (2013) Evaluation of combined efficacy of Pseudomonas fluorescens and Bacillus subtilis in managing tomato wilt caused by Fusarium oxysporum f. Sp. lycopersici (Fol). Plant Pathol J 12:154–161 Taechowisan T, Lu C, Shen Y, Lumyong S (2005) Secondary metabolites from endophytic Streptomyces aureofaciens CMUAc130 and their antifungal activity. Microbiology 151 (5):1691–1695 Taechowisan T, Chanaphat S, Ruensamran W, Phutdhawong WS (2013) Antibacterial activity of Decursin from Streptomyces sp. GMT-8; an endophyte in Zingiber officinale Rosc. J Appl Pharm Sci 3:74–78. https://doi.org/10.7324/JAPS.2013.31012

446

S. Sreejith et al.

Taghavi S, Garafola C, Monchy S, Newman L, Hoffman A, Weyens N, Barac T, Vangronsveld J, van der Lelie D (2009) Genome survey and characterization of endophytic bacteria exhibiting a beneficial effect on growth and development of poplar trees. Appl Environ Microbiol 75 (3):748–757 Tanaka K, Amaki Y, Ishihara A, Nakajima H (2015) Synergistic effects of [Ile7] surfactin homologues with bacillomycin D in suppression of gray mold disease by Bacillus amyloliquefaciens biocontrol strain SD-32. J Agric Food Chem 63:5344–5353. https://doi.org/ 10.1021/acs.jafc.5b01198 Tapi A, Chollet-Imbert M, Scherens B, Jacques P (2010) New approach for the detection of non-ribosomal peptide synthetase genes in Bacillus strains by polymerase chain reaction. Appl Microbiol Biotechnol 85(5):1521–1531 Tayung K, Sarkar M, Baruah P (2012) Endophytic fungi occurring in Ipomoea carnea tissues and their antimicrobial potentials. Braz Arch Biol Tech 55:653–660 Toral L, Rodríguez M, Béjar V, Sampedro I (2018) Antifungal activity of lipopeptides from Bacillus XT1 CECT 8661 against Botrytis cinerea. Front Microbiol 9:1315 Truyens S, Weyens N, Cuypers A, Vangronsveld J (2015) Bacterial seed endophytes: genera, vertical transmission and interaction with plants. Environ Microbiol Rep 7:40–50. https://doi. org/10.1111/1758-2229.12181 Verma SC, Ladha JK, Tripathi AK (2001) Evaluation of plant growth promoting and colonization ability of endophytic diazotrophs from deep water rice. J Biotechnol 91(2):127–141 Verma SK, Kingsley K, Bergen M et al (2018a) Bacterial endophytes from rice cut grass (Leersia oryzoides L.) increase growth, promote root gravitropic response, stimulate root hair formation, and protect rice seedlings from disease. Plant Soil 422(1–2):223–238. https://doi.org/10.1007/ s11104-017-3339-1 Verma S, Kingsley K, Bergen M et al (2018b) Fungal disease prevention in seedlings of rice (Oryza sativa) and other grasses by growth-promoting seed-associated endophytic bacteria from invasive Phragmites australis. Microorganisms 6:21. https://doi.org/10.3390/microorganisms6010021 Vincent D, Bedon F (2013) Secretomics of plant-fungus associations: more secrets to unravel. J Plant Biochem Physiol 1(5):1000e117. https://doi.org/10.4172/2329-9029.1000e117 Walitang DI, Kim K, Madhaiyan M et al (2017) Characterizing endophytic competence and plant growth promotion of bacterial endophytes inhabiting the seed endosphere of Rice. BMC Microbiol 17(1):209. https://doi.org/10.1186/s12866-017-1117-0 Wang P, Guo Q, Ma Y, Li S, Lu X, Zhang X, Ma P (2015) DegQ regulates the production of fengycins and biofilm formation of the biocontrol agent Bacillus subtilis NCD-2. Microbiol Res 178:42–50 Wearn JA, Sutton BC, Morley NJ, Gange AC (2012) Species and organ specificity of fungal endophytes in herbaceous grassland plants. J Ecol 100:1085–1092 White JF, Torres MS, Somu MP, Johnson H, Irizarry I, Chen Q, Zhang N, Walsh E, Tadych M, Bergen M (2014) Hydrogen peroxide staining to visualize intracellular bacterial infections of seedling root cells. Microsc Res Tech 77:566–573 White JF, Kingsley KI, Kowalski KP, Irizarry I, Micci A, Soares MA et al (2017) Disease protection and allelopathic interactions of seed-transmitted endophytic Pseudomonads of invasive reed grass (Phragmites australis). Plant Soil 422(1–2):195–208. https://doi.org/10.1007/ s11104-016-3169-6 Wisniewski-Dyé K, Borziak G, Khalsa-Moyers G, Alexandre LO, Sukharnikov K, Wuichet GB, Hurst WH, McDonald JS, Robertson V, Barbe A, Calteau Z, Rouy S, Mangenot C, PrigentCombaret P, Normand M, Boyer P, Siguier Y, Dessaux C, Elmerich G, Condemine G, Krishnen I, Kennedy AH, Paterson V, González P, Mavingui IB (2011) Zhulin Azospirillum genomes reveal transition of bacteria from aquatic to terrestrial environments. PLoS Genet 7:e1002430 Wu Z, Bañuelos GS, Lin ZQ, Liu Y, Yuan L, Yin X, Li M (2015) Biofortification and phytoremediation of selenium in China. Front Plant Sci 6:136 Xu M, Sheng J, Chen L, Men Y, Gan L, Guo S et al (2014) Bacterial community compositions of tomato (Lycopersicum esculentum Mill.) seeds and plant growth promoting activity of ACC

20

Agriculturally Important Biosynthetic Features of Endophytic Microorganisms

447

deaminase producing Bacillus subtilis (HYT-12-1) on tomato seedlings. World J Microbiol Biotechnol 30:835–845. https://doi.org/10.1007/s11274-013-1486-y Zarei M, Aminzadeh S, Zolgharnein H, Safahieh A, Daliri M, Noghabi KA, Ghoroghi A, Motallebi A (2011) Characterization of a chitinase with antifungal activity from a native Serratia marcescens B4A. Braz J Microbiol 42(3):1017–1029 Zhao K, Penttinen P, Guan T, Xiao J, Chen Q, Xu J et al (2011) The diversity and anti-microbial activity of endophytic actinomycetes isolated from medicinal plants in Panxi Plateau China. Curr Microbiol 62(1):182–190

Microbial Endophytes of Maize Seeds and Their Application in Crop Improvements

21

Sandip Chowdhury, Rusi Lata, Ravindra N. Kharwar, and Surendra K. Gond

Contents 21.1 21.2 21.3 21.4 21.5

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Maize . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diversity of Microbes Vectored Within Maize Seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transmission of Maize Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional Role of Maize Seed Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.5.1 Plant Growth Promotion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.5.2 Disease Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

450 451 451 455 457 457 457 460 460

Abstract

Maize is one of the main cereal crops grown all over the world. The presence of microbial endophytes which reside asymptomatically inside maize seeds may influence the yield and quality of crop. The present review concentrates on underexplored endophytes, such as seed-borne bacterial and fungal endophytes. The review encompasses the role of maize seed’s endophytes in enhancing crop efficiency, the nature of vertical transmission and secondary metabolites production, their belowground function, and the aboveground response. The diversity of endophytes in maize seed is discussed in detail focusing also on methodology applied for their isolation. This review may render help for the researchers working on the improvement of crops modulated through seed endophytes.

S. Chowdhury · R. Lata · S. K. Gond (*) Department of Botany, MMV, Banaras Hindu University, Varanasi, India R. N. Kharwar Department of Botany, Banaras Hindu University, Varanasi, India # Springer Nature Switzerland AG 2019 S. K. Verma, J. F. White, Jr (eds.), Seed Endophytes, https://doi.org/10.1007/978-3-030-10504-4_21

449

450

S. Chowdhury et al.

Keywords

Zea mays · Endophytism · Antimicrobial · Plant growth promotion · Gene expression

21.1

Introduction

Since beginning of the study, endophytes have been defined variously by different scientists and researchers. A considerable amount of work has already been done since 1905, which led to the development of the term “endophytism.” Endophytes have been known to reside asymptomatically in every part of almost all plant species. Seeds are indisputably very important in agricultural production (Mundt and Hinkle 1976; Kremer 1987; Schardl et al. 2004); however, the long-held concept that reproductive and disseminative organs of plants do not harbor microbes has completely been denied. Seeds of any plant are now considered as carriers of various beneficial bacteria, fungi, as well as pathogens (Liu et al. 2013). A number of studies have been conducted on microbial communities reside on the seed surface and within the seed body (Nelson 2004). Firmicutes were visualized inside Vitis vinifera, in the intercellular spaces of pulp cells and/or xylem of pulp and along some cell walls inside parts of seeds (Compant et al. 2011). Pseudomonas and Rahnella in the Norway spruce seeds were reported by Cankar et al. (2005). Isolates have been derived from surface-disinfected roots and immature seeds of Phaseolus vulgaris, and seed endophytes from rice have also been reported by many researchers (Mano et al. 2006). These microbes in association with plants significantly affect the plant health and soil fertility that have led to coevolutionary adaptations (Barea et al. 2005). The process of seed germination promotes the spread of these seed and soilborne microbial communities as shown in rice (Kaga et al. 2009), eucalyptus (Ferreira et al. 2008), and maize (Bacilio-Jiméne et al. 2001; Cottyn et al. 2001). In general, endophytic microbes offer a wide range of benefits to their host like plant growth promotion (Sturz et al. 2000), tolerance toward biotic and abiotic stresses (Bacon et al. 2015) through induction of plant defense mechanisms (Rojas et al. 2014), and production of anti-herbivory compounds (Sullivan et al. 2007). Besides their host’s benefit, endophytes are the source of biologically active secondary metabolites which may have several potential application in medical, agriculture, and industry (Strobel et al. 2004). For instance, endophytes modulate plant performance under thermal stress (Redman et al. 2002) and induce plant resistance resulting in a reduction of root-feeding nematodes and herbivores (Martínez-Medina et al. 2017; Cosme et al. 2016). Under ambient conditions, endophytes might have both positive (Newsham 2011) and negative effects on plant performance (Mayerhofer et al. 2012). Effects of endophytes on plants are likely more important under stress. In addition to their role in plant performance, many endophytes can survive and grow as saprophytes in soils (Peay et al. 2010) and include species that are primary decomposers of infected plant material (Song et al. 2017). Therefore, the exact functions of most endophyte species remain

21

Microbial Endophytes of Maize Seeds and Their Application in Crop Improvements 451

largely unknown (Newsham 2011), whereas their role in climate warming and plant range shifts is completely unexplored.

21.2

Maize

The origin of maize (Zea mays), also known as corn, has been traced back to 9000 years ago in southwest Mexico (Matsuoka et al. 2002). It has said to be originated from wild grass (teosintes) (Ranum et al. 2014) through seed enlargement, elimination of the protective hard fruit case surrounding the seed, enhancement of husk leaves, development of rachids, switching of seed placement on the plant, and reduced shoot branching (Wilkes 2004). It has been used since then by Native Americans as a better source of food. Due to its wider adaptability under various agroclimatic conditions, maize has been titled the queen of cereals and is stated as one of the most versatile crops. With the highest genetic yield potential among the cereals, it is cultivated in about 160 countries that have a wider diversity of soil, climate, biodiversity, and management practices. Covering about 179.72 million ha (2014–2015) of worlds total maize cultivable land, it contributes 36% (1014.37 million metric tons) in the global grain production. It is the major driver of the US economy contributing nearly 35.6% of the total production in the world. Contrastingly in India average productivity of maize is 2.63 metric tons/ha, and its share in global export of maize is 14% (2014–2015) (USDA 2016) (Tripathi et al. 2016). The management of crop and grain production is a necessity for food security of increasing human population.

21.3

Diversity of Microbes Vectored Within Maize Seeds

Maize is known to host a variety of microorganisms. While Fisher et al. (1992) reported non-mycorrhizal fungal endophytes from maize, natural associations with N2-fixing bacteria like Azospirillum have also been mentioned by Christiansen-Weniger and Vanderleyden (1994). Many endophytes from maize cultivars were isolated: Klebsiella (Chelius and Triplett 2000a; Dong et al. 2001), Pantoea, Herbaspirillum, Bacillus (Chelius and Triplett 2000b; Palus et al. 1996), etc. For isolation of seed endophytes from maize, different procedures have been used that differ in time given for surface sterilization of the seeds or the sterilizing agents and culture media used. Most of the researchers have selected NaOCl as sterilizing agent of maize seeds. The variation in the culture media used for isolation of maize seed endophytes may also be the probable reason for different bacterial groups (Table 21.1). A large number of maize varieties have evolved from the grass teosinte. Correlations were also established among maize varieties and endophytic community within them. It was questioned by Johnston-Monje and Raizada (2011) in their article whether microbial communities were conserved in domesticated maize as it has evolved from teosintes. They studied endophytic bacteria of ten different varieties of maize by culturing, cloning, and DNA fingerprinting using terminal restriction fragment length polymorphism (TRFLP) of 16S rDNA. The research

Maize cultivars: unknown cultivar, PR38F70, W22, and Pioneer inbred line Zea mays

Dry white maize Dry yellow maize Fresh yellow maize

3.

5.

4.

Unknown Italian corn cultivar

Variety of maize seed Eagle variety

2.

S. no. 1.

Pantoea ananatis, Microbacterium sp., Frigoribacterium sp., Bacillus sp., Paenibacillus sp., and Sphingomonas Bacillus sp., Methylobacterium, Tukamurella, Alcaligenes, Erwinia, Microbacterium, Rhodococcus Alternaria alternata, Fusarium verticillioides, Saccharomyces cerevisiae, Trichoderma koningii, Aspergillus flavus, Aspergillus niger Azospirillum sp., Bacillus, Cellulomonas sp., Micrococcus sp., Citrobacter sp., Pseudomonas sp., Staphylococcus sp., Kurtia sp., Microbacterium sp.

Endophytes isolated Chaetomium cochliodes Cladosporium cladosporioides Epicoccurn purpurasceris Fusarium ventricosum Fusarium oxysporum Penicillium spp. Verticillium lecanii Enterobacter cloacae

Table 21.1 The diversity of endophytes isolated from maize seed

Maize seeds soaked in 3.5% m/v NaOCl for 20 s Dipped for 30 s in 70% ethanol rinsed twice in distilled water and dried

The kernels were surface sterilized by shaking for 10 min in 5.25% NaOCl Rinsed three times in distilled water and germinated at room temperature Seeds were surface sterilized by soaking in NaOCl containing 40 μL/100 mL tween 80 –

Sterilization method used Seeds were immersed in 75% ethanol for 30 s, followed by 0.93– 1.3 M NaOCl for 3 min and again with 75% ethanol for 30 s

Sabouraud dextrose agar and potato dextrose agar for fungi Nutrient agar and MacConkey agar for bacteria

Plates with agar 0.7%

Tryptic soy agar

Potato dextrose agar

Culture media used Malt extract agar

Orole and Adejumo (2011)

Rosenblueth et al. (2010)

Rijavec et al. (2007)

Hinton and Bacon (1995)

References Fischer et al. (1992)

452 S. Chowdhury et al.

10.

9.

8.

7.

6.

Tropical corn (Zea mays indurata) Indian popcorn (Z. Mays indurata) Yellow dent (Zea mays indenata) Red dent (Z. mays indenata) Blue dent (Z. mays indenata) Ornamental corn (Z. mays indurata) Sweet corn (Zea mays saccharata)

Zea nicaraguensis, Gaspe Zea diploperennis, Zea mays parviglumis, Chapalote, Bolita, Jala, Gaspe yellow Flint, Pioneer 3751 Yuyu 23, Zhengdan 958, Jingdan 28 and Jingyu 11 PAU871, EMA-171 PAU-785, and Tendem cultivars Hybrid (Zea mays L., Nongda108)

Bacillus amyloliquefaciens Bacillus subtilis Bacillus subtilis B. amyloliquefaciens subsp. subtilis B. amyloliquefaciens subsp. subtilis B. amyloliquefaciens subsp. subtilis B. amyloliquefaciens

Undibacterium, Burkholderia, Limnobacter, Pantoea

Pantoea, Sphingomonas, Stenotrophomonas, Acinetobacter, Pseudomonas, Leclercia, Enterobacter Burkholderia cepacia

Arthrobacter Pantoea, Enterobacter

Yeast extract sucrose agar

(continued)

Gond et al. (2015a)

Liu et al. (2013)

LB media

Samples washed with sterile water, immersed in 70% alcohol for 3 min, washed with fresh NaOCl (2.5% available Cl ) for 5 min Rinsed with 70% alcohol for 30 s and finally washed 5–7 times with sterile water 4% NaOCl for 30 min and then washed three times with sterile distilled water

Montanez et al. (2012)

–

–

Liu et al. (2012)

JohnstonMonje and Raizada (2011)

Luria-Bertani media

LGI, R2A (Reasoner’s 2A agar), and PDA

Surface sterilization by ethanol and NaOCl

–

21 Microbial Endophytes of Maize Seeds and Their Application in Crop Improvements 453

Hybrids (Zea mays L.) (Jinghua8, Jingnongke728, Jingdan68, NK718, Jingke968, Jingke665)

30 maize varieties

14.

Variety of maize seed Indian fingers (Z. mays indurata) Teosinte (Zea maysmexicana) Zea mays Hybrid maize cultivars (Helmi, Morignon, Pelicon, and Peso)

13.

12.

S. no. 11.

Table 21.1 (continued)

Aeribacillus pallidus, Bacillus safensis, Enterobacter cloacae, Halomonas nitritophilus, Leuconostoc mesenteroides, P.agglomerans, Pseudomonas viridiflava, and Staphylococcus aureus B. subtilis, B. subtilis subsp. spizizenii, B. subtilis subsp. inaquosorum, B. safensis, B. amyloliquefaciens, B. flexus, B. firmus, B. licheniformis, B. pseudomycoides, B. aryabhattai, B. anthracis, Staphylococcus warneri, S. equorum subsp. linens, S. equorum, S. pasteuri, and Corynebacterium hansenii

Endophytes isolated Pantoea agglomerans Agrobacterium sp. Bacillus spp. (including, B. amyloliquefaciens and B. subtilis) Pantoea ananatis str.

Five seeds per genotype were treated with 0.1% of HgCl2 for 2 min. After that seeds were immersed in 95% ethanol for 4 min followed by repeated washings with sterile distilled water

Surface-sterilized with 70% ethanol for 3 min and 5% sodium hypochlorite for 5 min and followed by repeated washing with sterile distilled water (three times for 1 min) –

Sterilization method used 4% NaOCl for 30 min and then washed three times with sterile distilled water

Bodhankar et al. (2017)

Liu et al. (2017)

–

TSA and R2A for slow growers

SheibaniTezerji et al. (2015)

References Gond et al. (2015b)

TSA (tryptic soy agar)

Culture media used –

454 S. Chowdhury et al.

21

Microbial Endophytes of Maize Seeds and Their Application in Crop Improvements 455

Fig. 21.1 Diversity of maize seed endophytes

concluded the presence of the same bacterial genera across several genotypes of maize seeds (showed Enterobacter sp. and Pantoea sp. as the most common genera) along with some differences in the composition of the endophytic community. Liu et al. (2012) had also found similar bacterial taxa in genetically related maize hybrids. The ɣ-proteobacteria were the most abundant class of microbes observed by a phylogenetic study conducted by Johnston-Monje and Raizada (2011) which showed Enterobacter and Pantoea species as the most common genera. Figure 21.1 shows diversity of endophytes in maize seeds isolated by different researchers. The endophytic microbes belonging to different groups have been represented as percentage; however, the sample size from which isolation was carried out differed (Dunleavy 1989; Rijavec et al. 2007; Rosenblueth et al. 2010; Johnston-Monje and Raizada 2011; Liu et al. 2012).

