Natural Products and Molecular Genetics Underlying the Antifungal

Natural products and molecular genetics underlying the antifungal

activity of endophytic microbes

Walaa Kamel Moatey Mohamed Mousa

A Thesis

Presented to

The University of Guelph

In partial fulfilment of requirements

for the degree of

Doctor of Philosophy

Plant Agriculture

Guelph, Ontario, Canada

©Walaa K.M.M. Mousa, 2016

ABSTRACT

Natural products and molecular genetics underlying the antifungal activity of endophytic microbes

Walaa K. Mousa Advisory Committee:

University of Guelph Dr. Manish N. Raizada (Advisor) Dr. Ting Zhou (Co-advisor) Dr. Adrian Schwan Dr. Katarina Jordan

Microbes are robust and promiscuous machines for the biosynthesis of antimicrobial compounds which combat serious crop and human pathogens. A special subset of microbes that inhabit internal plant tissues without causing disease are referred to as endophytes. Endophytes can protect their hosts against pathogens. I hypothesized that plants which grow without synthetic pesticides, including the wild and ancient relatives of modern crops, and the marginalized crops grown by subsistence farmers, host endophytes that have co-evolved to combat host-specific pathogens. To test this hypothesis, I explored endophytes within the ancient Afro-Indian crop finger millet, and diverse maize/teosinte genotypes from the Americas, for anti-fungal activity against Fusarium graminearum. F. graminearum leads to devastating diseases in cereals including maize and wheat and is associated with accumulation of mycotoxins including deoxynivalenol (DON).

I have identified fungal and bacterial endophytes, their secreted natural products and/or genes with anti-Fusarium activity from both maize and finger millet. I have shown that some of these endophytes can efficiently suppress F. graminearum in planta and dramatically reduce DON during seed storage when introduced into modern maize and wheat. The most exciting discovery of my research is that an endophytic bacterium (strain M6, Enterobacter sp.), isolated from the roots of finger millet, builds a remarkable physical barrier consisting of bacterial micro-colonies that protect the host against

ii pathogen invasion. M6 creates an unusual root hair-endophyte stacking (RHESt) formation that prevents entry and/or traps the pathogen which is then killed. Tn5 mutant analysis demonstrated that the endophyte kills the fungal pathogen by using a c-di-GMP- dependent signaling network and diverse fungicides including phenazine. The endophyte has evolved an epistatic regulatory interaction to suppress an antibiotic released by Fusarium which would otherwise inhibit phenazine release into the RHESt. The end- result of this remarkable physico-chemical barrier is a reduction in levels of the mycotoxin DON, thus potentially protecting millions of subsistence farmers and their livestock. To the best of my knowledge, RHESt represents a novel plant defence mechanism and suggests the value of exploring the microbiomes of the world's ancient, orphan crops as source of endophytes with antimicrobial activity.

Keywords: Natural products, Antifungal, Genes, Endophytes, RHESt, Fusarium graminearum, Teosinte, Maize, Finger millet, Phoma sp., Fusarium sp. , Penicillium sp., Enterobacter sp., Citrobacter sp. and Paenibacillus polymyxa.

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ACKNOWLEDGMENTS

First and foremost, I would like to express my sincere thanks and gratitude to Dr. Manish Raizada for his unbelievable support, insightful discussions and sincere interest. I appreciate all his contributions of ideas, time and effort to make my Ph.D. experience very successful. I extend my thanks to all my committee members: Dr. Ting Zhou (co- advisor), Dr. Adrian Schwan and Dr. Katerina S. Jordan for their help and support and for letting me work in their laboratories.

I am very thankful to Dr. Michaela Strueder-Kypke, Molecular and Cellular Imaging Facility, University of Guelph, for her assistance in confocal microscopy imaging and insightful comments. I am thankful to Charles Shearer for his hard work to perform the greenhouse trials. I would like to thank Marina Attalla for helping in disease scoring. I thank Dr. Hanan Shehata for training on lab protocols and for her insightful comments. I am thankful to Gui-Ying Mei for assistance with the DON detoxification experiment. I would like to extend my sincere gratitude to all my colleagues in the Raizada lab for their advice and support. I am also grateful to Dr. Victor Limay-Rios for carrying out DON quantification assays and to Dr. Art Schaafsma and Dr. Ljiljana Tamburic-Ilincic for kindly providing maize and wheat seeds.