21.4

Transmission of Maize Endophytes

Transmission of endophytes occurs both horizontally and vertically (Fig. 21.2). Horizontal Transfer Sexual or asexual spores of endophytes enter their next host via rhizosphere, aerial tissues, stomata, floral parts, and insect. Horizontally transmitted endophytes are not host-specific and are highly diverse. They form localized infections and are found almost in all plants. For horizontal transfer, the soil is considered as the most important source of inoculum for endophytes (Hardoim et al. 2008). Bacterial endophytes are mostly transmitted horizontally. Vertical Transfer Endophytes are dispersed through seeds produced by host plants (Ernst et al. 2003; Schardl et al. 2004). Seeds serve the purpose of long-term conservation of the endophytic microbial community. Some plants have formed a mutual relationship with bacteria that are vertically transmitted via seed, providing continued transmission of beneficial symbionts, also similar in case of defensive mutualisms between plants and fungal endophytes (Hodgson et al. 2014). Bacon and Hinton (1996) reported seed-associated endophytic fungus (Fusarium moniliforme) which infects seedling from its systemic infection from the seed, usually by the second day followed by germination. The systemic infection of maize seeds produces maternal line vertical transmission from generation to generation. In

Spores or vegetative cells of endophyte get transferred to one host to another host

Horizontal Transmission

Endophytes get spreaded through out plant and reach to seeds

Direct entry of endophytes via natural opening present in plant

Fig. 21.2 Transmission of endophytic microbes

Entry of endophytes through wounds caused due to lateral root hair formation from soil

Hydathodes

Stomata

Floral opening

Endophytes stored in embryo of seed

Transfer to next generation via vegetative part of host plant

Vertical Transmission

Colonization of endophytes at the base of plant

As seed germinates the endophytes grows within emerging part of plant

456 S. Chowdhury et al.

21

Microbial Endophytes of Maize Seeds and Their Application in Crop Improvements 457

addition to the vertical transmission phase, maize is subject to infection throughout the growing season. When plant seeds germinate, the endophytes lying within are systematically transferred in the maternal line, and this continues from generation to generation. Johnston-Monje and Raizada (2011) suggested that some bacterial groups are conserved across generations despite human selection and crosscontinental migration. However, seeds, as earlier stated, are vectors for pathogens too, suppressing seed germination (Elmer 2001; Schardl et al. 2004). As belowground organisms have a limited dispersal capacity (Berg et al. 2010), this might reduce the success of range-expanding plant species. Similar to bacterial pathogen such as Pantoea stewartii, which systemically spreads in maize from the shoot vasculature through the chalaza and finally into the seed endosperm, bacterial seed endophytes are also transmitted through vascular connections from maternal plant (Block et al. 1998).

21.5

Functional Role of Maize Seed Endophytes

The existing study mainly focuses on some beneficial aspects of maize seedassociated endophytes (Table 21.2).

21.5.1 Plant Growth Promotion Endophytic microbes generally promote growth by producing plant growth hormones, solubilizing rock phosphate, and chelating toxic metals in the rhizosphere (Ahemad and Kibret 2014). All the endophytes isolated by Johnston-Monje and Raizada (2011) from different races of maize (teosintes, Mexican, and temperate varieties) showed growth promotion through hormone production (ACC deaminase, auxin) enzymes (pectinase, RNAse, cellulase), antagonistic activity, or through promotion of physiological growth such as changes in root and shoot length and biomass. Some isolates enhanced the growth of maize seedlings (Yates et al. 1997). Montanez et al. (2012) isolated several endophytic bacteria from 11 maize varieties. They obtained Burkholderia cepacia as a seed endophyte from maize variety PAU871. Biochemical characterization revealed that Burkholderia cepacia produced indoleacetic acid (40.1 μg ml 1) and phosphate solubilization capability of 2.1 PSI (phosphate solubilizing index).

21.5.2 Disease Resistance 21.5.2.1 Antagonism Through the Production of Antimicrobial Compounds The organic extracts from maize kernel fermentations by Acremonium zeae (a maize endophyte) exhibited significant antifungal activity against Aspergillus flavus and Fusarium verticillioides. It was revealed that the metabolites accounting for this

458

S. Chowdhury et al.

Table 21.2 Seed endophytes of maize and their functional roles S. no. 1.

2.

Endophyte Bacillus subtilis RRC101 Bacillus subtilis 26ss Acremonium zeae

3.

Pantoea sp.

Unknown cultivar, PR38F70, W22, and Pioneer inbred line

4.

Azospirillum lipoferum

–

5.

Enterobacter sp. Arthrobacter sp. Burkholderia sp. Pantoea sp. Staphylococcus sp. Bradyrhizobium sp. Deinococcus sp. Enterobacter sp. Klebsiella sp. Pseudomonas sp. Burkholderia sp. Cellulomonas sp. Enterobacter sp. Burkholderia sp. Enterobacter sp. Stenotrophomonas sp. Pantoea sp. Bacillus amyloliquefaciens subsp. subtilis

Zea nicaraguensis, Gaspe, Zea diploperennis, Zea mays parviglumis, Chapalote, Bolita, Jala, Gaspe yellow flint, Pioneer 3751

P-solubilization Nitrogen-free growth ACC deaminase activity Siderophore production Auxin production

Tropical corn

B. flexus NBRC 15715 Staphylococcus pasteuri ATCC 51129 and Staphylococcus equorum PA 231

30 maize varieties

Production of antifungal lipopeptides Upregulation of pathogenesisrelated genes against fungal pathogens Ammonia production Ammonia production (can act as toxic by-product for pathogens)

6.

7.

Variety of maize Unknown Italian cultivar

Maize cultivated within the USA

Function in host Control against Fusarium moniliforme Production of antifungal pyrrocidines A and B Antifungal activity against Lecanicillium aphanoladii Production of ABA and gibberellins

References Bacon et al. (2001) Donald et al. (2005) Rijavec et al. (2007) Cohen et al. (2009) JohnstonMonje and Raizada (2011)

Gond et al. (2015a)

Bodhankar et al. (2017)

(continued)

21

Microbial Endophytes of Maize Seeds and Their Application in Crop Improvements 459

Table 21.2 (continued) S. no. 8.

9.

Endophyte Pseudomonas aeruginosa

Enterobacter asburiae

Variety of maize –

Function in host Production of HCN, siderophore, ammonia, IAA, gibberellins and cytokinins Production of HCN, siderophore, ammonia, IAA, gibberellins and cytokinins

References Sandhya et al. (2017)

activity were antibiotics pyrrocidines A and B (Donald et al. 2005). Fusarium graminearum is known to cause Gibberella ear rot (GER) in modern maize and also produces the mycotoxin, deoxynivalenol (DON) (Miedaner et al. 2010). Three potent endophytes from teosintes suppressed F. graminearum in vitro and GER in a modern maize hybrid and also lowered the production of DON (deoxynivalenol) mycotoxin during storage. Pantoea polymyxa strains produced the previously characterized anti-Fusarium compound, fusaricidin (Mousa et al. 2015). Some Bacillus spp. isolated from different varieties of maize has shown strong antifungal activity against fungal pathogens including Fusarium moniliforme (Gond et al. 2015b). Acremonium zeae, an endophyte of maize, has a protective mechanism for maize seeds which display a significant antifungal activity against Aspergillus flavus and Fusarium verticillioides (Donald et al. 2005).

21.5.2.2 Defense Gene Expression An endophytic Bacillus amyloliquefaciens subsp. subtilis isolated from surfacesterilized seedlings of tropical corn was reported to produce antifungal lipopeptides and enhance defense gene expression (PR-1 and PR-10) (Gond et al. 2015a). Plant defense pathways triggered by endophytic bacterium appeared to be salicylic acid independent. 21.5.2.3 Abiotic Stress Tolerance Gond et al. (2015a) isolated Bacillus sp. from eight modern corn varieties, and only Pantoea agglomerans and Agrobacterium sp. were isolated from teosinte. P. agglomerans was shown to enhance salt tolerance under hypersaline conditions in the tropical corn seedlings. It was revealed by gene expression analysis that P. agglomerans upregulated the aquaporin gene family especially plasma membrane integral protein type 2 (PIP2–1) genes in inoculated plants under salt stress. Under increased soil Cd stress, Z. mays root-associated dark septate endophyte (DSE), Exophiala pisciphila, showed enhanced antioxidant enzyme activity (Wang et al. 2016). Three key genes (pathways) involved in uptake, detoxification, and transport of Cd were identified as downregulation of ZIP, upregulation of PCS

460

S. Chowdhury et al.

and MTP upon inoculation with DSE, and exposed to subsequent high Cd concentrations.

21.6

Conclusion

Seeds are carrier of both microphyte parental genes (Guan 2009) and a variety of beneficial bacteria and pathogens. These microorganisms originate from various microbial communities born on the seed surface (Nelson 2004). The microorganisms survive around the plant surface like rhizobacteria has been well established as plant growth promoters. Based on the above discussions, we may conclude that seeds of maize also harbor a diverse range of microbial endophytic community. These microbes are culturable and have been isolated on different culture media, but there must be some nonculturable also for which another approach like nextgeneration sequencing (NGS) could be applied. The endophytic community travels from one generation to another generation of maize via systemic infection in seed. These endophytic microbes protect plants not only against pathogens but also under abiotic stresses (Lata et al. 2018). Selected endophytic microbe of maize seed may be applied for crop improvement. Biotechnological approaches and genetic engineering may help to utilize these endophytic microbes for plant growth promotion in host as well as nonhost crop plants. Acknowledgment Authors are thankful to the Department of Botany, MMV, Banaras Hindu University for providing necessary facility. RL acknowledges UGC New Delhi, for Junior Research Fellowship. Financial support from SERB, New Delhi (EEQ/2016/000555), is greatly acknowledged. RNK expresses his thanks to SERB (DST), New Delhi, for project (SB/EMEQ-121/2014) and to Head & Coordinator, CAS and DST-FIST in Botany, Institute of Science, BHU, Varanasi, for facilities.

References Ahemad M, Kibret M (2014) Mechanisms and applications of plant growth promoting rhizobacteria: current perspective. J King Saudi Univ Sci 26(1):1–20 Bacilio-Jiménez M, Aguilar-Flores S, del Valle MV et al (2001) Endophytic bacteria in rice seeds inhibit early colonization of roots by Azospirillum brasilense. Soil Biol Biochem 33(2):167–172 Bacon CW, Hinton DM (1996) Symptomless endophytic colonization of maize by Fusarium moniliforme. Can J Bot 74:1195–1202 Bacon CW, Yates IE, Hinton DM et al (2001) Biological control of Fusarium moniliforme in maize. Environ Health Perspect 109(Suppl 2):325 Bacon CW, Palencia ER, Hinton DM (2015) Abiotic and biotic plant stress-tolerant and beneficial secondary metabolites produced by endophytic Bacillus species. In: Arora N (ed) Plant microbes symbiosis: applied facets. Springer, New Delhi, pp 163–177 Barea JM, Pozo MJ, Azcon R et al (2005) Microbial co-operation in the rhizosphere. J Exp Bot 56 (417):1761–1778 Berg MP, Kiers E, Driessen G et al (2010) Adapt or disperse: understanding species persistence in a changing world. Glob Chang Biol 16(2):587–598

21

Microbial Endophytes of Maize Seeds and Their Application in Crop Improvements 461

Block CC, Hill JH, McGee DC (1998) Seed transmission of Pantoea stewartii in field and sweet corn. Plant Dis 82(7):775–780 Bodhankar S, Grover M, Hemanth S et al (2017) Maize seed endophytic bacteria: dominance of antagonistic, lytic enzyme-producing Bacillus spp. 3 Biotech 7(4):232 Cankar K, Kraigher H, Ravnikar M et al (2005) Bacterial endophytes from seeds of Norway spruce (Piceaabies L. Karst). FEMS Microbiol Lett 244(2):341–345 Chelius MK, Triplett EW (2000a) Immunolocalization of dinitrogenase reductase produced by Klebsiella pneumoniae in association with Zea mays L. Appl Environ Microbiol 66(2):783–787 Chelius MK, Triplett EW (2000b) Diazotrophic endophytes associated with maize. In: Triplett EW (ed) Prokaryotic nitrogen fixation: a model system for the analysis of a biological process. Horizon Scientific Press, Norfolk, pp 779–792 Christiansen-Weniger C, Vanderleyden J (1994) Ammonium-excreting Azospirillum sp. become intracellularly established in maize (Zea mays) para-nodules. Biol Fertil Soils 17(1):1–8 Cohen AC, Travaglia CN, Bottini R et al (2009) Participation of abscisic acid and gibberellins produced by endophytic Azospirillum in the alleviation of drought effects in maize. Botany 87(5):455–462 Compant S, Mitter B, Colli-Mull JG et al (2011) Endophytes of grapevine flowers, berries, and seeds: identification of cultivable bacteria, comparison with other plant parts, and visualization of niches of colonization. Microb Ecol 62(1):188–197 Cosme M, Lu J, Erb M et al (2016) A fungal endophyte helps plants to tolerate root herbivory through changes in gibberellin and jasmonate signaling. New Phytol 211(3):1065–1076 Cottyn B, Regalado E, Lanoot B et al (2001) Bacterial populations associated with rice seed in the tropical environment. Phytopathology 91(3):282–292 Donald TW, Shoshannah ROTH, Deyrup ST et al (2005) A protective endophyte of maize: Acremonium zeae antibiotics inhibitory to Aspergillus flavus and Fusarium verticillioides. Mycol Res 109(5):610–618 Dong Y, Glasner JD, Blattner FR et al (2001) Genomic interspecies microarray hybridization: rapid discovery of three thousand genes in the maize endophyte, Klebsiella pneumoniae 342, by microarray hybridization with Escherichia coli K12 open reading frames. Appl Environ Microbiol 67(4):1911–1921 Dunleavy JM (1989) Curtobacterium plantarum sp. nov. is ubiquitous in plant leaves and is seed transmitted in soybean and corn. Int J Syst Evol Microbiol 39(3):240–249 Elmer WH (2001) Seeds as vehicles for pathogen importation. Biol Invasions 3:263–271 Ernst M, Mendgen KW, Wirsel SG (2003) Endophytic fungal mutualists: seed-borne Stagonospora spp. enhance reed biomass production in axenic microcosms. Mol Plant-Microbe Interact 16(7):580–587 Ferreira A, Quecine MC, Lacava PT et al (2008) Diversity of endophytic bacteria from eucalyptus species seeds and colonization of seedlings by Pantoea agglomerans. FEMS Microbiol Lett 287:8–14 Fisher PJ, Petrini O, Scott HL (1992) The distribution of some fungal and bacterial endophytes in maize (Zea mays L.). New Phytol 122(2):299–305 Gond SK, Bergen MS, Torres MS et al (2015a) Endophytic Bacillus spp. produce antifungal lipopeptides and induce host defense gene expression in maize. Microbiol Res 172:79–87 Gond SK, Torres MS, Bergen MS et al (2015b) Induction of salt tolerance and up-regulation of aquaporin genes in tropical corn by rhizobacterium Pantoea agglomerans. Lett Appl Microbiol 60(4):392–399 Guan KL (2009) Seed physiological ecology (in Chinese). Chinese Agricultural Press, Beijing, pp 1–7 Hardoim PR, van Overbeek LS, van Elsas JD (2008) Properties of bacterial endophytes and their proposed role in plant growth. Trends Microbiol 16(10):463–471 Hinton DM, Bacon CW (1995) Enterobacter cloacae is an endophytic symbiont of corn. Mycopathologia 129(2):117–125 Hodgson S, Cates C, Hodgson J et al (2014) Vertical transmission of fungal endophytes is widespread in forbs. Ecol Evol 4(8):1199–1208 Johnston-Monje D, Raizada MN (2011) Plant and endophyte relationships: nutrient management. In: Comprehensive biotechnology, 2nd edn. Academic Press, Burlington, pp 713–727

462

S. Chowdhury et al.

Kaga H, Mano H, Tanaka F et al (2009) Rice seeds as sources of endophytic bacteria. Microb Environ 24:154–162 Kremer RJ (1987) Identity and properties of bacteria inhabiting seeds of selected broadleaf weed species. Microb Ecol 14(1):29–37 Lata R, Chowdhury S, Gond SK, White JFJ (2018) Induction of abiotic stress tolerance in plants by endophytic microbes. Lett Appl Microbiol 66(4):268–276 Liu Y, Zuo S, Xu L et al (2012) Study on diversity of endophytic bacterial communities in seeds of hybrid maize and their parental lines. Arch Microbiol 194(12):1001–1012 Liu Y, Zuo S, al ZY (2013) Investigation on diversity and population succession dynamics of endophytic bacteria from seeds of maize (Zea mays L., Nongda108) at different growth stages. Ann Microbiol 63(1):71–79 Liu Y, Wang R, Li Y et al (2017) High-throughput sequencing-based analysis of the composition and diversity of endophytic bacterial community in seeds of “Beijing” hybrid maize planted in China. Plant Growth Regul 81(2):317–324 Mano H, Tanaka F, Watanabe A et al (2006) Culturable surface and endophytic bacterial flora of the maturing seeds of rice plants (Oryza sativa) cultivated in a paddy field. Microb Environ 21:86–100 Martínez-Medina A, Fernandez I, Lok GB et al (2017) Shifting from priming of salicylic acid to jasmonic acid regulated defences by Trichoderma protects tomato against the root knot nematode Meloidogyne incognita. New Phytol 213(3):1363–1377 Matsuoka Y, Vigouroux Y, Goodman MM et al (2002) A single domestication for maize shown by multilocus microsatellite genotyping. Proc Natl Acad Sci USA 99:6080–6084 Mayerhofer MS, Kernaghan G, Harper KA (2012) The effects of fungal root endophytes on plant growth: a meta-analysis. Mycorrhiza 23:119–128 Miedaner T, Bolduan C, Melchinger AE (2010) Aggressiveness and mycotoxin production of eight isolates each of Fusarium graminearum and Fusarium verticillioides for ear rot on susceptible and resistant early maize inbred lines. Eur J Plant Pathol 127(1):113–123 Montanez A, Blanco AR, Barlocco C et al (2012) Characterization of cultivable putative endophytic plant growth promoting bacteria associated with maize cultivars (Zea mays L.) and their inoculation effects in vitro. Appl Soil Ecol 58:21–28 Mousa WK, Shearer CR, Limay-Rios V et al (2015) Bacterial endophytes from wild maize suppress Fusarium graminearum in modern maize and inhibit mycotoxin accumulation. Front Plant Sci 6:805 Mundt JO, Hinkle NF (1976) Bacteria within ovules and seeds. Appl Environ Microbiol 32:694–698 Nelson EB (2004) Microbial dynamics and interactions in the spermosphere. Annu Rev Phytopathol 42:271–309 Newsham KK (2011) A meta-analysis of plant responses to dark septate root endophytes. New Phytol 190(3):783–793 Orole OO, Adejumo TO (2011) Bacterial and fungal endophytes associated with grains and roots of maize. J Ecol Nat Environ 3(9):298–303 Palus JA, Borneman J, Ludden PW et al (1996) Isolation and characterization of endophytic diazotrophs from Zea mays L. and Zea luxurians Iltis and Doebley. Plant Soil 186:135–142 Peay KG, Bidartondo MI, Elizabeth Arnold A (2010) Not every fungus is everywhere: scaling to the biogeography of fungal–plant interactions across roots, shoots and ecosystems. New Phytol 185 (4):878–882 Ranum P, PeñaRosas JP, GarciaCasal MN (2014) Global maize production, utilization, and consumption. Ann N Y Acad Sci 1312(1):105–112 Redman RS, Sheehan KB, Stout RG et al (2002) Thermotolerance generated by plant/fungal symbiosis. Science 298(5598):1581–1581 Rijavec T, Lapanje A, Dermastia M et al (2007) Isolation of bacterial endophytes from germinated maize kernels. Can J Microbiol 53:802–808 Rojas CM, Senthil-Kumar M, Tzin V et al (2014) Regulation of primary plant metabolism during plant-pathogen interactions and its contribution to plant defense. Front Recent Dev Plant Sci 5:17

21

Microbial Endophytes of Maize Seeds and Their Application in Crop Improvements 463

Rosenblueth M, López-López A, Martínez J, Rogel MA, Toledo I, Martínez-Romero E (2010) Seed bacterial endophytes: common genera, seed-to-seed variability and their possible role in plants. In: XXVIII international horticultural congress on science and horticulture for people (IHC2010): international symposium on 938, pp 39–48 Sandhya V, Shrivastava M, Ali SZ et al (2017) Endophytes from maize with plant growth promotion and biocontrol activity under drought stress. Russ Agric Sci 43(1):22–34 Schardl CL, Leuchtmann A, Spiering MJ (2004) Symbioses of grasses with seedborne fungal endophytes. Annu Rev Plant Biol 55:315–340 Sheibani-Tezerji R, Naveed M, Jehl MA et al (2015) The genomes of closely related Pantoea ananatis maize seed endophytes having different effects on the host plant differ in secretion system genes and mobile genetic elements. Front Microbiol 6:440 Song Z, Kennedy PG, Liew FJ et al (2017) Fungal endophytes as priority colonizers initiating wood decomposition. Funct Ecol 31(2):407–418 Strobel G, Daisy B, Castillo U et al (2004) Natural products from endophytic microorganisms. J Nat Prod 67(2):257–268 Sturz AV, Christie BR, Nowak J (2000) Bacterial endophytes: potential role in developing sustainable systems of crop production. Crit Rev Plant Sci 19(1):1–30 Sullivan TJ, Rodstrom J, Vandop J et al (2007) Symbiont-mediated changes in Lolium arundinaceum inducible defenses: evidence from changes in gene expression and leaf composition expression and leaf composition. New Phytol 176:673–679 Tripathi A, Joshi N, Kumar A (2016) Maize production technologies in India-a review. Octa J Environ Res 4(3):234–251 Wang JL, Li T, Liu GY et al (2016) Unraveling the role of dark septate endophyte (DSE) colonizing maize (Zea mays) under cadmium stress: physiological, cytological and genic aspects. Sci Rep 6:22028 Wilkes G (2004) Corn, strange and marvelous: but is a definitive origin known? In: Smith CW, Betran J, Runge ECA (eds) Corn: origin, history, technology, and production. Wiley, Hoboken, NJ, pp 3–63 Yates IE, Bacon CW, Hinton DM (1997) Effects of endophytic infection by Fusarium moniliforme on corn growth and cellular morphology. Plant Dis 81:723–728

Colonization of Seeds by Soilborne Fungi: Linking Seed Dormancy-Defense Syndromes, Evolutionary Constraints, and Fungal Traits

22

Simon Maccracken Stump, Carolina Sarmiento, Paul-Camilo Zalamea, James W. Dalling, Adam S. Davis, Justin P. Shaffer, and A. Elizabeth Arnold Contents 22.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2 Case Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2.1 Experimental Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2.2 Data Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2.4 Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.3 Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

466 469 470 471 473 475 478 479

S. M. Stump (*) Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ, USA School of Forestry and Environmental Studies, Yale University, New Haven, CT, USA e-mail: [email protected] C. Sarmiento · P.-C. Zalamea Smithsonian Tropical Research Institute, Panama, Republic of Panama J. W. Dalling Smithsonian Tropical Research Institute, Panama, Republic of Panama Department of Plant Biology, University of Illinois, Urbana, IL, USA A. S. Davis Department of Crop Sciences, University of Illinois, Urbana, IL, USA J. P. Shaffer School of Plant Sciences, University of Arizona, Tucson, AZ, USA A. Elizabeth Arnold Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ, USA School of Plant Sciences, University of Arizona, Tucson, AZ, USA # Springer Nature Switzerland AG 2019 S. K. Verma, J. F. White, Jr (eds.), Seed Endophytes, https://doi.org/10.1007/978-3-030-10504-4_22

465

466

S. M. Stump et al.

Abstract

The diverse soilborne fungi that recruit to seeds after dispersal include some of the most important agents of seed mortality, as well as strains that enhance germination or inhabit seeds without detriment. Ecological factors that influence seed colonization are not well understood yet are fundamental to the interactions between soilborne fungi and seeds that ultimately influence plant demography and community structure. Here we present current perspectives on seed defense syndromes and related frameworks for predicting colonization success of fungi, with a focus on seeds of tropical pioneer trees. We present a case study that tests whether fungal host range can be predicted by field observations of host use, seed defense syndromes, or phylogenetic relatedness of fungi or hosts. We show that phylogenetic relatedness of hosts, but not fungi, is a strong predictor of fungal colonization of seeds. We posit that the impacts of individual fungi and microbial consortia on seed viability and germination may in turn reflect fungal interactions with the suites of plant defenses codified recently under the broad framework of seed dormancy-defense syndromes. Our findings set the stage for experiments that track colonization, germination, and seedling establishment in the field, important for understanding impacts of fungi on the recruitment of tropical trees. Keywords

Barro Colorado Island · Clonostachys · Effective specialization · Fusarium · Lasiodiplodia · Phylogenetic signal · Pioneer trees · Trichoderma

22.1

Introduction

Fungi are important drivers of plant distributions, demography, and fitness, influencing the growth, survival, reproduction, and nutrient uptake of the plants with which they interact (Kirkpatrick and Bazzaz 1979; Harley and Smith 1983; Agrios 2005; Augspurger and Wilkinson 2007). Insights into their ecology and natural history in agricultural and agroforestry systems are important for clarifying the economic aspects of plant-fungal interactions and their response to altered ecosystems (e.g., Parker and Smith 1990; Agrios 2005; Gilbert 2005; DesprezLoustau et al. 2007; Barrett et al. 2009; Bonfante and Anca 2009). Despite a growing interest in plant-fungal interactions in natural systems, substantial gaps in knowledge remain regarding the diversity, composition, functional traits, and importance of the fungi that affiliate with plants in unmanaged plant communities. These gaps are especially profound for one of the most important but least-studied guilds of fungi: those that interact with seeds in the soil. Together, the diverse soilborne fungi that recruit to seeds after dispersal include some of the most important agents of seed mortality, as well as fungal strains that enhance germination or coexist with seeds without detriment (see Gallery et al. 2007; Kluger et al. 2008; Zalamea et al. 2015; Sarmiento et al. 2017; Shaffer et al. 2018). These impacts are especially profound in earth’s most diverse terrestrial

22

Colonization of Seeds by Soilborne Fungi: Linking Seed Dormancy-Defense. . .

467

ecosystems—tropical forests—where soilborne fungi have emerged as major determinants of seed fate (Gallery et al. 2010; Dalling et al. 1998; Sarmiento et al. 2017; Zalamea et al. 2018). As a prelude to the effects of pathogens and mutualists that interact with seedlings at establishment and in early phases of growth (e.g., Mangan et al. 2010; Bashyal et al. 2014), seed-associated fungi act as a primary filter that determines the capacity of tropical seeds to survive and germinate (Zalamea et al. 2015). Understanding the factors that shape fungal colonization of seeds, and their subsequent impacts on seed germination and viability, is important for developing predictions about host range, host specificity, and the roles of fungi in shaping the dynamics of natural and humanmaintained ecosystems from the earliest stages of tree recruitment. Recent studies suggest that many tropical seed-associated fungi are generalists in terms of their ability to colonize diverse hosts (e.g., Gallery et al. 2007; Kluger et al. 2008). However, host-specific effects on seed viability and germination are common and important (Sarmiento et al. 2017). Such functional or effective specialization plays out in the form of differential impacts of individual fungi on diverse plant species and, in turn, differential responses of individual plant species to diverse fungi (Sarmiento et al. 2017). To date most of the analyses of interactions between tropical seeds and soilborne fungi have focused on germination and seed viability of species of tropical pioneer trees (e.g., Sarmiento et al. 2017; Shaffer et al. 2018). Pioneer trees are compelling for the study of seed-fungal interactions because the small seeds of such early successional trees frequently persist in soils after dispersal for periods ranging from weeks to decades, only germinating when conditions become appropriate (e.g., when canopy gaps form; Schupp et al. 1989; Dalling et al. 1998). Because the time between gap formation events in a given site can be on the order of many years, many pioneer trees have seed traits that allow them to persist in the soil seed bank for years to decades (Dalling and Brown 2009). Recent work has shown that seeds of pioneer trees possess suites of defensive traits that are relevant for their interactions with fungi (Zalamea et al. 2018; see also Dalling et al. 2011). These “dormancy-defense syndromes” (DDS) represent constitutive physical and chemical defenses that, in particular combinations, can be linked directly to seed dormancy classes (Zalamea et al. 2018). Broadly, tropical pioneer trees can have seeds that are ephemeral in the soil (i.e., are quiescent, do not display strong physical or chemical defenses, and typically germinate without dormancy when conditions are right). Other species have seeds that are permeable and chemically well-defended (e.g., with phenols), corresponding to physiological dormancy (Zalamea et al. 2018). As a third strategy, some species have seeds that are impermeable and exhibit robust physical defenses, corresponding to physical dormancy (Zalamea et al. 2018). Strikingly, physical and chemical defenses of tropical seeds do not display univariate trade-offs, instead working in concert and linked directly to dormancy classes (Zalamea et al. 2018). The DDS framework fosters a predictive approach whereby seed dormancy class can be used as a proxy for estimating strategies of seed defense against fungi. Such predictions can be tested experimentally. Sarmiento et al. (2017) and Shaffer et al.

468

S. M. Stump et al.

(2018) have shown that seeds of tropical pioneer trees respond differently to particular fungi, with the next step being an explicit linkage of the outcome of such interactions to the DDS model. An important first step is to explore the factors that shape the earliest phases of interactions between soilborne fungi and seeds—that is, the process and dynamics of seed colonization, when fungal hyphae first contact seed surfaces and colonize seed interiors. Factors that influence colonization of seeds have not been identified for tropical seed-associated fungi but may include seed traits that reflect the evolutionary placement and relatedness of host species or functional traits relevant to DDS that do not necessarily reflect phylogenetic relatedness. Plants that are closely related to one another typically share more fungal associates with one another than with evolutionarily distant plants, suggesting that traits reflecting phylogenetic relatedness are important in determining host ranges of fungi (Webb et al. 2002; Blomberg et al. 2003; Gilbert and Parker 2016). In line with that prediction, common garden experiments with nine species of tropical pioneer trees in a lowland tropical forest in Panama revealed that the communities of fungi that infect seed interiors after burial in soil (mimicking dispersal) are structured much more strongly by host taxon (e.g., host species) than by burial duration, burial location, or seed viability (Sarmiento et al. 2017). This suggests that the early phases of colonization should reflect the evolutionary relatedness of hosts. However, some functional traits of seeds are relatively decoupled from phylogeny, instead appearing to reflect trait convergence. For example, different species in the genus Trema produce seeds that represent different dormancy classes (Zalamea et al. 2018). In some cases such functional traits might vary with phylogenetic relatedness, but in tropical pioneer trees, members of different families have converged on particular DDS (Zalamea et al. 2018). Here we provide a case study in which we examine seed colonization by soilborne fungi. Our aim is to quantify the host range of fungal strains isolated originally from seeds of tropical pioneer trees, which they colonized in soil in the experiments described by Zalamea et al. (2015) and Sarmiento et al. (2017). We evaluate whether host range can be predicted by field observations of host use, seed dormancy-defense syndromes, or phylogenetic relatedness of fungi or hosts. On the basis of previous work, we predicted that host range observed in the field would represent a subset of the potential host range of each strain, that strains would be host-generalists with regard to seed colonization, and that the earliest phase of seed colonization would reflect seed traits in a manner consistent with the DDS framework. Consistent with our first prediction, we show that each fungus colonized multiple tree species beyond those observed in field surveys. In line with our second prediction, individual strains differed in their capacity to colonize different tree species. In contrast to our third prediction, we found that phylogenetic relatedness of hosts was a stronger predictor of fungal colonization than seed dormancy class alone. This suggests that the ultimate filter of community composition in a given seed may be the host taxon, consistent with the results shown by Sarmiento et al. (2017). In turn, effective specialization, which results in differential impacts of fungi on seed viability and germination in different tree species, may be structured by factors relevant to

22

Colonization of Seeds by Soilborne Fungi: Linking Seed Dormancy-Defense. . .

469

dormancy-defense syndromes of host plants and the functional traits of fungi themselves.

22.2

Case Study

Our case study was conducted in conjunction with a common garden experiment in lowland tropical forest on Barro Colorado Island (BCI), Panama, where we are the seed dormancy-defense syndrome hypothesis (Zalamea et al. 2015, 2018; Sarmiento et al. 2017). The study examines the defensive traits and microbial associations of seeds of 18 species of pioneer trees. Briefly, seeds of each species were collected from multiple maternal sources, surface-sterilized, and buried in mesh bags to exclude macroscopic predators in common gardens at five locations on BCI. Bags were retrieved at timepoints ranging from 0 to 30 months, after which seeds were assessed for viability, germinability, seed coat integrity, and microbial infection (Zalamea et al. 2015, 2018; Sarmiento et al. 2017). The experimental design permits seed traits to be linked to microbial infection at the level of individual seeds, providing an opportunity to estimate the observed host range of each fungus and their effect on seed survival (Sarmiento et al. 2017). Fungi were isolated on 2% malt extract agar (MEA) from surface-sterilized seeds that had been buried in forest soil. The viability of each of those seeds was scored by tetrazolium staining (Peters 2000), such that each fungal isolate could be traced to a given tree species, experimental garden, seed burial duration, and seed viability class. Each strain was vouchered at the University of Arizona Robert L. Gilbertson Mycological Herbarium (ARIZ) and sequenced bidirectionally for a ca. 1000 base pair fragment comprising the nuclear ribosomal internal transcribed spacers and 5.8S gene (ITSrDNA) and ca. 600 base pairs of the nuclear ribosomal large subunit (LSUrDNA) (Sarmiento et al. 2017). These data were used to establish operational taxonomic units (OTUs) at 95%, 97%, 99%, and 100% sequence similarity, and taxonomic analyses were placed strains to the genus level and above (Sarmiento et al. 2017). For the present case study, eight isolates were selected to represent a phylogenetically diverse pool of strains that contain both distantly and closely related taxa (Table 22.1). Together the focal strains represent four genera and four families of Ascomycota and a range of observed abundance, host range, and host effects (Sarmiento et al. 2017). Our selection included two pairs of isolates that are 99% similar in the ITSrDNA-LSUrDNA region, and a pair of isolates that are 95% similar (Table 22.1). We present results that use 99% ITSrDNA-LSUrDNA similarity as our OTU designation, though results with other OTU cutoffs gave qualitatively similar results. These fungi were used to inoculate seeds of five species of pioneer trees in vitro: Apeiba membranacea (Malvaceae, physical dormancy), Ficus insipida (Moraceae, quiescent), Zanthoxylum ekmanii (Rutaceae, physiological dormancy), Trema micrantha “brown” (Cannabaceae, quiescent), and Trema micrantha “black” (Cannabaceae, physiological dormancy; see Dalling et al. 1997; Silvera et al. 2003; Pizano et al. 2010). These species co-occur on BCI (Dalling et al. 1997) and are being

470

S. M. Stump et al.

Table 22.1 Seed-associated fungi used in case study to assess seed colonization in in vitro trials Isolate frequency (%) 1.6

Viability score 0.09

0.06

0.33

AM, AS, CI, CL, CP, CG LS, TB, ZE

Fusarium sp. 2 Fusarium sp. 3 Lasiodiplodia sp.

0.02

0

1.2

Trichoderma sp.

OTU identification Clonostachys sp. Fusarium sp. 1

Focal isolate (s) PS0504

Original source TB

PS0018

AM

TB

PS0943 PS0547

AS TB

0.32

AS, CP, JC, LL, TB, ZE

PS0993

AS

2.8

0.63

PS0042

AM

5.2

0.63

AS, AM, CI, CL, CP, CV, FI, HA, JC LL, LS, TB, ZE AS, AM, CI, CL, CP, FI, HA, LL, OP, TB, ZE

PS1042 PS0037

AS AM

Observed associations AS, LL, TB

Table lists the genus-level identification of each operational taxonomic units (OTU, based on 99% ITSrDNA-LSUrDNA sequence similarity); the isolation frequency for each OTU (based on the number of isolates among a total of 5323 isolates collected; Sarmiento et al. (2017)); the proportion of seeds from which the OTU was isolated that were viable; the host range (observed associations) for field collections of each OTU; the focal isolates used in these experiments; and the species from which each focal isolate was originally obtained Plant species names: AS, Annona spraguei; AM, Apeiba membranacea; CG, Colubrina glandulosa; CI, Cecropia insignis; CL, Cecropia longipes; CP, Cecropia peltata; CV, Cochlospermum vitifolium; LL, Lindackeria laurina; LS, Luehea seemannii; HA, Hieronyma alchorneoides; FI, Ficus insipida; JC, Jacaranda copaia; OP, Ochroma pyramidale; TB, Trema micrantha “black”

studied as part of the larger experiment described above (Sarmiento et al. 2017; Zalamea et al. 2018).

22.2.1 Experimental Procedures Fresh, mature fruits or recently fallen seeds of each focal species were collected from multiple adult trees per species at BCI. Seeds were removed from the fruits, cleaned, allowed to dry, and stored using standard protocols for each species (Zalamea et al. 2018). Our methods followed Sarmiento et al. (2017) and Shaffer et al. (2018). Briefly, we exposed 5 sets of 20 seeds of each species to each fungal isolate. Prior to inoculation, seeds were surface-sterilized by sequential immersion (95% EtOH, 10 s; 0.7% NaClO, 2 min; 70% EtOH, 2 min). This procedure removes surface microbes, but does not affect germination or viability (Gallery et al. 2007; Sarmiento et al. 2017). Seeds then were placed on a lawn of actively growing fungal mycelium (ca. 11–13 days old) on 2% MEA in 60 mm Petri dishes (20 seeds/dish). Dishes were wrapped with Parafilm and incubated in the dark at ambient temperatures (consistent

22

Colonization of Seeds by Soilborne Fungi: Linking Seed Dormancy-Defense. . .

471

with outdoor temperatures; ca. 26
C) for 5–7 days. Control seeds were surfacesterilized, placed into Petri dishes containing 2% MEA but no fungal growth, and incubated as above. Overall, 4500 seeds were included in the case study. After incubation, seeds were examined for visible colonization by fungi by scoring the number of seeds per plate with evident hyphal growth on their seed coats. Fungi on seeds were judged to be consistent with the inoculated strains (rather than contaminants) by visual inspection of morphological characteristics. To test whether colonization was an indication of internal infection, we surfacesterilized a subset of seeds and transferred them to sterile Petri plates lined with sterile filter paper. We moistened the filter paper with sterile water, sealed the plates with Parafilm, and placed them in a shadehouse at ambient outdoor temperature (Gallery et al. 2007). Fungi reappeared in each Petri plate, providing evidence of internal infection.