I would like to express my thanks, appreciation and love for my professors and colleagues at the Pharmacognosy Department, School of Pharmacy, Mansoura University, for their great advice and support.

I am thankful to my family for their love and support and for providing me with the highest possible education. Special thanks to my husband Moustafa Ibrahim and my daughters Hanen and Jana for their continuous motivation and inspiration.

I am grateful to the Egyptian government for providing me with the generous funding that made this work possible.

TABLE OF CONTENTS Abstract Acknowledgments iv List of Tables viii List of Figures x List of Abbreviations xv Chapter 1: Thesis outline and general introduction 1.1 Thesis Outline 1 1.2 General introduction 3 1.3 Hypothesis 16 1.4 Objectives 16

Chapter 2: Natural disease control in cereal grains (literature review) 2.1 Abstract 18 2.2 Introduction 19 2.3 Overview of disease management strategies 20 2.4 Introduction to soil and plant associated microbes 21 2.5 Overview of biocontrol mechanisms 21 2.5.1 Competition 22 2.5.2 Antibiosis 23 2.5.3 Induced host resistance 27 2.5.4 Disease suppressive soil 29 2.5.5 Hyperparasitism 30 2.5.6 Transgenic plants 31 2.6 Microbes that combine multiple mechanisms for biocontrol 32 2.7 Examples of commercial biocontrol products 32 2.8 Discussion 33

Chapter 3: The diversity of anti-microbial secondary metabolites produced by fungal endophytes: An interdisciplinary perspective (review article) 3.1 Abstract 36 3.2 Introduction 37 3.3 Diverse classes of fungal antimicrobial secondary metabolites 38 3.3.1 Terpenoid compounds 38 3.3.2 Alkaloids 50 3.3.3 Phenolic compounds 55 3.3.4 Aliphatic compounds 59

3.3.5 Polyketides 61 3.3.6 Peptides 68 3.4 Conclusion and future directions 70

Chapter 4: Biodiversity of genes encoding anti-microbial traits within plant associated microbes (review article) 4.1 Abstract 74 4.2 Introduction 75 4.3 Biosynthetic genes encode diverse classes of anti-microbial 76 compounds 4.3.1 Polyketides 81 4.3.2 Non-ribosomal peptides 87 4.3.3 Terpenoids 95 4.3.4 Alkaloids 101 4.3.5 Heterocyclic nitrogenous compounds 105 4.3.6 Volatile compounds 107 4.3.7 Bacteriocins 108 4.3.8 Enzymes 110 4.4 Discussion 113 4.5 Gaps and Future Perspectives 122

Chapter 5: An endophytic fungus isolated from finger millet (Eleusine coracana) produces anti-fungal natural products 5.1 Abstract 127 5.2 Introduction 128 5.3 Material and Methods 130 5.4 Results 136 5.5 Discussion 159 5.6 Study Limitations and Future Experiments 164

Chapter 6: Characterization of antifungal natural products isolated from endophytic fungi of finger millet (Eleusine coracana) 6.1 Abstract 168 6.2 Introduction 169 6.3 Material and Methods 170 6.4 Results 176 6.5 Discussion 188

Chapter 7: Root hair-endophyte stacking (RHESt) in an ancient Afro- Indian crop creates an unusual physico-chemical barrier to trap pathogen(s) 7.1 Abstract 193 7.2 Introduction 194 7.3 Material and Methods 195 7.4 Results 202 7.5 Discussion 228

Chapter 8: Bacterial endophytes from wild maize suppress Fusarium graminearum in modern maize and inhibit mycotoxin accumulation 235 8.1 Abstract 236 8.2 Introduction 237 8.3 Material and Methods 247 8.4 Results 276 8.5 Discussion

Chapter 9: An endophytic bacterium isolated from wild maize suppresses Fusarium head blight and DON mycotoxin in modern wheat 9.1 Abstract 283 9.2 Introduction 284 9.3 Material and Methods 286 9.4 Results 291 9.5 Discussion 302

Chapter 10: General Discussion 10.1 Summary of the major finding in the thesis 307 10.2 Significance and impact of the study 309 10.3 Conclusion 311

Chapter 11: References 312

Chapter 12: Appendices 369

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LIST OF TABLES Table 4.1:Table Summary of anti-microbial compounds belonging to genetic 77 pathways that show evidenceof horizontal gene transfer (HGT).