22.2.2 Data Analyses Except when otherwise noted, colonization was examined using generalized linear models with a quasibinomial error family, implemented with glm() in R (R Development Core Team 2009). A quasibinomial error family was used because our data showed more between-plate variation than expected under a binomial distribution. The per-dish colonization fraction was used as the response variable. Values of 0% or 100% were amended by adding one success and one failure to each plate, as the logit transformation needed for a binomial or quasibinomial error family is undefined at 0 and 1. We measured whether fungi had host-specific colonization rates by determining the significance of (seed species)  (fungal OTU) interactions. Generalized linear models require that a particular fungal OTU and seed species be chosen as the null case, against which interactions are judged (Crawley 2007). The no-fungus control was an obvious choice for a fungal null case, as the null hypotheses were that fungi did not colonize seeds differently. However, there was no obvious choice for a null seed species, and the choice altered which interactions were significant. To account for this, we assessed the number of significant interactions taking every seed species as the null case. We tested two hypotheses about the host range of fungi: (1) fungi cannot infect seeds outside of their observed host range, and (2) fungi are capable of infecting seeds outside of their observed host range, although colonization rates are low. The first hypothesis was tested by observing whether fungi colonized seed species that were not one of their known associations. Known associations were defined by the observation of that OTU in a seed of that species in the field experiments detailed by Sarmiento et al. (2017) (Table 22.1). We tested the second hypothesis by determining if seed colonization was higher on species known previously to be associated with that OTU vs. species for which such associations had not been observed in the field (Table 22.1). We used a generalized linear mixed model with known association as a fixed effect and fungal OTU and seed species as random effects (to account

472

S. M. Stump et al.

for fungi being differently able to colonize seeds and seeds being differently protected against fungal colonization). We also included an interaction between fungal OTU and known association, in case some fungi were more able to colonize seeds outside of their known host range. We tested for evolutionary constraints on host range and host affinity among fungi using three methods. First, we tested for evolutionary constraints over short timescales by testing whether colonization differed within fungal OTU. We did this by testing for significant (seed species)  (fungal isolate) interactions among our 99% similar isolates (i.e., within Fusarium sp. 1 and Lasiodiplodia sp.) or among those representing our 95% similar species pair (i.e., Fusarium sp. 2 and Fusarium sp. 3). Second, we tested for evolutionary constraints at intermediate timescales by testing if within-OTU differences in colonization were smaller than between-OTU differences in colonization. The difference in colonization between isolates was quantified using two dissimilarity indices. First, mean dissimilarity (djk) between two isolates j and k is d jk ¼ I j  I k where I j and I k are the mean colonization fraction of isolates j and k, respectively, across all seed species. Second, relative dissimilarity (rjk) is r jk ¼

rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi X    2 I sj  I j  I sk  I k s

where Isj and Isk are the proportion of seeds of seed species s that were colonized by fungal isolate j and k in vitro, and the summation is over all seed species. Thus, if strain j had a higher colonization rate on all seeds than strain k, but each had relatively similar colonization once the mean difference was removed (e.g., both specialized on physically dormant seeds), then djk would be large and rjk would be small. We tested whether within-OTU dissimilarity was smaller than between-OTU dissimilarity using a one-tailed randomization test (n ¼ 1,000,000), implemented in R. To calculate p-values, we factored out cases where the randomized within-OTU dissimilarities were the same as the actual within-OTU dissimilarities. Finally, we tested whether host range and affinity are conserved among fungi over longer timescales by testing for phylogenetic constraints in the colonization fraction on seeds of each plant species, the mean colonization fraction across all species, and the relative colonization fraction (i.e., colonization fraction of a given species—mean colonization overall). A phylogenetic tree for the fungi examined here was generated using LSUrDNA data (obtained by Sarmiento et al. 2017). Sequences were aligned using Muscle (Edgar 2004), and a tree was inferred in RAxML (Stamatakis 2006). Phylogenetic constraints were assessed with Blomberg’s K (Blomberg et al. 2003) and Pagel’s λ (Pagel 1992), as each produces slightly different outcomes (Godoy et al. 2014). Significance was assessed using randomization (n ¼ 1,000,000) and likelihood ratio tests, respectively, implemented using phylosig in R (R Development Core Team 2009). We tested whether fungal colonization reflects plant relatedness or dormancydefense syndromes by testing how much variation in fungal colonization could be

22

Colonization of Seeds by Soilborne Fungi: Linking Seed Dormancy-Defense. . .

473

explained by clade or dormancy class. There were 40 ways that the 5 plant species could be categorized into 2 or 3 groups (i.e., there were 10 3-1-1 groupings, 10 3-2-0 groupings, 5 4-1-0 groupings, and 15 2-2-1 groupings). One of these groupings was by plant order (F. insipida, T. micrantha “brown,” and T. micrantha “black” are Rosales, A. membranacea is Malvales, and Z. ekmanii is Sapindales). Another was by dormancy class (F. insipida and T. micrantha “brown” are quiescent, T. micrantha “black” and Z. ekmanii are physiologically dormant, and A. membranacea is physically dormant), as dormancy class can provide insight into the seed defense syndrome (Dalling et al. 2011; Zalamea et al. 2018). We assessed how well each of the 40 groupings fit the data using a generalized linear model with a binomial error family. Our model had fungal OTU, seed species, and group identity as fixed effects, along with the (seed species)  (group identity) interaction. The grouping with the lowest Akaike information criterion (AIC, Akaike 1974) was considered to be the best. We compared the ranking of clade and dormancy class among all possible groupings.

22.2.3 Results Seeds of five species of tropical pioneer trees were colonized by all focal fungi in in vitro inoculation trials (Fig. 22.1). Fungal colonization success varied as a function of fungal strains and plant species (Fig. 22.1). However, even accounting for this, there were on average 8 significant (seed species)  (fungal OTU) interactions ( p < 0.05) and 4 highly significant interactions ( p < 0.01), out of a possible 24. Thus, if one selected two fungal isolates and one seed species at random, there was about a one in three chances that those isolates had significantly different colonization rates on that seed (and one in six chances that they differed at the p < 0.01 level). Similarly, if two seed species and one fungal isolate were selected at random, there was about a one in three chances that those that isolate had different colonization rates on each seed. Our results were similar if we

Fig. 22.1 Fraction of seeds colonized by fungi. Each bar represents the fraction of seeds colonized in each OTU-seed species pairing (20 seeds per plate, for a total of 100 seeds). Error bars represent 1 standard error. Bars marked with “þ” indicate OTU that were isolated from seeds of that species in field surveys, and bars marked with “–” have not yet been isolated from seeds of that species. Clo, Clonostachys sp.; Fus, Fusarium sp; Las, Lasiodiplodia sp.; Tri, Trichoderma sp.

474

S. M. Stump et al.

Fungal colonization

1

0.75

*

*

*

*

Las strain 1

*

* 0.5

Fus 1 strain 1 Fus 1 strain 2

*

Fus 2

0.25

0

Las strain 2

Fus 3

+ + + + – – + +– – – – + + + ++ +

A. membranacea

F. insipida

T. micrantha "black"

– –– ––– T.micrantha "brown"

+ + + +–+ Z. ekmanii

Fig. 22.2 Fraction of seeds colonized by fungi as a function of seed species and fungal isolate. Each bar represents 100 seeds. Error bars represent 1 standard error. Bars marked with “þ” indicate OTU that were isolated from seeds of that species in field surveys, and bars marked with “–” have not yet been isolated from seeds of that species. Any significant ( p < 0.05) within-OTU differences in colonization or germination are marked with a star. Las strain 1 (PS0042) and Las strain 2 (PS1042) are 99% similar in their ITSrDNA-LSUrDNA. Fus 1 strain 1 (PS0018) and Fus 1 strain 2 (PS0943) are 99% similar in their ITSrDNA-LSUrDNA. Fus 2 and 3 (Fusarium sp. 3 and 4) are 95% similar in their ITSrDNA-LSUrDNA

focused on individual isolates instead of OTU: an average of 12.4 interactions were significant ( p < 0.05), and an average of 8.8 interactions were significant ( p < 0.01), out of a possible 32. All fungi were able to colonize seeds outside of their previously observed associations (Fig. 22.1). When average colonization fraction was accounted for, fungi showed no difference in their ability to colonize seeds in their known host range vs. seeds of other species ( p ¼ 0.74). Our results suggested a limited amount of phylogenetic constraint on host range among fungi. Isolates within the same OTU (both at 99% and 95% of sequence similarity) differed in their ability to colonize seeds of at least one plant species (Fig. 22.2). Colonization patterns for a given isolate were marginally more similar to that of isolates of the same OTU than isolates of different OTU (relative dissimilarity; p ¼ 0.06 for 99% OTU and p ¼ 0.07 for 95% OTU). However, mean dissimilarity, djk, was not significantly lower within-OTU vs. between them ( p ¼ 0.15 for 99% OTU and p ¼ 0.13 for 95% OTU). Mean colonization across all seed species showed phylogenetic constraint using both focal statistics ( p ¼ 0.055 for K and p ¼ 0.02 for λ, Table 22.2). We found evidence for phylogenetic constraint in the relative ability to colonize Z. ekmanii (i.e., similar fungi had similar colonization rates on Z. ekmanii, p ¼ 0.048 for K and p ¼ 0.061 λ, Table 22.2). In other cases, the colonization fraction showed a significant constraint for only one of the two statistics. Plant order was the best grouping (AIC ¼ 1088) for explaining fungal colonization (Fig. 22.3). The next four best groupings also grouped species into monophyletic groups: they placed Z. ekmanii and A. membranacea in their own group and then contained every possible permutation of T. micrantha “brown,” T. micrantha “black,”

22

Colonization of Seeds by Soilborne Fungi: Linking Seed Dormancy-Defense. . .

475

Table 22.2 Phylogenetic conservatism in fungal colonization Seed species A. membranacea—mean F. insipida—mean T. micrantha “black”—mean T. micrantha “brown”—mean Z. ekmanii—mean A. membranacea—relative F. insipida—relative T. micrantha “black”—relative T. micrantha “brown”—relative Z. ekmanii—relative Overall mean

K estimate 8.16  105 5.65  104 1.46  104 1.68  103 1.26  104 9.27  105 5.40  104 7.39  105 1.55  104 3.76  104 2.46  104

p-value 0.307 0.032 0.156 0.090 0.196 0.245 0.051 0.238 0.139 0.048 0.055

λ estimate 0.204 0.744 0.614 0.906 0.000 0.000 0.000 0.295 0.856 0.959 0.772

p-value 0.696 0.110 0.066 0.001 1.000 1.000 1.000 0.434 0.011 0.061 0.024

Phylogenetic constraint was assessed using Blomberg’s K (Blomberg et al. 2003) and Pagel’s λ (Pagel 1992). Mean colonization fraction on a particular seed species is the fraction of seeds colonized across all five replicates. Mean overall colonization is the mean colonization across the entire study (i.e., all five replicates of all five tree species). Relative colonization is the mean colonization on a particular species minus the mean overall colonization and is used to disentangle overall colonization ability from the ability to colonize particular seeds. Significant or marginally significant ( p < 0.07) values are shown in bold. Significant constraints indicate that related fungi are more likely to have a similar colonization fraction on a given seed species, a similar colonization fraction overall, or a similar relative colonization fraction on a given seed species

and F. insipida. Dormancy class was the 11th best grouping, with an AIC of 1250 (Fig. 22.3).

22.2.4 Perspectives Seed-associated fungi play a critical role in the demography of tropical trees (Kirkpatrick and Bazzaz 1979; Harley and Smith 1983; Arnold et al. 2003; Agrios 2005; Augspurger and Wilkinson 2007; Sarmiento et al. 2017). However, basic details about the host range and specificity of seed-associated fungi are rarely known. Our experiments complement previous observations about seed fate and seed-fungal associations in a lowland tropical forest (Sarmiento et al. 2017; Zalamea et al. 2018). Our results suggest that the fungi we considered had a wide host range in terms of colonization but had isolate-specific colonization rates on different hosts. We found that all fungal OTU were able to colonize species they had not been associated within field surveys, suggesting that potential host range is larger than observed host range. We found that host range was evolutionarily labile. Finally, we found that host taxonomy was a strong predictor of fungal colonization patterns. Because infection persisted among the subset of seeds that was surface-sterilized after allowing for colonization by fungi, our results imply that colonization indicated internal infection. This suggests that the fungi we considered have a wide potential host range, even if their observed host range appears narrow.

476

S. M. Stump et al. 8 7

*

**

Frequency

6 5 4 3 2 1 0

1100 1150 1120 1250 1300 1350 1400 1450 1500 1550

Grouping AIC Fig. 22.3 Predictors of fungal colonization on seeds. A histogram displays the AIC values of a generalized linear model of every possible fungal grouping. There are 40 possible ways to group 5 fungi into 2–3 groups. The “plant order” grouping (Z. ekmanii by itself, A. membranacea by itself, and a group of T. micrantha “brown,” T. micrantha “black,” and F. insipida) is the best grouping (AIC 1088), indicated by an arrow and one star (*). The dormancy class grouping (F. insipida paired with T. micrantha “brown,” Z. ekmanii paired with T. micrantha “black,” and A. membranacea by itself) is the 11th best grouping (AIC 1250), indicated by the arrow and two stars (**). The second, third, fourth, and fifth best grouping paired Z. ekmanii with A. membranacea and then included every possible permutation of the other species

Potential host range could be wider than the observed host range for several reasons. First, some seed-fungal associations may be rare and thus simply have not yet been catalogued in field surveys. Alternatively, these seed-fungus associations may be short-lived, rare, or absent under natural conditions because of competition with other microbes (Barrett et al. 2009). This could be tested in part by inoculating seeds with fungal consortia and testing if colonization is reduced in some cases. A third alternative is that in vitro colonization may not be indicative of the ability to infect seeds under natural conditions, where factors such as soil chemistry, or remnants of fruit on seed surfaces (here removed by treatment before inoculation), could be important. These questions could be addressed with a colonization experiment performed under more natural conditions. For example, instead of placing seeds on agar that have been colonized by fungus, we could place seeds in soil that has been sterilized and then inoculated with a small amount of fungi. The observation that the actual host range of plant-associated fungi is often more narrow than their potential host range has been documented previously for a number of pathogenic fungi, including fungi associated with seeds (Beckstead et al. 2014, see also de Vienne et al. 2009). However, while many studies have documented this trend, relatively few have explored for predictors for novel plant-fungus associations (de Vienne et al. 2009). It is becoming clear that phylogenetic relatedness of hosts is a strong factor in determining whether they are likely to share certain pests (reviewed

22

Colonization of Seeds by Soilborne Fungi: Linking Seed Dormancy-Defense. . .

477

in Gilbert and Parker 2016). Here, we add merit to the importance of host relatedness in predicting susceptibility to seed-associated fungi by showing that plant order best explained seed colonization by fungi (Fig. 22.3). Overall, results of our case study suggest that host range and affinity of seed-infecting fungi are evolutionarily labile: there was little evidence of phylogenetic constraint on specialization (Table 22.2), and even fungal strains that were 99% similar at the ITSrDNA-LSUrDNA locus differed in colonization on seeds of different plant species (Fig. 22.2). It is possible that these differences are due to within-OTU variation in loci that code for functional traits. Alternatively, these fungal isolates may differ in other factors, such as infection with endohyphal bacteria (Hoffman and Arnold 2010; Shaffer et al. 2016). Given how quickly microbes adapt to new hosts under experimental conditions (e.g., Little et al. 2006; Wallis et al. 2007; Agudelo-Romero et al. 2008), it seems reasonable that specialization at the species level could change quickly over evolutionary time. Interestingly, our results suggest that generalized colonization ability was phylogenetically conserved among fungi, even if species-specific colonization rates were not: mean colonization showed phylogenetic constraint among both statistics, whereas the majority of colonization on individual plant species showed no significant constraint (Table 22.2). This result would be unlikely if the ability to colonize each seed species evolved independently. The evolution of host-specific virulence often comes at a cost of virulence on another host (Ebert 1998); it is thus reasonable to suspect that the ability to colonize particular hosts is more labile than the ability to colonize any host. Alternatively, the effect we detected may reflect a phylogenetic signal in the environmental conditions that promote colonization by particular strains or taxa. Infection is partly a function of environmental conditions (Parker and Gilbert 2004; Barrett et al. 2009), and different symbiont species can be differently infectious under different conditions (Whipps 1987; Pažoutová et al. 2000). Perhaps the phylogenetic signal was observed because the fungal isolates with high in vitro colonization were most infectious under the temperature, moisture, and nutrient conditions in our study, and their sister taxa had similar environmental requirements. This could be tested by doing another inoculation experiment under different conditions and testing whether colonization fractions change but phylogenetic signal holds. Although related fungi did not necessarily colonize the same species of plants, related plants were colonized similarly by fungal isolates: plant taxonomy was the strongest predictor of fungal colonization (Fig. 22.3). We know of only one study that examined both phylogenetic constraint in host defenses and pathogen virulence, and they found that pathogens were far less phylogenetically constrained than their hosts in terms of associations (Mariadassou et al. 2010). This result should perhaps not be surprising, given that the generation time of fungi is far shorter than that of trees (Gilbert and Parker 2016). Overall, dormancy class (and thus DDS) did not emerge as clear predictor of fungal colonization. We anticipate that DDS may be particularly important at the next phase of seed-fungal interactions: that is, we expect that host taxon is the first major filter that will select communities of seed-associated fungi, as suggested by Sarmiento et al. (2017). Subsequently, those that survive in seeds and function, individually or in consortia, to

478

S. M. Stump et al.

influence seed viability and germination may be influenced by the defense and dormancy traits codified as DDS. In that situation we would anticipate that fungi would be differentially sensitive to phenols and other seed chemical defenses, especially those mobilized by seed imbibition and the physiological cascades associated with germination.

22.3

Future Directions

The case study presented here represents an early step in documenting the natural history of seed-associated fungi in tropical soils, with special attention to the earliest phases of fungal contact with and establishment in seeds. In general, our results suggest fungal host range in nature is limited more by competition or environmental conditions, rather than an inability to infect certain seeds. The ability of fungi to colonize seeds was more labile than the ability of seeds to protect themselves from fungal colonization. Our study points to the primary role of host taxonomy in determining colonization success but suggests also the need to learn more about fungal functional traits. Although phylogenetically diverse, the fungi that colonize seeds of tropical trees are especially common among genera such as Fusarium and Xylaria (see Shaffer et al. 2016), suggesting the potential to evaluate functional traits in robust phylogenetic contexts at the genus level. In future work we advocate exploring the relevance of DDS for predicting the responses of seeds to infection by individual fungi and consortia and cataloguing fungal traits to understand how growth rate, enzyme production, and nutrient scavenging may influence the host breadth, colonization efficiency, and impacts on seed fate of soilborne fungi. Recent attention to the capacity of some soilborne fungi to harbor endohyphal bacterial symbionts that influence seed colonization by fungi and their subsequent impacts on seed viability and germination (Shaffer et al. 2018) speak to important and under-explored roles of such traits in driving the dynamics of seeds in soil seed banks, in turn relevant to the dynamics of earth’s most diverse terrestrial ecosystems. Acknowledgments We thank Margaret Wilch, Kayla Garcia, Daniel Roche, Abby Robison, and the support staff on BCI for assistance, guidance, and logistical support during the experiment. We thank Peter Chesson, M. Natalia Umaña, and Meghan Krishnadas for helpful discussion and advice on statistics. This work was funded by NSF DEB-1119758 to AEA and NSF DEB-1120205 to JWD and ASD. PCZ was supported in part by a grant from the Simons Foundation to the Smithsonian Tropical Research Institute (429440, WTW). SMS was supported by NSF DGE-0841234 and the University of Arizona and gratefully acknowledges the Department of Ecology and Evolutionary Biology and the Institute for the Environment for funding support. We thank the Smithsonian Tropical Research Institute and the Republic of Panama for the opportunity to conduct research there.

22

Colonization of Seeds by Soilborne Fungi: Linking Seed Dormancy-Defense. . .

479

References Agrios GN (2005) Plant pathology, vol 5E. Academic Press, Cambridge Agudelo-Romero P, de la Iglesia F, Elena SF (2008) The pleiotropic cost of host-specialization in tobacco etch potyvirus. Infect Genet Evol 8:806–814. https://doi.org/10.1016/j.meegid.2008.07.010 Akaike H (1974) A new look at the statistical model identification. IEEE Trans Autom Control 19:716–723. https://doi.org/10.1109/TAC.1974.1100705 Arnold AE, Mejía LC, Kyllo D et al (2003) Fungal endophytes limit pathogen damage in a tropical tree. Proc Natl Acad Sci USA 100:15649–15654. https://doi.org/10.1073/pnas.2533483100 Augspurger CK, Wilkinson HT (2007) Host specificity of pathogenic pythium species: implications for tree species diversity. Biotropica 39:702–708. https://doi.org/10.1111/j.1744-7429.2007.00326.x Barrett LG, Kniskern JM, Bodenhausen N (2009) Continua of specificity and virulence in plant hostpathogen interactions: causes and consequences. New Phytol 183:513–529. https://doi.org/10.1111/ j.1469-8137.2009.02927.x Bashyal B, Aggarwal R, Banerjee S, Gupta S, Sharma S (2014) Pathogenicity, ecology and genetic diversity of the Fusarium spp. associated with an emerging bakanae disease of rice (Oryza sativa L.) in India. In: Kharwar R, Upadhyay R, Dubey N, Raghuwanshi R (eds) Microbial Diversity and Biotechnology in Food Security. Springer, New Delhi Beckstead J, Meyer SE, Reinhart KO et al (2014) Factors affecting host range in a generalist seed pathogen of semi-arid shrublands. Plant Ecol 215:427–440. https://doi.org/10.1007/s11258-0140313-3 Blomberg SP, Garland T, Ives AR (2003) Testing for phylogenetic signal in comparative data: behavioral traits are more labile. Evolution 57:717–745. https://doi.org/10.1111/j.0014-3820.2003. tb00285.x Bonfante P, Anca IA (2009) Plants, mycorrhizal fungi, and bacteria: a network of interactions. Annu Rev Microbiol 63:363–383. https://doi.org/10.1146/annurev.micro.091208.073504 Crawley MJ (2007) The R book, vol 1E. Wiley, Hoboken Dalling JW, Brown TA (2009) Long-term persistence of pioneer species in tropical rain forest soil seed banks. Am Nat 173:531–535. https://doi.org/10.1086/597221 Dalling JW, Swaine MD, Garwood NC (1997) Soil seed bank community dynamics in seasonally moist lowland tropical forest, Panama. J Trop Ecol 13:659–680. https://doi.org/10.1017/ S0266467400010853 Dalling JW, Swaine MD, Garwood NC (1998) Dispersal patterns and seed bank dynamics of pioneer trees in moist tropical forest. Ecology 79:564–578. https://doi.org/10.1890/0012-9658(1998)079[ 0564:DPASBD]2.0.CO;2 Dalling JW, Davis AS, Schutte BJ et al (2011) Seed survival in soil: interacting effects of predation, dormancy and the soil microbial community. J Ecol 99:89–95. https://doi.org/10.1111/j.1365-2745. 2010.01739.x de Vienne DM, Hood ME, Giraud T (2009) Phylogenetic determinants of potential host shifts in fungal pathogens. J Evol Biol 22:2532–2541. https://doi.org/10.1111/j.1420-9101.2009.01878.x Desprez-Loustau ML, Robin C, Buee M et al (2007) The fungal dimension of biological invasions. Trends Ecol Evol 22:472–480. https://doi.org/10.1016/j.tree.2007.04.005 Ebert D (1998) Evolution - experimental evolution of parasites. Science 282:1432–1435. https://doi.org/ 10.1126/science.282.5393.1432 Edgar RC (2004) Muscle: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797. https://doi.org/10.1093/nar/gkh340 Gallery RE, Dalling JW, Arnold AE (2007) Diversity, host affinity, and distribution of seed-infecting fungi: a case study with Cecropia. Ecology 88:582–588. https://doi.org/10.1890/05-1207 Gallery RE, Moore DJP, Dalling JW (2010) Interspecific variation in susceptibility to fungal pathogens in seeds of 10 tree species in the neotropical genus cecropia. J Ecol 98:147–115. https://doi.org/10.1111/j.1365-2745.2009.01589.x

480

S. M. Stump et al.

Gilbert GS (2005) The dimension of plant disease in tropical forests. In: Burlesem DFRP, Pinard MA, Sue E (eds) Biotic interactions in the tropics: their role in the maintenance of species diversity. Cambridge University Press, Cambridge, pp 141–164 Gilbert GS, Parker IM (2016) The evolutionary ecology of plant disease: a phylogenetic perspective. Annu Rev Ecol Syst 54:549–578. https://doi.org/10.1146/annurev-phyto-102313-045959 Godoy O, Kraft NJ, Levine JM (2014) Phylogenetic relatedness and the determinants of competitive outcomes. Ecol Lett 17:836–844. https://doi.org/10.1111/ele.12289 Harley JL, Smith SE (1983) Mycorrhizal symbiosis. Academic Press, Cambridge Hoffman MT, Arnold AE (2010) Diverse bacteria inhabit living hyphae of phylogenetically diverse fungal endophytes. Appl Environ Microbiol 76:4063–4075. https://doi.org/10.1128/AEM.02928-09 Kirkpatrick B, Bazzaz F (1979) Influence of certain fungi on seed germination and seedling survival of four colonizing annuals. J Appl Ecol 16:515–527. https://doi.org/10.2307/2402526 Kluger CG, Dalling JW, Gallery RE et al (2008) Host generalists dominate fungal communities associated with seeds of four neotropical pioneer species. J Trop Ecol 24:351–354. https://doi.org/ 10.1017/S0266467408005026 Little TJ, Watt K, Ebert D (2006) Parasite-host specificity: experimental studies on the basis of parasite adaptation. Evolution 60:31–38. https://doi.org/10.1111/j.0014-3820.2006.tb01079.x Mariadassou M, Robin S, Vacher C (2010) Uncovering latent structure in valued graphs: a variational approach. Ann Appl Stat 4:715–742. https://doi.org/10.1214/10-AOAS361 Mangan SA, Schnitzer SA, Herre EA, Mack KML, Valencia MC, Sanchez EI, Bever JD (2010) Negative plant-soil feedback predicts tree-species relative abundance in a tropical forest. Nature 446:752–755. https://doi.org/10.1038/nature09273 Pagel MD (1992) A method for the analysis of comparative data. J Theor Biol 156:431–442. https:// doi.org/10.1016/S0022-5193(05)80637-X Parker IM, Gilbert GS (2004) The evolutionary ecology of novel plant-pathogen interactions. Annu Rev Ecol Evol Syst 35:675–700. https://doi.org/10.1146/annurev.ecolsys.34.011802.132339 Parker GA, Smith JM (1990) Optimality theory in evolutionary biology. Nature 348:27–33. https:// doi.org/10.1038/348027a0 Pažoutová S, Olšovská J, Linka M et al (2000) Chemoraces and habitat specialization of Claviceps purpurea populations. Appl Environ Microb 66:5419–5425. https://doi.org/10.1128/AEM.66. 12.5419-5425.2000 Peters J (2000) Tetrazolium testing handbook: contribution no. 29 to the handbook on seed testing. Association of Official Seed Analysts Pizano C, Mangan SA, Herre EA et al (2010) Above- and belowground interactions drive habitat segregation between two cryptic species of tropical trees. Ecology 92:47–56. https://doi.org/10. 1890/09-1715.1 R Development Core Team (2009) R: a language and environment for statistical computing. R Foundation for Statistical Computing Sarmiento C, Zalamea PC, Dalling JW et al (2017) Soilborne fungi have host affinity and hostspecific effects on seed germination and survival in a lowland tropical forest. Proc Natl Acad Sci USA 114:11458–11463. https://doi.org/10.1073/pnas.1706324114 Shaffer JP, Sarmiento C, Zalamea P-C, Gallery RE, Davis AS, Baltrus DA, Arnold AE (2016) Diversity, specificity, and phylogenetic relationships of endohyphal bacteria in fungi that inhabit tropical seeds and leaves. Front Ecol Evol 4:116. https://doi.org/10.3389/fevo.2016.00116 Shaffer JP, Zalamea PC, Sarmiento C et al (2018) Context-dependent and variable effects of endohyphal bacteria on interactions between fungi and seeds. Fungal Ecol 36:117 Schupp EW, Howe HF, Augspurger CK, Levey DJ (1989) Arrival and survival in tropical treefall gaps. Ecology 70:562–564. https://doi.org/10.2307/1940206 Silvera K, Skillman JB, Dalling JW (2003) Seed germination, seedling growth and habitat partitioning in two morpho-types of the tropical pioneer tree Trema micrantha; in a seasonal forest in Panama. J Trop Ecol 19:27–34. https://doi.org/10.1017/S0266467403003043

22

Colonization of Seeds by Soilborne Fungi: Linking Seed Dormancy-Defense. . .

481

Stamatakis A (2006) Raxml-vi-hpc: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688–2690. https://doi.org/10.1093/ bioinformatics/btl446 Wallis CM, Stone AL, Sherman DJ et al (2007) Adaptation of plum pox virus to a herbaceous host (Pisum sativum) following serial passages. J Gen Virol 88:2839–2845. https://doi.org/10.1099/vir. 0.82814-0 Webb CO, Ackerly DD, McPeek MA et al (2002) Phylogenies and community ecology. Annu Rev Ecol Syst 33:475–505. https://doi.org/10.1146/annurev.ecolsys.33.010802.150448 Whipps JM (1987) Effect of media on growth and interactions between a range of soil-borne glasshouse pathogens and antagonistic fungi. New Phytol 107:127–142. https://doi.org/10.1111/j.1469-8137. 1987.tb04887.x Zalamea P, Sarmiento C, Arnold AE et al (2015) Do soil microbes and abrasion by soil particles influence persistence and loss of physical dormancy in seeds of tropical pioneers? Front Plant Sci 5:1–15. https://doi.org/10.3389/fpls.2014.00799 Zalamea PC, Dalling JW, Sarmiento C et al (2018) Dormancy-defense syndromes and tradeoffs between physical and chemical defenses in seeds of pioneer species. Ecology 99:1988–1998. https://doi.org/10.1002/ecy.2419

Seed Endophytes in Crop Plants: Metagenomic Approaches to Study the Functional Roles and Interactions

23

Priyanka Verma

Contents 23.1 23.2 23.3 23.4 23.5 23.6

Seed Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanism of Endophytes Living Inside Seed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interaction of Seed Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identification of Cultivable Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genome Sequence Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.6.1 Metagenome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.6.2 Proteome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.6.3 Transcriptome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.6.4 Microarray . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.6.5 Metaproteogenomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7 Potential Applications of Endophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7.1 Phytostimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7.2 Pigment Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7.3 Enzyme Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7.4 Antimicrobial Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7.5 Source of Bioactive and Novel Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7.6 Reciprocal Interactions Between Above- and Belowground Communities . . . 23.7.7 Biocontrol Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7.8 Nutrient Cycling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7.9 Bioremediation/Biodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.7.10 Production of Volatile Organic Compounds and Their Benefits . . . . . . . . . . . . 23.8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

484 485 486 489 490 490 491 492 493 494 495 495 495 496 496 496 497 497 498 498 498 499 500 500

Abstract

Seed endophytes play a key role in increasing plant health and growth in both managed and natural ecosystems. These can be applied in agricultural production or for the phytoremediation of pollutants. However, because of their capacity to P. Verma (*) Department of Microbiology, Eternal University, Sirmaur, Himachal Pradesh, India # Springer Nature Switzerland AG 2019 S. K. Verma, J. F. White, Jr (eds.), Seed Endophytes, https://doi.org/10.1007/978-3-030-10504-4_23

483

484

P. Verma

confer plant beneficial effects, efficient colonization of the plant environment is of utmost importance. The majority of endophytes derives from the soil environment. They may migrate to the rhizosphere and subsequently the rhizoplane of their hosts before they are able to show beneficial effects. These endophytes can also penetrate plant roots, and may move to aerial plant parts. A better understanding on colonization processes has been obtained mostly by microscopic visualization as well as by analyzing the characteristics of mutants carrying disfunctional genes potentially involved in colonization. In this chapter we describe the different metagenomic approaches of seed colonization and survey the known mechanisms responsible for endophytic competence. The understanding of seed colonization processes is important to better predict how endophytes interact with seeds. Keywords

Seed endophytes · Agriculture · Metagenomic approaches · Phytoremediation

23.1

Seed Endophytes

The first description of symbiosis is “the living together of dissimilar organisms” (De Bary 1879); an array of symbiotic lifestyles have been defined based on fitness benefits or impacts to macroscopic hosts and microscopic symbionts (Lewis 1985). Collectively, more than 100 years of research suggests that most, if not all, plants in natural ecosystems are symbiotic with mycorrhizal fungi and/or fungal endophytes (Petrini 1986). These fungal symbionts can have profound effects on plant ecology, fitness, and evolution (Brundrett 2006), shaping plant communities (Clay and Holah 1999) and manifesting strong effects on the community structure and diversity of associated organisms (e.g., bacteria, nematodes, and insects) (Omacini et al. 2001). The fossil record indicates that plants have been associated with endophytic (Krings et al. 2007) and mychorrhizal (Redecker et al. 2000) fungi for >400 Myr and were likely associated when plants colonized land, thus playing a long and important role in driving the evolution of life on land. Endophytes are bacterial or fungal microorganisms that colonize healthy plant tissue intercellularly and/or intracellularly without causing any apparent symptoms of disease (Wilson 1995). They are ubiquitous, colonize in all plants, and have been isolated from almost all plants examined till date. Their association can be obligate or facultative and causes no harm to the host plants. They exhibit complex interactions with their hosts which involves mutualism and antagonism (Carroll 1988, 1991; Gehring et al. 1997; Johnson et al. 1997; Parker 1999, 1995). Plants strictly limit the growth of endophytes, and these endophytes use many mechanisms to gradually adapt to their living environments (Dudeja et al. 2012). In order to maintain stable symbiosis, endophytes produce several compounds that promote growth of plants and help them adapt better to the environment (Das 2009; Lee et al. 2004). Improvement of endophyte resources could bring us a variety of benefits, such as novel and effective bioactive compounds that cannot be synthesized by chemical reactions. For

23

Seed Endophytes in Crop Plants: Metagenomic Approaches to Study the. . .

485

this, there should be a better understanding about endophytes and their significance and roles. Understanding the biology of plants and their microbial ecology is important. As evidenced by more number of publications on endophytes in recent years, many studies have been performed for evaluating their colonization pattern of vegetative tissues as well as their effects on plant growth. This book chapter puts forward the concept about seed endophytes and their importance to the hosts and to the environment.

23.2

Mechanism of Endophytes Living Inside Seed

The existence of endophytic bacteria inside different plant tissues is a welldocumented phenomenon (Fisher et al. 1992; Gagné et al. 1987; Gardner et al. 1982; McInroy and Kloepper 1995; Patriquin et al. 1983; Sharrock et al. 1991). In general, endophytes have been defined as bacteria that are able to colonize living plant tissues without harming the plant or gaining benefit other than securing residency (Schaad et al. 2001). Several studies have shown that the interaction between plants and some endophytic bacteria was associated with beneficial effects such as plant growth promotion and biocontrol potential against plant pathogens (Bashan et al. 1990; Chen et al. 1996; Döbereiner 1992; Hallmann et al. 1995; Lalande et al. 1989; Pleban et al. 1995; Qiu et al. 1990). Although the precise location and spread of endophytic bacteria in plant tissues have been described by several authors (Gagné et al. 1987; Jacobs et al. 1985; Mahaffee et al. 1997; Old and Nicolson 1978; Patriquin and Döbereiner 1978; Quadt-Hallmann and Kloepper 1996), little is known about the mechanisms by which endophytes enter the plant. Huang (1986) summarized the avenues of entry for different plant pathogenic bacteria. Such pathways included stomata (Roos and Hattingh 1983), lenticels (Fox et al. 1972), and wounds including broken trichomes, areas of emerging lateral roots (Jacobs et al. 1985; Mahaffee et al. 1997), and the germinating radicle (Gagné et al. 1987). Bacteria may also enter through undifferentiated meristematic root tissue (Hollis 1951; Mahaffee et al. 1997). The lack of penetration structures renders bacteria unable to exert mechanical or physical forces to penetrate intact epidermal cells (Goodman 1982). However, bacteria may enter intact plant tissue by invagination of the root hair cell wall, by penetration of the junction between root hair and adjacent epidermal cells, or by enzymatic processes involving degradation of cell wall-bound polysaccharides (Huang 1986). Alternatively, it may be proposed that bacteria enter the epidermis through passive plant uptake with transpiration. Further spread inside the plants may occur via intercellular spaces or conducting elements. Endophytic colonization of cotton by Enterobacter asburiae JM22 and Pseudomonas fluorescens 89B-61 has recently been demonstrated after bacteria were applied to seeds or leaves (Mahaffee et al. 1997; Musson et al. 1995; Quadt-Hallmann et al. 1997; Quadt-Hallmann and Kloepper 1996). Strain JM22 colonized different plant species such as cucumber, bean, and cotton systemically and moved from the roots to

486

P. Verma

the stem, cotyledons, and finally leaves. Initially, bacteria were concentrated in grooves between epidermal cells on the root surface, within intercellular spaces, including spaces close to the conducting elements, and inside single root epidermal cells (Quadt-Hallmann and Kloepper 1996). Strain 89B-61 also demonstrated internal colonization of cotton root tissues, but bacteria were observed only in intercellular spaces close to the root surface with no evidence of further internal spread (QuadtHallmann et al. 1997). While the systemic colonist JM22 consistently lacked biocontrol activity, the plant growth promoting rhizobacterial (PGPR) strain and local cortical colonist 89B-61 exhibited biological control activity, including induced systemic resistance, against various pathogens in greenhouse experiments and field trials (Chen et al. 1995; Wei et al. 1991, 1996). Endophytes are microorganisms that live within plant tissues without causing symptoms of disease. They are important components of plant microbiomes. Endophytes interact with, and overlap in function with, other core microbial groups that colonize plant tissues, e.g., mycorrhizal fungi, pathogens, epiphytes, and saprotrophs. Some fungal endophytes affect plant growth and plant responses to pathogens, herbivores, and environmental change; others produce useful or interesting secondary metabolites. Workers have developed GFP cassettes for chromosomal integration and expression of gfp in a variety of bacteria (Tombolini and Jansson 1998; Tombolini et al. 1997; Xi et al. 1999). Bacterial cells with chromosomal integration of gfp can be identified by epifluorescence microscopy or confocal laser scanning microscopy (Germaine 2007; Villacieros et al. 2003). Germaine (2007) investigated a number of green fluorescent protein GFP labeled poplar endophytes for their colonization ability and also explored different methods of inoculation; a simple “stick dipping” method was found to be very efficient, leading to extensive colonization of the specific tissues of poplar plants (Fig. 23.1).

23.3

Interaction of Seed Endophytes

Endophytes are the microorganisms that reside within various tissues of the host plant in a commensal or beneficial manner. They can be considered as promising source of natural metabolites holding plethora of potential benefits in medical field (Kaul et al. 2012; Mousa and Raizada 2013; Premjanu and Jayanthy 2012; Strobel and Daisy 2003; Strobel et al. 2004). A large number of compounds with significant bioactivities have been isolated from endophytes (Kaul et al. 2012, 2014; Strobel et al. 2004). The scientific community has explored a number of medicinal plants until now, for their endophytic repository. In spite of a long list of reports on bioactive compounds from endophytes, commercial production of such compounds is still in its infancy (Kusari et al. 2014). Moreover, endophytes also have the ability to benefit the host plants with biotic and abiotic stress tolerance as well as improved nutrient acquisition and plant growth promotion (Johnson et al. 2003; Rodriguez et al. 2008). Such an ability can be exploited as a novel strategy to mitigate the repercussions of world climate change on agricultural crops and land. Therefore, in order to realize the potential of endophytes in pharmaceutical and agricultural industry, integrative understanding of all aspects of endophytism is essential.

23

Seed Endophytes in Crop Plants: Metagenomic Approaches to Study the. . .

487

Fig. 23.1 Schematic diagram of the different plant–bacterial endophyte interactions that have been studied and their applications. Source: Ryan et al. (2008)

Earlier, scientist community focused on unraveling the endophytic diversity and their metabolite potential, but nowadays there is more thrust on deep understanding of the host plant—endophytes niche. Suryanarayanan (2013) has highlighted some gray areas of endophytism which need to be addressed, viz., endophytes-host plant interactions, interactions among different endophytes of the same host plant, relation of endophytes with non-endophytic groups of the plant microbiome, life strategies of endophytes with respect to their saprotrophic and pathogenic counterparts, etc. Modern methodologies would be helpful to fill these gaps and thus enhance our knowledge about endophytism (Suryanarayanan 2013). Cultivable endophytic bacteria have been isolated from the seeds of coffee (Posada and Vega 2006), Norway spruce (Cankar et al. 2005), rice (Tripathi et al. 2006), and rapeseed (Granér et al. 2003). However, not much is known about their ecological function. Some of these bacteria were found to have antifungal activity (Mukhopadhyay et al. 1996), but pathogenic bacteria were also found to inhabit the seeds (Grum et al. 1998; Schaad et al. 1995). For example, the infection of carrot seed by Xanthomonas campestris pv. carotae (Kuan et al. 1985) involves the bacterium gaining access to an internal part of the seed, for example, the embryo, as was also reported for X. campestris pv. manihotis (Elango and Lozano 1980). Erwinia stewartii targets the endosperm (Rand and Cash 1921), while X. campestris pv. malvacearum entered the seed coat (Brinkerhoff and Hunter 1963). Barac et al. (2004) infected lettuce plants with X. campestris pv. vitians, which causes bacterial leaf spot; they

488

P. Verma

Table 23.1 Microbial endophytes colonization from different host seeds S. N. 1.

Host seed Phaseolus vulgaris seed

Microbial endophyte Rhizobium endophyticum

2.

Seeds of Norway spruce

3.

Seeds of herbaceous plants and the overwintered cereal seed the cereal, cucumber, persimmon, soybean, and squash

4.

Seeds of the parasol tree

5.

Surface sterilized seeds

6. 7.

Seed-borne pathogens from cereals Scots pine buds

8. 9.

Carrot seed Seeds of yellow lupine

10. 11.

Seeds of Nicotiana tabacum Rice seeds

Rahnella aquatilis Pseudomonas sp. Pseudomonas putida Bacillus brevis Bacillus megaterium Bacillus circulans Bacillus pumilis Bacillus subtilis Streptococcus faecium Streptomyces albolongus Streptomyces sp. Bacillus, Erwinia, Flavobacterium, Pseudomonas Cytophaga, Leuconostoc, Micrococcus Xanthomonas Pseudomonas syringae Xanthomonas campestris Methylobacterium pseudomonas Xanthomonas campestris Pseudomonas Rahnella Sanguibacter sp. Methylobacterium spp. Sphingomonas spp., Bacillus spp. Curtobacterium spp.

References López-López et al. (2010) Cankar et al. (2005)

Mundt and Hinkle (1976)

Mundt and Hinkle (1976) Bacon and Hinton (1997), Granér et al. (2003), Mundt and Hinkle (1976)

Grum et al. (1998), Schaad et al. (1995) Pirttilä et al. (2000) Kuan et al. (1985) Barac et al. (2004) Mastretta et al. (2009) Mano et al. (2006)

concluded that the pathogen had the capacity to enter and translocate within the vascular system of lettuce plants without inducing visible disease symptoms. Seeds produced from diseased lettuce plants were externally contaminated at a level of about the 2% incidence of X. campestris pv. vitians, but internally the seeds were not infected. In this case it seems that the pathogen was stopped at the seed surface, which could suggest a kind of communication between bacteria and plant host. Bacterial cell-to-cell or bacteria host communication was hypothesized by EspinosaUrgel et al. (2000) when they restored the seed adhesion capacity of Pseudomonas putida KT2440 by mutating the ddcA of this strain, which codes for a putative membrane polypeptide. Some microbial endophytes represent seed as a host in Table 23.1.

23

Seed Endophytes in Crop Plants: Metagenomic Approaches to Study the. . .

23.4

489

Identification of Cultivable Endophytes

With recent advances and developments in biotechnology, more studies at the molecular level are done with endophytes, which include metagenomic studies, the use of molecular markers, molecular cloning, and genetic expression studies. Denaturing gradient gel electrophoresis (DGGE) profiles of 16S rRNA gene fragments amplified from total plant DNA were applied to find nonculturable endophytes through comparison of the profile with the bands obtained from the culturable endophytes from citrus plants (Araújo et al. 2002). Bacterial automated ribosomal intergenic spacer analysis (B-ARISA) and pyrosequencing were used to characterize the endophyte community of a Solanum tuberosum cultivar (Manter et al. 2010). B-ARISA profiles revealed a significant difference in the endophytic community between cultivars, and canonical correspondence analysis showed a significant correlation between the community structure and plant biomass. Pyrosequencing was used to determine the bacterial operational taxonomic units (OTUs) richness. Metagenomics approach is another method used to find the microorganisms from different environments which cannot be cultured easily. This approach was used to find the 1-aminocyclopropane-1-carboxylate deaminase gene (acdS) operon from an uncultured endophytic microorganism colonizing Solanum tuberosum L. (Nikolic et al. 2011). Nikolic et al. (2011) concluded that metagenomic analysis can complement PCR-based analysis and yield information on whole gene operons. Little variation within the endophytic population diversity in Festuca eskia was found, regardless of provenance altitude and site and/or endophytes infection frequency using sequence-tagged sites (STS) and simple sequence repeats (SSR) markers (Glienke-Blanco et al. 2002). SSR marker was also used to study the genetic variation among two isolated endophytes Neotyphodium sibiricum and N. gansuense from the host plant Achnatherum sibiricum (Zhang et al. 2010). Significant linkage disequilibrium of SSR loci suggested that both fungal species primarily spread through clonal growth through plant seeds, and variation in diversity and presence of hybrids in both species showed that, while clonal reproduction was prevalent, occasional recombination may also be occurring. Through molecular cloning and genetic expression analysis of geranylgeranyl diphosphate (GGPP) synthase, it was found that ltmG, ltmM, and ltmK are members of a group of genes employed for production of lolitrem (a potent tremorgen) biosynthesis in fungi Epichloe festucae and Neotyphodium lolii (Young et al. 2005). Genome research in endophytes has progressed to the extent that complete genome of Enterobacter sp. 638, an endophytic plant growth promoting gammaproteobacterium, that was isolated from the stem of poplar, a potentially important biofuel feed stock plant, was sequenced (Taghavi et al. 2010). Sequencing showed the possession of a 4,518,712 bp chromosome and a 157,749 bp plasmid (pENT6381). Different sets of genes specific to the plant niche adaptation of this bacterium were identified by genome annotation and comparative genomics. This includes genes that code for putative proteins involved in survival in the rhizosphere (to cope with oxidative stress or uptake of nutrients released by plant roots), root adhesion,

490

P. Verma

colonization/establishment inside the plant, plant protection against fungal and bacterial infections, and improved plant growth and development.

23.5

Functional Characteristics

System biology science embraces four key technologies viz. genomics, transcriptomics, proteomics, and metabolomics. All approaches (including metagenomics, metatranscriptomics, etc.) are in a state of continual improvement. While the individual type data are useful, they are even more valuable when used in combination. Genomics accesses a wealth of hidden actions and interactions in a microenvironment in the form of molecular machinery but much still remains unknown. On the other hand, transcriptome studies reveal gene expression without considering protein level regulation or protein turnover, etc. Proteomics reveals functional gene products that may better reflect plant endophyte interactions. However, proteomic studies require collation of protein data with predicted protein data (derived from genomic studies) (Hettich et al. 2013). Transcriptomic and proteomic studies are ineffective without genome studies. Complementation of metagenomics data with metatranscriptomic and metaproteomic data would generate a more complete view of the activities of endophytes in plants. All techniques are interdependent and the data generated from one complement the other; combined data generated from different modern “omics” tools could prove essential to solving the riddle of endophytism.

23.6

Genome Sequence Analysis

The basic physiological aspect of the endophyte host interaction is poorly understood. Therefore, identification, isolation, and characterization of genes involved in such beneficial interactions are critically important for the effective manipulation of the mutualistic association between the two. Endophyte genome analysis has provided a new tool to closely view the endophytism and to reveal the requisite features to harbor plants as a habitat. It has revealed the genes, important for the endophytic life style, as found common in the endophyte genomes such as genes for nitrogen fixation, phytohormone production (IAA, GA, etc.), mineral acquisition (Fe, P, etc.), stress tolerance, adhesion, and other colonization-related genes (Firrincieli et al. 2015; Fouts et al. 2008; Martínez-García et al. 2015). These traits explain the role of microbes in nutrient cycling as well as their ability to colonize plant endosphere. Whole genome analysis of endophytic microbes has revealed the genetic features that directly or indirectly influence the various bioactivities as well as colonizing preferences. It aids in the identification of particular genes involved in mechanism of antibiotic resistance, antibiotic production, plant growth promotion, endophytic secretory system, surface attachment and insertion elements, transport system, and other related metabolic mechanisms. Genome sequences improve the data analysis in metaomics (metagenomics, transcriptomics, and proteomics) studies of plant-

23

Seed Endophytes in Crop Plants: Metagenomic Approaches to Study the. . .

491

Fig. 23.2 Genome sequence analysis to improve the data analysis in metaomics

associated microbes. Host plant genome evolution is also affected by endophytic colonization (Guo et al. 2015), whereas Zgadzaj et al. (2015) reported that host genetic factors control establishment of both endophyte and the symbiont within root nodules; therefore host genome studies are also important for a clearer view (Fig. 23.2).

23.6.1 Metagenome Metagenomics uses sequences from microbes in particular niches, without the need for isolation of individual species. To gain an understanding of the contributions of endophytes to host plants, it is necessary to show how endophytes help hosts metabolically. The failure of many endophytes to grow readily in culture makes it difficult to do experiments to evaluate benefits to hosts (Dinsdale et al. 2008). A metagenomics approach is useful to examine effects on plants without culturing microbes (Dinsdale et al. 2008) and reveals information about the entire microbial community. DNA is extracted from the entire population and analyzed for its content of specific gene markers, depending on the groups of microbes being examined. Sessitsch et al. (2012) showed putative functional features of root symbionts of rice using metagenome analysis. They reported many metabolic adaptations of microbes to the root microhabitat, indicating a high potential of the endophytic community in

492

P. Verma

plant growth promotion, enhancement of host stress resistance, protection from pathogens, and bioremediation potential. Functional diversity has been maintained among microbial communities inhabiting different environments. Comparative metagenomics approach can be successfully used to study functional diversity among endophytes of same or different host plants. Dinsdale et al. (2008) used the comparative metagenomics approach to describe the variations in functional potential of nine different microbiomes. High-throughput sequencing called next-generation sequencing (NGS) has made metagenomic studies comparatively easier and catalyzed the rapid, unprecedented characterization studies of microbiomes (Akinsanya et al. 2015). It has equipped researchers with a wide ranging tool for quick and affordable study of DNA sequences from an environmental sample (Jones 2010). Four hundred and fifty-four sequencing has provided a convenient means for the characterization of fungal communities (Jumpponen et al. 2010). Toju et al. (2013) described the community composition of root-associated fungi in a temperate forest in Japan. They demonstrated the coexistence of mycorrhizal fungi and endophytic fungi in roots of different plant species using 454 pyrosequencing techniques. Coexistence involves interactions between the two microbes that may be examined using metaproteomics, metatranscriptomics, or metaproteogenomics approach. There are limitations to NGS technologies; high ratio of sequences with no homolog in public databases is one of the major limitations of metagenomic studies (Hert et al. 2008; Jones 2010).

23.6.2 Proteome Genomic analyses of microbial communities are becoming frequently used, but through genomic analyses it is frequently difficult to show the function of microbial communities. Proteomics is defined as the large-scale study of different proteins expressed by an organism, whereas metaproteomics involves identification of the functional expression of the metagenome and elucidation of the metabolic activities occurring within a community at the moment of sampling. It is also known as whole community proteomics. Maron et al. (2007) have stressed on the relevance of metaproteome analysis in identification of new functional, stress-related genes and in relating genomic diversity with the functionality of the microbes inhabiting complex environments. Mass spectrometry (MS) has become the dominant tool for almost all proteomic measurements. Metaproteomics exploits the power of high performance MS for extensive characterization of the complete suite of proteins expressed by a microbial community in an environmental sample. Total proteins can be extracted from microenvironment either by direct or indirect lysis (Maron et al. 2007). Through a cell lysis strategy, total protein content may be extracted from the plant under non-stress and stress conditions, and the protein fingerprint may be analyzed to assess functions. Conversely, using indirect lysis method, total protein content can be extracted from pre-isolated endophytes under different stress conditions, and comparison of

23

Seed Endophytes in Crop Plants: Metagenomic Approaches to Study the. . .

493

the protein fingerprints obtained after 2D-gel electrophoresis analysis can be used to reveal the role of endophytes under different stress conditions (Bhuyan et al. 2015). Otherwise, total protein content of host plants in presence and absence of endophytes can also be assessed to identify actual specific proteins involved in interactions between the two. Lery et al. (2011) found 78 differentially expressed proteins between sugarcane infected with Gluconacetobacter and control cultures of the bacterium using mass spectrometry. Proteome studies are not complete without genomic information. Protein extraction and sample preparation are often hampered due to the presence of interfering substances, including polyphenols, polysaccharides, lipids, acids, and other metabolites.

23.6.3 Transcriptome Transcriptomics has been found as a feasible approach to study the microbial communities associated with different plants (Molina et al. 2012; Sheibani-Tezerji et al. 2015). It involves the comparative analysis of transcriptomes of groups of interacting species and helps to understand the response of microbial communities toward changing environments. While genome- and metagenome-based studies enumerate the presence or absence of specific genes, expression studies of specific genes in different microenvironments are essential to understand the endophytic phenomenon. Deep analysis of the differentially expressed genes in the host plant as well as symbiotic microbes would provide insight into the basic nature and mechanism of mutualistic relationships between the two. Dual RNA-seq transcriptional profiling gives better idea of gene expression in both the partners of symbiosis at a time. Camilios-Neto et al. (2014) have used dual RNA-seq technology for transcriptional profiling of wheat roots colonized by Azospirillum brasilense and observed upregulation of nutrient acquisition and cell cycle genes. RNA-seq allows detection of more differentially expressed genes than microarray alone. Despite of more advantages of RNA-seq, microarray is still more commonly used tool for transcriptional profiling because of the high cost and relatively difficult data storage and analysis in RNA sequence technology. Metatranscriptomic analysis of soybean plant has revealed the presence of a number of small RNA sequences unrelated to soybean genome. Interestingly comparative analysis of the obtained sequences established the presence of various pathogenic, symbiotic, and free-living microbes in different samples of soybean plant (Molina et al. 2012). Comparative transcriptome analysis of endophyte-free and endophyte-infected plants directs us toward understanding the basis of endophyte-mediated disease resistance and plant growth promotion properties. Comparative studies regarding differential expression profiles of endophytes within and outside host plant can be helpful to identify interaction factors involved in maintaining the relationship. Conversely, differential expression of different host plant genes in presence and absence of endophytes can also be studied. SSH, microarray analysis, and SOLiD-

494

P. Verma

SAGE-like techniques can be successfully used for differential expression analysis (Ambrose and Belanger 2012; Dinkins et al. 2010; Johnson et al. 2003). SOLiDSAGE transcriptome analysis of endophyte-free and Epichloe festucae-infected Festuca rubra has revealed about 200 plant-associated genes that expressed differentially between the two plant samples (Ambrose and Belanger 2012).

23.6.4 Microarray Microarray technique has equipped the modern genome-based studies with the tools for genome-specific gene expression studies, endophyte gene profiling, exploration of host plant–symbiont interactions, and many others for transcriptome analysis (Felitti et al. 2006). Barnett et al. (2004) used the dual genome Symbiosis Chip-based tool to study symbiotic. Symbiosis Chip-based studies allow simultaneous analysis of gene expression in both partners of the association and can easily be used to study the endophyte host interactions. Barnett et al. (2004) studied the coordinate differentiation and response generated from signal exchange between the two symbiotic partners simultaneously, viz., α-proteobacterium Sinorhizobium meliloti and its legume partner Medicago truncatula during nodule development. They designed a custom Affymetrix Gene Chip with the complete S. meliloti genome and 10,000 probe sets for M. truncatula. Genomic interspecies microarray hybridization technique has proved to be useful in the characterization of previously untouched genomes, provided that the genome of a close relative has already been fully sequenced (Dong et al. 2001). Microarray technique allows the identification of a number of genes in an uncharacterized genome without the need for genome sequencing. This technique finds more applications with sequencing of endophytic genomes (Table 23.1). Genes have been discovered efficiently in maize endophyte K. pneumoniae 342 by hybridizing the DNA from KP342 to a microarray containing 96% of the annotated ORFs from Escherichia coli K12 (Dong et al. 2001). Microarray studies can be used to study the transcriptional changes induced by entry of endophytes in plants. These studies provide a new insight into the biology of endophyte host interaction and represent a step forward toward identification of host genes required for successful endophyte infestation. Felitti et al. (2006) described the potential of Epichloe and Neotyphodium endophyte cDNA microarrays (NchipTM and EndochipTM microarrays) for genome-wide transcriptome analysis. Microarray analysis of transcriptome of endophytic Pseudomonas-infected Arabidopsis revealed the upregulation of phytohormone production and nodule formation genes, whereas ethylene-responsive genes were found to be downregulated (Wang et al. 2005). Reference selection is a critical step in microarray studies as non-specific references may generate ambiguous results. However, nonavailability or limited access to the specific gene expression/profiling databases has restricted such studies.

23

Seed Endophytes in Crop Plants: Metagenomic Approaches to Study the. . .

495

23.6.5 Metaproteogenomics Metaproteogenomics links the proteome and the genome of the environmental samples and allows identification of more proteins (functions) than proteomics alone. It involves combinatorial study of metagenome and metaproteome of same sample. Knief et al. (2012) have used metaproteogenomic approach to study microbial communities in the phyllosphere and rhizosphere of rice. The results showed that despite the presence of nifH genes in both microenvironments, expression was found in rhizosphere only. If such an approach could be applied to study the endosphere, more significant data regarding the endophyte functionality can be collected. Characterization of the metaproteogenome is expected to provide data linking genetic and functional diversity of microbial communities. Proteins involved in plant endophyte interactions that could not be studied in cultivated isolates are new targets for functional studies. Plant-associated bacterial protein secretion system can be successfully used for determining plant bacterial interactions (Downie 2010). Delmotte et al. (2009) have successfully used community proteogenomics to identify the unique traits of phyllosphere bacteria. Bacterial proteogenomic pipeline and other tools are available for proteogenomic analysis studies (Uszkoreit et al. 2014). The technique offers insights into possible strategies adopted for endophytic lifestyle. The combined metagenome and metaproteome analysis would allow one to overcome the limitations of protein identifications as in metaproteomic approach due to nonavailability of closely related reference genomes.

23.7

Potential Applications of Endophytes

23.7.1 Phytostimulation Plants require 16 essential elements like C, H, N, O, and P and 11 more. These essential elements are available to plants for their growth and development in chemical form, which they obtain from atmosphere, soil, water, and organic matter. Endophytes also play an important role in the uptake of these nutrients. They elicit different modes of action in tall fescue adaptation to P deficiency (Malinowski et al. 2000) and induce increased uptake of N (Arachevaleta et al. 1989). Endophytic bacteria produce a wide range of phytohormones, such as auxins, cytokinins, and gibberellic acids. Burkholderia vietnamiensis, a diazotrophic endophytic bacterium isolated from wild cottonwood (Populus trichocarpa), produced indole acetic acid (IAA), which promotes the growth of the plant (Xin et al. 2009). This was confirmed by comparison between uninoculated control and plants inoculated with B. vietnamiensis on nitrogenfree media, in which inoculated plants gained more dry weight and more nitrogen content. A new strain of fungus Cladosporium sphaerospermum isolated from the roots of Glycine max (L) Merr. showed the presence of higher amounts of bioactive GA3, GA4, and GA7, which induced maximum plant growth in both rice and soybean varieties (Hamayun et al. 2009).

496

P. Verma

23.7.2 Pigment Production An orange pigment identified as quercetin glycoside was isolated from an endophytic fungus belonging to Penicillium sp. (Nair and Padmavathy 2014). This was the first report on quercetin glycoside produced by endophytic fungus. Endophytic fungus strain named SX01, later identified as Penicillium purpurogenum, from the twigs of Ginkgo biloba L., was able to produce abundant soluble red pigments which could be used as natural food colorant (Qiu et al. 2010). A pigment isolated from the endophytic fungus Monodictys castaneae was found to inhibit few human pathogenic bacteria Staphylococcus aureus, Klebsiella pneumoniae, Salmonella typhi, and Vibrio cholerae and was proved to be more active than streptomycin (Visalakchi and Muthumary 2009).

23.7.3 Enzyme Production Many commercially important enzymes are produced by several soil microorganisms. The hunt for other potential sources had led to the discovery of a few vital enzymes being produced by endophytes. Endophytic fungi like Acremonium terricola, Aspergillus japonicus, Cladosporium cladosporioides, Cladosporium sphaerospermum, Fusarium lateritium, Monodictys castaneae, Nigrospora sphaerica, Penicillium aurantiogriseum, Penicillium glandicola, Pestalotiopsis guepinii, Phoma tropica, Phomopsis archeri, Tetraploa aristata, and Xylaria sp. and many other unidentified species in Opuntia ficus-indica Mill. have indicated their promising potential for deployment in biotechnological processes involving production of pectinases, cellulases, xylanases, and proteases (Bezerra et al. 2012). An endophyte, Acremonium zeae, isolated from maize produced the enzyme hemicellulase extracellularly (Bischoff et al. 2009). This hydrolytic enzyme from A. zeae may be suitable for application in the bioconversion of lignocellulosic biomass into fermentable sugars.

23.7.4 Antimicrobial Activity Most of the endophytes isolated from plants are known for their antimicrobial activity. They help in controlling microbial pathogens in plants and/or animals. Endophytes isolated from medicinal plants showed bioactivity for broad spectrum of pathogenic microorganisms (Devaraju and Satish 2011; Selim et al. 2011; Sette et al. 2006). A total of 37 endophytes were isolated all together from Tectona grandis L. and Samanea saman Merr. of which 18 could produce inhibitory substances effective against Bacillus subtilis, Staphylococcus aureus, and Escherichia coli and 3 isolates inhibited growth of Candida albicans in vitro (Chareprasert et al. 2006). Kumar et al. (2011) assayed the bioactivity of the endophytic microorganisms like Dothideomycetes sp., Alternaria tenuissima, Thielavia subthermophila, Alternaria sp., Nigrospora oryzae, Colletotrichum truncatum, and Chaetomium sp. isolated

23

Seed Endophytes in Crop Plants: Metagenomic Approaches to Study the. . .

497

from the medicinal plant, Tylophora indica, against Sclerotinia sclerotiorum and Fusarium oxysporum which were found to inhibit their growth.

23.7.5 Source of Bioactive and Novel Compounds Endophytes are capable of synthesizing bioactive compounds that are used by plants for defense against pathogens, and some of these compounds have proven to be useful for novel drug discovery. Recent studies have reported hundreds of natural products including alkaloids, terpenoids, flavonoids, and steroids, from endophytes. Most of the bioactive compounds isolated from endophytes are known to have functions of antibiotics, immune suppressants, anticancer agents, biological control agents, and so forth (Joseph and Priya 2011). Maytansinoids, like rifamycin and geldanamycin, which structurally belong to the ansamycin family of polyketide macrolactams are products of three closely related plant families (Celastraceae, Rhamnaceae, and Euphorbiaceae), mosses, and certain bacteria such as Actinosynnema pretiosum. It was hypothesized that microbes in the rhizosphere might be involved in the biosynthesis of plant maytansinoids (Pullen et al. 2003). Several endophytic actinomycetes were isolated from Trewia nudiflora, of which Streptomyces sp. 5B and Streptomyces sp. M27 m3 were proved to have the potential of producing ansamycins (Arachevaleta et al. 1989). One novel chlorine-containing ansamycin, namely, naphthomycin K, which was isolated from the endophytic strain Streptomyces sp. CS of the maytansinoid producer medicinal plant Maytenus hookeri, showed evident cytotoxic activity against P388 and A-549 cell lines but no inhibitory activities against Staphylococcus aureus and Mycobacterium tuberculosis (Lu and Shen 2007). Siderophores are biologically active compound with function of chelating iron ions in living organisms. They have found extensive applications in the field of agriculture and medicine. They are also a component of virulence of microorganisms infecting man, animals, and plants (Neilands 1995). Five different strains of Phialocephala fortinii, a dark septate fungal, were studied, and all of them excreted three siderophores, namely, ferricrocin, ferrirubin, and ferrichrome C, whose production was dependent on pH and iron (III) concentration of the culture medium (Bartholdy et al. 2001). P. fortinii can thus be used for large-scale production of these siderophores. Taxol is a drug used to cure breast cancer, ovarian cancer, and lung cancer. An endophytic microorganism Metarhizium anisopliae, isolated from Taxus chinensis, was found to produce Taxol in abundance in vitro (Liu et al. 2009). Colletotrichum gloeosporioides isolated from the leaves of a medicinal plant, Justicia gendarussa, also produces Taxol (Gangadevi and Muthumary 2008).

23.7.6 Reciprocal Interactions Between Above- and Belowground Communities The microbial community responses in soils conditioned by plants of the annual grass Lolium multiflorum with contrasting levels of infection with the endophyte

498

P. Verma

Neotyphodium occultans were explored (Casas et al. 2011). Soil conditioning by highly infected plants affected soil catabolic profiles and tended to increase soil fungal activity. A shift in bacterial community structures was detected, while no changes were observed for fungi. Soil responses became evident even without changes in host plant biomass or soil organic carbon or total nitrogen content, suggesting that the endophyte modified host rhizo depositions during the conditioning phase. A few researchers have reported changes in the rhizosphere chemistry and enzymatics activity mediated by endophyte presence in perennial host grasses (Casas et al. 2011; Van Hecke et al. 2005).

23.7.7 Biocontrol Agents Endophytic microorganisms are regarded as an effective biocontrol agent, alternative to chemical control. Endophytic fungi have been described to play an important role in controlling insect herbivory not only in grasses but also in conifers (Posada and Vega 2006). An endophytic fungi Beauveria bassiana known as an entomopathogen was found to control the borer insects in coffee seedlings (Posada and Vega 2006) and sorghum (Tefera and Vidal 2009).

23.7.8 Nutrient Cycling Nutrient cycling is a very important process that happens continuously to balance the existing nutrients and make it available for every component of the ecosystem. Biodegradation of the dead biomasses becomes one major step in it to bring the utilized nutrients back to the ecosystem which in turn again becomes available to the organisms. This becomes a cyclic chain process. A lot of saprophytic organisms play a major role in it. Few studies have shown that endophytes have important role in biodegradation of the litter of its host plants (Fukasawa et al. 2009; Korkama-Rajala et al. 2008; Kumaresan 2002; Müller et al. 2001; Osono 2003, 2006; Osono and Hirose 2009; Promputtha et al. 2010). During biodegradation of the litter, the endophytic microbes colonize initially within the plants (Thormann et al. 2003) and facilitate the saprophytic microbes to act on through antagonistic interaction and thus increasing the litter decomposition (Fryar et al. 2001; Terekhova and Semenova 2005). In another study, it was demonstrated that all endophytes had the ability to decompose organic components, including lignin, cellulose, and hemicellulose; however, the preferences of various groups of endophytes with respect to organic compounds differed (He et al. 2012).

23.7.9 Bioremediation/Biodegradation Endophytes have a powerful ability to breakdown complex compounds. Bioremediation is a method of removal of pollutants and wastes from the environment by the

23

Seed Endophytes in Crop Plants: Metagenomic Approaches to Study the. . .

499

use of microorganisms. It relies on the biological processes in microbes to breakdown these wastes. This is made possible due to the great microbial diversity. A group of researchers studied the role of endophytes in bioremediation in Nicotiana tabacum plants (Mastretta et al. 2009). Inoculation of Nicotiana tabacum with endophytes resulted in improved biomass production under conditions of Cadmium (Cd) stress, and the total plant Cd concentration was higher compared to noninoculated plants. These results demonstrated the beneficial effects of seed endophytes on metal toxicity and accumulation. To explore the endophytic diversity for the breakdown of plastic, several dozen endophytic fungi were screened for their ability to degrade the synthetic polymer polyester polyurethane (PUR) (Russell et al. 2011). Though several organisms demonstrated the ability to efficiently degrade PUR in both solid and liquid suspensions, robust activity was observed among several isolates in the genus Pestalotiopsis. Two Pestalotiopsis microspora isolates were uniquely able to grow on PUR as the sole carbon source under both aerobic and anaerobic conditions. Molecular characterization of this activity suggested that an enzyme serine hydrolase is responsible for degradation of PUR (Russell et al. 2011).

23.7.10 Production of Volatile Organic Compounds and Their Benefits Hypoxylon sp. which is an endophytic fungus isolated from Persea indica produced an impressive spectrum of volatile organic compounds (VOCs), most notably 1, 8- cineole, 1-methyl-1,4-cyclohexadiene and tentatively identified alpha-methylene-alphafenchocamphorone, among many others, most of which are unidentified. It displayed maximal VOC antimicrobial activity against Botrytis cinerea, Phytophthora cinnamomi, Cercospora beticola, and Sclerotinia sclerotiorum suggesting that the VOCs may play some role in the biology of the fungus and its survival in its host plant (Tomsheck et al. 2010). They unequivocally demonstrated that 1, 8-cineole (a monoterpene) is produced in addition by this Hypoxylon sp., which represents a novel and important source of this compound. This monoterpene is an octane derivative and has potential use as a fuel additive as do the other VOCs of this organism. This study thus shows that fungal sourcing of this compound and other VOCs as produced by Hypoxylon sp. greatly expands their potential applications in medicine, industry, and energy production. An unusual Phomopsis sp. was isolated as endophyte of Odontoglossum sp. (Orchidaceae), produced a unique mixture of volatile organic compounds (VOCs) including sabinene (a monoterpene with a peppery odor), 1-butanol, 3-methyl; benzene ethanol; 1-propanol, 2-methyl, and 2- propanone (Singh et al. 2011). The gases of Phomopsis sp. possess antifungal properties, and an artificial mixture of the VOCs mimicked the antibiotic effects of this organism with the greatest bioactivity against a wide range of plant pathogenic test fungi including Pythium, Phytophthora, Sclerotinia, Rhizoctonia, Fusarium, Botrytis, Verticillium, and Colletotrichum. As with many VOC-producing endophytes, this Phomopsis sp. did survive and grow in the presence of the inhibitory gases of Muscodor albus, an endophytic fungus. The authors in Singh et al. (2011) had hypothesized

500

P. Verma

that there was a possible involvement of VOC production by the fungus and its role in the biology/ecology of the fungus–plant–environmental relationship.

23.8

Conclusions

Exploitation of endophyte–plant interactions can result in the promotion of plant health and can play a significant role in low-input sustainable agriculture applications for both food and nonfood crops. With the availability of complete genome sequences of key endophytic bacteria, the gene governing colonization and establishment of endophytic bacteria in planta can be identified. This information will form the foundation for transcriptome and proteome analysis currently optimized in studying other plant–microbe interactions. Incorporating this information with well-established techniques such as IVET and recently advanced “omic” technologies offers the ability to search for genes on a global scale that are found to be induced or repressed during colonization of plant tissues. An understanding of the mechanisms enabling these endophytic bacteria to interact with plants will be essential to fully achieve the biotechnological potential of efficient plant–bacterial partnerships for a range of applications. Deep understanding of endophyte host interactions is the need of the hour in order to realize the use of endophytes as plant growth promoters. By using a multidisciplinary approach, factors inevitable for both the establishment as well as maintenance of symbiotic association between the two can be better understood. The complementary information generated from modern “omics” studies in association with other system biology techniques are inevitable to build up models to predict and explain endophyte-mediated processes. This will also prove to be quite useful in revealing and better understanding of the network of the complex interactions of endophytes with the host plant and also other associated microbes. Plant–pathogen interaction studies can be used as a base model to understand plant–endophyte relationship. This can make us understand the role of such diverse microbial communities in the plant microbiome as well as in natural ecosystem, so that their biotechnological potential can be harnessed more efficiently and sustainably. Acknowledgment The authors are grateful to the Department of Microbiology, Akal College of Basic Science, Eternal University, Himachal Pradesh, for providing the facilities and financial support.

References Akinsanya MA, Goh JK, Lim SP, Ting ASY (2015) Metagenomics study of endophytic bacteria in Aloe vera using next-generation technology. Genomics Data 6:159–163 Ambrose KV, Belanger FC (2012) SOLiD-SAGE of endophyte-infected red fescue reveals numerous effects on host transcriptome and an abundance of highly expressed fungal secreted proteins. PLoS One 7:e53214

23

Seed Endophytes in Crop Plants: Metagenomic Approaches to Study the. . .

501

Arachevaleta M, Bacon C, Hoveland C, Radcliffe D (1989) Effect of the tall fescue endophyte on plant response to environmental stress. Agron J 81:83–90 Araújo WL, Marcon J, Maccheroni W, van Elsas JD, van Vuurde JW, Azevedo JL (2002) Diversity of endophytic bacterial populations and their interaction with Xylella fastidiosa in citrus plants. Appl Environ Microbiol 68:4906–4914 Bacon C, Hinton D (1997) Isolation and culture of endophytic bacteria and fungi. ASM Press, Washington, DC Barac T et al (2004) Engineered endophytic bacteria improve phytoremediation of water-soluble, volatile, organic pollutants. Nat Biotechnol 22:583 Barnett MJ, Toman CJ, Fisher RF, Long SR (2004) A dual-genome symbiosis chip for coordinate study of signal exchange and development in a prokaryote–host interaction. Proc Natl Acad Sci 101:16636–16641 Bartholdy B, Berreck M, Haselwandter K (2001) Hydroxamate siderophore synthesis by Phialocephala fortinii, a typical dark septate fungal root endophyte. Biometals 14:33–42 Bashan Y, Harrison SK, Whitmoyer RE (1990) Enhanced growth of wheat and soybean plants inoculated with Azospirillum brasilense is not necessarily due to general enhancement of mineral uptake. Appl Environ Microbiol 56:769–775 Bezerra J, Santos M, Svedese V, Lima D, Fernandes M, Paiva L, Souza-Motta C (2012) Richness of endophytic fungi isolated from Opuntia ficus-indica mill. (Cactaceae) and preliminary screening for enzyme production. World J Microbiol Biotechnol 28:1989–1995 Bhuyan S, Bandyopadhyay P, Yadava P (2015) Extraction of proteins for two-dimensional gel electrophoresis and proteomic analysis from an endophytic fungus. Protoc Exch. doi https://doi. org/10.1038/protex.2015.084 Bischoff KM, Wicklow DT, Jordan DB, de Rezende ST, Liu S, Hughes SR, Rich JO (2009) Extracellular hemicellulolytic enzymes from the maize endophyte Acremonium zeae. Curr Microbiol 58:499–503 Brinkerhoff L, Hunter R (1963) Internally infected seed as a source of inoculum for the primary cycle of bacterial blight of cotton. Phytopathology 53:1397–1401 Brundrett MC (2006) Understanding the roles of multifunctional mycorrhizal and endophytic fungi. In: Microbial root endophytes. Springer, Heidelberg, pp 281–298 Camilios-Neto D et al (2014) Dual RNA-seq transcriptional analysis of wheat roots colonized by Azospirillum brasilense reveals up-regulation of nutrient acquisition and cell cycle genes. BMC Genomics 15:378 Cankar K, Kraigher H, Ravnikar M, Rupnik M (2005) Bacterial endophytes from seeds of Norway spruce (Picea abies L. Karst). FEMS Microbiol Lett 244:341–345 Carroll G (1988) Fungal endophytes in stems and leaves: from latent pathogen to mutualistic symbiont. Ecology 69:2–9 Carroll GC (1991) Fungal associates of woody plants as insect antagonists in leaves and stems. In: Barbosa P, Krischik VA, Jones CG (eds) Microbial mediation of plant-herbivore interactions. John Wiley and Son, New York, pp 253–271 Casas C, Omacini M, Montecchia MS, Correa OS (2011) Soil microbial community responses to the fungal endophyte Neotyphodium in Italian ryegrass. Plant Soil 340:347–355 Chareprasert S, Piapukiew J, Thienhirun S, Whalley AJ, Sihanonth P (2006) Endophytic fungi of teak leaves Tectona grandis L. and rain tree leaves Samanea saman Merr. World J Microbiol Biotechnol 22:481–486 Chen C, Bauske E, Musson G, Rodriguez-Kabana R, Kloepper J (1995) Biological control of Fusarium wilt on cotton by use of endophytic bacteria. Biol Control 5:83–91 Chen Y, Mei R, Lu S, Liu L, Kloepper J (1996) The use of yield increasing bacteria (YIB) as plant growth-promoting rhizobacteria in Chinese agriculture. In: Utkhede RS, Gupta VK (eds) Management of soil born diseases. Kalyani Publishers, Ludhiana, pp 165–184 Clay K, Holah J (1999) Fungal endophyte symbiosis and plant diversity in successional fields. Science 285:1742–1744

502

P. Verma

Das A (2009) Symbiosis: the art of living. In: Varma A, Kharkwal AC (eds) Symbiotic fungi principles and practice. Springer, Berlin De Bary A (1879) Die erscheinung der symbiose. Verlag von Karl J Trübner, Strasbourg Delmotte N et al (2009) Community proteogenomics reveals insights into the physiology of phyllosphere bacteria. Proc Natl Acad Sci 106:16428–16433 Devaraju R, Satish S (2011) Endophytic mycoflora of Mirabilis jalapa L. and studies on antimicrobial activity of its endophytic Fusarium sp. Soc Appl Sci 2:75–79 Dinkins RD, Barnes A, Waters W (2010) Microarray analysis of endophyte-infected and endophytefree tall fescue. J Plant Physiol 167:1197–1203 Dinsdale EA et al (2008) Functional metagenomic profiling of nine biomes. Nature 452:629 Döbereiner J (1992) Recent changes in concepts of plant bacteria interactions: endophytic N2 fixing bacteria. Ciência Cult 44:310–313 Dong Y, Glasner JD, Blattner FR, Triplett EW (2001) Genomic interspecies microarray hybridization: rapid discovery of three thousand genes in the maize endophyte, Klebsiella pneumoniae 342, by microarray hybridization with Escherichia coli K-12 open reading frames. Appl Environ Microbiol 67:1911–1921 Downie JA (2010) The roles of extracellular proteins, polysaccharides and signals in the interactions of rhizobia with legume roots. FEMS Microbiol Rev 34:150–170 Dudeja S, Giri R, Saini R, Suneja-Madan P, Kothe E (2012) Interaction of endophytic microbes with legumes. J Basic Microbiol 52:248–260 Elango F, Lozano J (1980) Transmission of Xanthomonas manihotis in seed of cassava (Manihot esculenta). Plant Dis 64:784–786 Espinosa-Urgel M, Salido A, Ramos J-L (2000) Genetic analysis of functions involved in adhesion of Pseudomonas putida to seeds. J Bacteriol 182:2363–2369 Felitti S et al (2006) Transcriptome analysis of Neotyphodium and Epichloë grass endophytes. Fungal Genet Biol 43:465–475 Firrincieli A et al (2015) Genome sequence of the plant growth promoting endophytic yeast Rhodotorula graminis WP1. Front Microbiol 6:978 Fisher P, Petrini O, Scott HL (1992) The distribution of some fungal and bacterial endophytes in maize (Zea mays L.). New Phytol 122:299–305 Fouts DE et al (2008) Complete genome sequence of the N2-fixing broad host range endophyte Klebsiella pneumoniae 342 and virulence predictions verified in mice. PLoS Genet 4:e1000141 Fox R, Manners J, Myers A (1972) Ultrastructure of tissue disintegration and host reactions in potato tubers infected by Erwinia carotovora var. atroseptica. Potato Res 15:130–145 Fryar S, Yuen T, Hyde K, Hodgkiss I (2001) The influence of competition between tropical fungi on wood colonization in streams. Microb Ecol 41:245–251 Fukasawa Y, Osono T, Takeda H (2009) Effects of attack of saprobic fungi on twig litter decomposition by endophytic fungi. Ecol Res 24:1067 Gagné S, Richard C, Rousseau H, Antoun H (1987) Xylem-residing bacteria in alfalfa roots. Can J Microbiol 33:996–1000 Gangadevi V, Muthumary J (2008) Isolation of Colletotrichum gloeosporioides, a novel endophytic taxol-producing fungus from the leaves of a medicinal plant, Justicia gendarussa. Mycol Balc 5:1–4 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 Gehring CA, Cobb NS, Whitham TG (1997) Three-way interactions among ectomycorrhizal mutualists, scale insects, and resistant and susceptible pinyon pines. Am Nat 149:824–841 Germaine K (2007) Construction of endophytic xenobiotic degrader bacteria for improving the phytoremediation of organic pollutants. PhD thesis, Institute of Technology Carlow, Carlow Glienke-Blanco C, Aguilar-Vildoso CI, Vieira MLC, Barroso PAV, Azevedo JL (2002) Genetic variability in the endophytic fungus Guignardia citricarpa isolated from citrus plants. Genet Mol Biol 25:251–255 Goodman R (1982) The infection process. Phytopathogenic prokaryotes 1:31–62

23

Seed Endophytes in Crop Plants: Metagenomic Approaches to Study the. . .

503

Granér G, Persson P, Meijer J, Alström S (2003) A study on microbial diversity in different cultivars of Brassica napus in relation to its wilt pathogen, Verticillium longisporum. FEMS Microbiol Lett 224:269–276 Grum M, Camloh M, Rudolph K, Ravnikar M (1998) Elimination of bean seed-borne bacteria by thermotherapy and meristem culture. Plant Cell Tissue Organ Cult 52:79–82 Guo L et al (2015) A host plant genome (Zizania latifolia) after a century-long endophyte infection. Plant J 83:600–609 Hallmann J, Kloepper J, Rodriguez-Kabana R, Sikora R (1995) Endophytic rhizobacteria as antagonists of Meloidogyne incognita on cucumber. Phytopathology 85:136 Hamayun M et al (2009) Cladosporium sphaerospermum as a new plant growth-promoting endophyte from the roots of Glycine max (L.) Merr. World J Microbiol Biotechnol 25:627–632 He X et al (2012) Diversity and decomposition potential of endophytes in leaves of a Cinnamomum camphora plantation in China. Ecol Res 27:273–284 Hert DG, Fredlake CP, Barron AE (2008) Advantages and limitations of next-generation sequencing technologies: a comparison of electrophoresis and non-electrophoresis methods. Electrophoresis 29:4618–4626 Hettich RL, Pan C, Chourey K, Giannone RJ (2013) Metaproteomics: harnessing the power of high performance mass spectrometry to identify the suite of proteins that control metabolic activities in microbial communities. ACS Publications, Washington, DC Hollis JP (1951) Bacteria in healthy potato tissue. Phytopathology 41:350–366 Huang J-S (1986) Ultrastructure of bacterial penetration in plants. Annu Rev Phytopathol 24:141–157 Jacobs MJ, Bugbee WM, Gabrielson DA (1985) Enumeration, location, and characterization of endophytic bacteria within sugar beet roots. Can J Bot 63:1262–1265 Johnson NC, Graham JH, Smith F (1997) Functioning of mycorrhizal associations along the mutualism–parasitism continuum. New Phytol 135:575–585 Johnson LJ, Johnson RD, Schardl CL, Panaccione DG (2003) Identification of differentially expressed genes in the mutualistic association of tall fescue with Neotyphodium coenophialum. Physiol Mol Plant Pathol 63:305–317 Jones WJ (2010) High-throughput sequencing and metagenomics. Estuar Coasts 33:944–952 Joseph B, Priya RM (2011) Bioactive compounds from Endophytes and their potential in American. J Biochem Mol Biol 1:291–309 Jumpponen A, Jones KL, Mattox JD, Yaege C (2010) Massively parallel 454-sequencing of fungal communities in Quercus spp. ectomycorrhizas indicates seasonal dynamics in urban and rural sites. Mol Ecol 19:41–53 Kaul S, Gupta S, Ahmed M, Dhar MK (2012) Endophytic fungi from medicinal plants: a treasure hunt for bioactive metabolites. Phytochem Rev 11:487–505 Knief C et al (2012) Metaproteogenomic analysis of microbial communities in the phyllosphere and rhizosphere of rice. ISME J 6:1378 Korkama-Rajala T, Müller MM, Pennanen T (2008) Decomposition and fungi of needle litter from slow-and fast-growing Norway spruce (Picea abies) clones. Microb Ecol 56:76 Krings M, Taylor TN, Hass H, Kerp H, Dotzler N, Hermsen EJ (2007) Fungal endophytes in a 400-million-yr-old land plant: infection pathways, spatial distribution, and host responses. New Phytol 174:648–657 Kuan T-L, Minsavage G, Gabrielson R (1985) Detection of Xanthomonas campestris pv. Carotae in carrot seed. Plant Dis 69:758–760 Kumar S, Kaushik N, Edrada-Ebel R, Ebel R, Proksch P (2011) Isolation, characterization, and bioactivity of endophytic fungi of Tylophora indica. World J Microbiol Biotechnol 27:571–577 Kumaresan V (2002) Endophytes assemblages in young mature and senescent leaves of Rhizophora apiculata: evidence for the role of endophytes in mangrove litter degradation. Fungal Divers 9:81–91 Kusari S, Singh S, Jayabaskaran C (2014) Biotechnological potential of plant-associated endophytic fungi: hope versus hype. Trends Biotechnol 32:297–303

504

P. Verma

Lalande R, Bissonnette N, Coutlée D, Antoun H (1989) Identification of rhizobacteria from maize and determination of their plant-growth promoting potential. Plant Soil 115:7–11 Lee S, Flores-Encarnacion M, Contreras-Zentella M, Garcia-Flores L, Escamilla J, Kennedy C (2004) Indole-3-acetic acid biosynthesis is deficient in Gluconacetobacter diazotrophicus strains with mutations in cytochrome c biogenesis genes. J Bacteriol 186:5384–5391 Lery LM, Hemerly AS, Nogueira EM, von Krüger WM, Bisch PM (2011) Quantitative proteomic analysis of the interaction between the endophytic plant-growth-promoting bacterium Gluconacetobacter diazotrophicus and sugarcane. Mol Plant-Microbe Interact 24:562–576 Lewis D (1985) Symbiosis and mutualism: crisp concepts and soggy semantics. In: Boucher DH (ed) The biology of mutualism. Oxford University Press, Oxford Liu K, Ding X, Deng B, Chen W (2009) Isolation and characterization of endophytic taxolproducing fungi from Taxus chinensis. J Ind Microbiol Biotechnol 36:1171 López-López A, Rogel MA, Ormeno-Orrillo E, Martínez-Romero J, Martínez-Romero E (2010) Phaseolus vulgaris seed-borne endophytic community with novel bacterial species such as Rhizobium endophyticum sp. nov. Syst Appl Microbiol 33:322–327 Lu C, Shen Y (2007) A novel ansamycin, naphthomycin K from Streptomyces sp. J Antibiot 60:649 Mahaffee W et al (1997) Spatial and temporal colonization of Phaseolus vulgaris by the bacterial endophytes Pseudomonas fluorescens strain 89B-27 and Enterobacter asburiae strain JM22. Appl Environ Microbiol. https://doi.org/10.1128/AEM.02846-17 Malinowski DP, Alloush GA, Belesky DP (2000) Leaf endophyte Neotyphodium coenophialum modifies mineral uptake in tall fescue. Plant Soil 227:115–126 Mano H, Tanaka F, Watanabe A, Kaga H, Okunishi S, Morisaki H (2006) Culturable surface and endophytic bacterial flora of the maturing seeds of rice plants (Oryza sativa) cultivated in a paddy field. Microbes Environ 21:86–100 Manter DK, Delgado JA, Holm DG, Stong RA (2010) Pyrosequencing reveals a highly diverse and cultivar-specific bacterial endophyte community in potato roots. Microb Ecol 60:157–166 Maron P-A, Ranjard L, Mougel C, Lemanceau P (2007) Metaproteomics: a new approach for studying functional microbial ecology. Microb Ecol 53:486–493 Martínez-García PM, Ruano-Rosa D, Schilirò E, Prieto P, Ramos C, Rodríguez-Palenzuela P, Mercado-Blanco J (2015) Complete genome sequence of Pseudomonas fluorescens strain PICF7, an indigenous root endophyte from olive (Olea europaea L.) and effective biocontrol agent against Verticillium dahliae. Stand Genomic Sci 10:10 Mastretta C et al (2009) Endophytic bacteria from seeds of Nicotiana tabacum can reduce cadmium phytotoxicity. Int J Phytoremediation 11:251–267 McInroy JA, Kloepper JW (1995) Survey of indigenous bacterial endophytes from cotton and sweet corn. Plant Soil 173:337–342 Molina LG, Cordenonsi da Fonseca G, GLd M, de Oliveira LFV, JBd C, Kulcheski FR, Margis R (2012) Metatranscriptomic analysis of small RNAs present in soybean deep sequencing libraries. Genet Mol Biol 35:292–303 Mousa WK, Raizada MN (2013) The diversity of anti-microbial secondary metabolites produced by fungal endophytes: an interdisciplinary perspective. Front Microbiol 4:65 Mukhopadhyay K, Garrison NK, Hinton DM, Bacon CW, Khush GS, Peck HD, Datta N (1996) Identification and characterization of bacterial endophytes of rice. Mycopathologia 134:151–159 Müller MM, Valjakka R, Suokko A, Hantula J (2001) Diversity of endophytic fungi of single Norway spruce needles and their role as pioneer decomposers. Mol Ecol 10:1801–1810 Mundt JO, Hinkle NF (1976) Bacteria within ovules and seeds. Appl Environ Microbiol 32:694–698 Musson G, McInroy J, Kloepper J (1995) Development of delivery systems for introducing endophytic bacteria into cotton. Biocontrol Sci Tech 5:407–416 Nair DN, Padmavathy S (2014) Impact of endophytic microorganisms on plants, environment and humans. Sci World J 2014:250693 Neilands J (1995) Siderophores: structure and function of microbial iron transport compounds. J Biol Chem 270:26723–26726

23

Seed Endophytes in Crop Plants: Metagenomic Approaches to Study the. . .

505

Nikolic B, Schwab H, Sessitsch A (2011) Metagenomic analysis of the 1-aminocyclopropane-1carboxylate deaminase gene (acdS) operon of an uncultured bacterial endophyte colonizing Solanum tuberosum L. Arch Microbiol 193:665–676 Old KM, Nicolson TH (1978) The root cortex as part of a microbial continuum. In: Loutit MW, Miles JAR (eds) Microbial ecology. SpringerVerlag, Berlin, pp 291–294 Omacini M, Chaneton EJ, Ghersa CM, Müller CB (2001) Symbiotic fungal endophytes control insect host–parasite interaction webs. Nature 409:78 Osono T (2003) Effects of prior decomposition of beech leaf litter by phyllosphere fungi on substrate utilization by fungal decomposers. Mycoscience 44:0041–0045 Osono T (2006) Role of phyllosphere fungi of forest trees in the development of decomposer fungal communities and decomposition processes of leaf litter. Can J Microbiol 52:701–716 Osono T, Hirose D (2009) Effects of prior decomposition of Camellia japonica leaf litter by an endophytic fungus on the subsequent decomposition by fungal colonizers. Mycoscience 50:52–55 Parker MP (1995) Plant fitness variation caused by different mutualist genotypes. Ecology 76:1525–1535 Parker MA (1999) Mutualism in metapopulations of legumes and rhizobia. Am Nat 153:S48–S60 Patriquin D, Döbereiner J (1978) Light microscopy observations of tetrazolium-reducing bacteria in the endorhizosphere of maize and other grasses in Brazil. Can J Microbiol 24:734–742 Patriquin D, Döbereiner J, Jain D (1983) Sites and processes of association between diazotrophs and grasses. Can J Microbiol 29:900–915 Petrini O (1986) Taxonomy of endophytic fungi of aerial plant tissues. In: Fokkema NJ, Van Den Huevel J (eds) Microbiology of the phyllosphere. Cambridge University Press, Cambridge, pp 175–187 Pirttilä AM, Laukkanen H, Pospiech H, Myllylä R, Hohtola A (2000) Detection of intracellular bacteria in the buds of scotch pine (Pinus sylvestris L.) by in situ hybridization. Appl Environ Microbiol 66:3073–3077 Pleban S, Ingel F, Chet I (1995) Control of Rhizoctonia solani and Sclerotium rolfsii in the greenhouse using endophytic Bacillus spp. Eur J Plant Pathol 101:665–672 Posada F, Vega FE (2006) Inoculation and colonization of coffee seedlings (Coffea arabica L.) with the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales). Mycoscience 47:284–289 Premjanu N, Jayanthy C (2012) Endophytic fungi a repository of bioactive compounds-a review. Intl J Inst Phar Life Sci 2:135–162 Promputtha I, Hyde KD, McKenzie EH, Peberdy JF, Lumyong S (2010) Can leaf degrading enzymes provide evidence that endophytic fungi becoming saprobes? Fungal Divers 41:89–99 Pullen CB et al (2003) Occurrence and non-detectability of maytansinoids in individual plants of the genera Maytenus and Putterlickia. Phytochemistry 62:377–387 Qiu X, Pei Y, Wang Y, Zhang F (1990) Isolation of pseudomonads from cotton plants and their effect on seedling diseases. Acta Phytophylacica Sinica 17:303–306 Qiu M, Xie R, Shi Y, Chen H, Wen Y, Gao Y, Hu X (2010) Isolation and identification of endophytic fungus SX01, a red pigment producer from Ginkgo biloba L. World J Microbiol Biotechnol 26:993–998 Quadt-Hallmann A, Kloepper J (1996) Immunological detection and localization of the cotton endophyte Enterobacter asburiae JM22 in different plant species. Can J Microbiol 42:1144–1154 Quadt-Hallmann A, Hallmann J, Kloepper J (1997) Bacterial endophytes in cotton: location and interaction with other plant-associated bacteria. Can J Microbiol 43:254–259 Rand FV, Cash LC (1921) Stewart’s disease of corn. J Agric Res 21:263–264 Redecker D, Kodner R, Graham LE (2000) Glomalean fungi from the Ordovician. Science 289:1920–1921 Rodriguez RJ et al (2008) Stress tolerance in plants via habitat-adapted symbiosis. ISME J 2:404 Roos IM, Hattingh M (1983) Scanning electron microscopy of Pseudomonas syringae pv, morsprunorum on sweet cherry leaves. J Phytopathol 108:18–25

506

P. Verma

Russell JR et al (2011) Biodegradation of polyester polyurethane by endophytic fungi. Appl Environ Microbiol 77:6076–6084 Ryan RP, Germaine K, Franks A, Ryan DJ, Dowling DN (2008) Bacterial endophytes: recent developments and applications. FEMS Microbiol Lett 278:1–9 Schaad NW, Cheong S, Tamaki S, Hatziloukas E, Panopoulos NJ (1995) A combined biological and enzymatic amplification (BIO-PCR) technique to detect Pseudomonas syringae pv. Phaseolicola in bean seed extracts. Phytopathology 85:243–246 Schaad NW, Jones JB, Chun W (2001) Laboratory guide for the identification of plant pathogenic bacteria, vol 3. APS Press, Urbana Selim K, El-Beih A, AbdEl-Rahman T, El-Diwany A (2011) Biodiversity and antimicrobial activity of endophytes associated with Egyptian medicinal plants. Mycosphere 2:669–678 Sessitsch A et al (2012) Functional characteristics of an endophyte community colonizing rice roots as revealed by metagenomic analysis. Mol Plant-Microbe Interact 25:28–36 Sette L, Passarini M, Delarmelina C, Salati F, Duarte M (2006) Molecular characterization and antimicrobial activity of endophytic fungi from coffee plants. World J Microbiol Biotechnol 22:1185–1195 Sharrock K, Parkes S, Jack H, Rees-George J, Hawthorne B (1991) Involvement of bacterial endophytes in storage rots of buttercup squash (Cucurbita maxima D. hybrid ‘Delica’). N Z J Crop Hortic Sci 19:157–165 Sheibani-Tezerji R, Rattei T, Sessitsch A, Trognitz F, Mitter B (2015) Transcriptome profiling of the endophyte Burkholderia phytofirmans PsJN indicates sensing of the plant environment and drought stress. MBio 6:e00621–e00615 Singh SK, Strobel GA, Knighton B, Geary B, Sears J, Ezra D (2011) An endophytic Phomopsis sp. possessing bioactivity and fuel potential with its volatile organic compounds. Microb Ecol 61:729–739 Strobel G, Daisy B (2003) Bioprospecting for microbial endophytes and their natural products. Microbiol Mol Biol Rev 67:491–502 Strobel G, Daisy B, Castillo U, Harper J (2004) Natural products from endophytic microorganisms. J Nat Prod 67:257–268 Suryanarayanan TS (2013) Endophyte research: going beyond isolation and metabolite documentation. Fungal Ecol 6:561–568 Taghavi S et al (2010) Genome sequence of the plant growth promoting endophytic bacterium Enterobacter sp. 638. PLoS Genet 6:e1000943 Tefera T, Vidal S (2009) Effect of inoculation method and plant growth medium on endophytic colonization of sorghum by the entomopathogenic fungus Beauveria bassiana. BioControl 54:663–669 Terekhova V, Semenova T (2005) The structure of micromycete communities and their synecologic interactions with basidiomycetes during plant debris decomposition. Microbiology 74:91–96 Thormann MN, Currah RS, Bayley SE (2003) Succession of microfungal assemblages in decomposing peatland plants. Plant Soil 250:323–333 Toju H, Yamamoto S, Sato H, Tanabe AS, Gilbert GS, Kadowaki K (2013) Community composition of root-associated fungi in a Q uercus-dominated temperate forest:“codominance” of mycorrhizal and root-endophytic fungi. Ecol Evol 3:1281–1293 Tombolini R, Jansson JK (1998) Monitoring of GFP-tagged bacterial cells. In: Bioluminescence methods and protocols. Springer, New York, pp 285–298 Tombolini R, Unge A, Davey ME, de Bruijn FJ, Jansson JK (1997) Flow cytometric and microscopic analysis of GFP-tagged Pseudomonas fluorescens bacteria. FEMS Microbiol Ecol 22:17–28 Tomsheck AR et al (2010) Hypoxylon sp., an endophyte of Persea indica, producing 1, 8-cineole and other bioactive volatiles with fuel potential. Microb Ecol 60:903–914 Tripathi AK, Verma SC, Chowdhury SP, Lebuhn M, Gattinger A, Schloter M (2006) Ochrobactrum oryzae sp. nov., an endophytic bacterial species isolated from deep-water rice in India. Int J Syst Evol Microbiol 56:1677–1680

23

Seed Endophytes in Crop Plants: Metagenomic Approaches to Study the. . .

507

Uszkoreit J, Plohnke N, Rexroth S, Marcus K, Eisenacher M (2014) The bacterial proteogenomic pipeline. BMC Genomics 15:S19 Van Hecke MM, Treonis AM, Kaufman JR (2005) How does the fungal endophyte Neotyphodium coenophialum affect tall fescue (Festuca arundinacea) rhizodeposition and soil microorganisms? Plant Soil 275:101–109 Villacieros M et al (2003) Colonization behaviour of Pseudomonas fluorescens and Sinorhizobium meliloti in the alfalfa (Medicago sativa) rhizosphere. Plant Soil 251:47–54 Visalakchi S, Muthumary J (2009) Antimicrobial activity of the new endophytic Monodictys castaneae SVJM139 pigment and its optimization. Afr J Microbiol Res 3:550–556 Wang Y, Ohara Y, Nakayashiki H, Tosa Y, Mayama S (2005) Microarray analysis of the gene expression profile induced by the endophytic plant growth-promoting rhizobacteria, Pseudomonas fluorescens FPT9601-T5 in Arabidopsis. Mol Plant-Microbe Interact 18:385–396 Wei G, Kloepper JW, Tuzun S (1991) Induction of systemic resistance of cucumber to Colletotrichum orbiculare by select strains of plant growth-promoting rhizobacteria. Phytopathology 81:1508–1512 Wei G, Kloepper J, Tuzun S (1996) Induced systemic resistance to cucumber diseases and increased plant growth by plant growth-promoting rhizobacteria under field conditions. Phytopathology 86:121 Wilson D (1995) Endophyte: the evolution of a term, and clarification of its use and definition. Oikos 73:274–276 Xi C, Lambrecht M, Vanderleyden J, Michiels J (1999) Bi-functional gfp-and gusA-containing mini-Tn5 transposon derivatives for combined gene expression and bacterial localization studies. J Microbiol Methods 35:85–92 Xin G, Zhang G, Kang JW, Staley JT, Doty SL (2009) A diazotrophic, indole-3-acetic acidproducing endophyte from wild. Biol Fertil Soils 45:669–674 Young C, Bryant M, Christensen M, Tapper B, Bryan G, Scott B (2005) Molecular cloning and genetic analysis of a symbiosis-expressed gene cluster for lolitrem biosynthesis from a mutualistic endophyte of perennial. Mol Gen Genomics 274:13–29 Zgadzaj R et al (2015) A legume genetic framework controls infection of nodules by symbiotic and endophytic bacteria. PLoS Genet 11:e1005280 Zhang X, Ren A, Ci H, Gao Y (2010) Genetic diversity and structure of Neotyphodium species and their host Achnatherum sibiricum in a natural grass–endophyte system. Microb Ecol 59:744–756