Table 4.2: Summary of anti-microbial compounds belonging to genetic 79 pathways, grouped by different evolutionary levels of diversification.

Table 5.1: Effect of endophyte extracts on the growth of a diversity of 140 fungal species.

Table 8.1: Effect of the candidate bacterial endophytes on the growth of 251 diverse crop fungal pathogens in vitro.

Table 8.2: Suppression of Gibberella Ear Rot by the candidate endophytes 272 in two replicate greenhouse trials.

Table 8.3: Reduction of DON mycotoxin accumulation during storage 274 following treatment with the candidate endophytes.

Table 9.1: Suppression of Fusarium Head Blight by anti-Fusarium 295 endophytes in two replicate greenhouse trials.

Table 9.2: Reduction of DON mycotoxin accumulation during storage 300 following treatment with anti-Fusarium endophytes.

Table A5.1 18S rDNA sequences and BLAST analysis of fungal 371 endophytes isolated from finger millet in this study.

Table A5.2 Taxonomic identification of the endophytic fungi isolated from 373 finger millet.

Table A5.3 List of retention times and expected masses of compounds 374 from extract of WF4 fungus fermented on rice.

Table A5.4 List of retention times and expected masses of compounds 375 from extract of WF4 fungus fermented on millet.

Table A5.5 NMR data for atomic assignments of compounds isolated from 377 WF4.

Table A6.1 NMR data for atomic assignments of compounds isolated from 383 finger millet fungi.

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Table A7.1 Gene-specific primers used in quantitative real-time PCR 390 analysis.

Table A7.2 Taxonomic classification of finger millet bacterial endophytes 391 based on 16S rDNA sequences and BLAST analysis.

Table A7.3 Effect of endophyte strain M6 isolated from finger millet on the 392 growth of diverse fungal pathogens in vitro.

Table A7.4 Suppression of F. graminearum disease symptoms in maize 393 and wheat by endophyte M6 in replicated greenhouse trials.

Table A7.5 Reduction of DON mycotoxin accumulation during prolonged 394 seed storage following treatment with endophyte M6.

Table A7.6 Complete list of strain M6 Tn5 insertion mutants showing loss 395 of antifungal activity against F. graminearum in vitro.

Table A7.7 M6 wild type nucleotide coding sequences corresponding to 396 Tn5 insertion mutants that showed loss of antifungal activity against F. graminearum in vitro.

Table A8.1 Taxonomic classification of maize bacterial endophytes based 410 on 16S rDNA and 23S rDNA sequences and BLAST analysis.

Table A8.2 Sequences of fusA orthologs from Paenibacillus endophytic 413 species identified in this study.

Table A8.3 Origin of the endophytes screened in this study including the 414 maize genotypes and tissues from which they were isolated, and the best BLAST matches to the GenBank database.

LIST OF FIGURES Figure 1.1: The fungal pathogen, Fusarium graminearum and its 8 mycotoxins.

Figure 1.2: Finger millet fields. 11

Figure 1.3: Finger millet farmers. 12

Figure 1.4: Illustration of Zea mays ssp. Parviglumis. 14

Figure 1.5: Illustration of Zea diploperennis. 15

Figure 3.1: Structures of sesquiterpene derivatives of fungal endophyte 42 origin.

Figure 3.2: Structures of diterpene and triterpene derivatives of fungal 47 endophyte origin.

Figure 3.3: Structures of additional terpenoid derivatives of fungal 49 endophyte origin.

Figure 3.4: Structures of alkaloidal derivatives of fungal endophyte origin. 54

Figure 3.5: Structures of phenylpropanoid derivatives of fungal endophyte 58 origin.

Figure 3.6: Structures of aliphatic derivatives of fungal endophyte origin. 60

Figure 3.7: Structures of polyketide derivatives of fungal endophyte 66 origin.

Figure 3.8: Structures of peptides derivatives of fungal endophyte origin. 69

Figure 4.1: Structures of polyketide compounds featured in this review. 86

Figure 4.2: Structures of non-ribosomal peptide compounds featured in 93 this review.

Figure 4.3: Diagram illustrating how a diversity of fusaricidins are 94 produced from a single allele of fusA which encodes the non-

ribosomal peptide synthase (NRPS). Figure 4.4: Structures of terpenoid compounds featured in this review. 96

Figure 4.5: Comparative analysis of the trichothecene biosynthetic gene 98 clusters.

Figure 4.6: Structures of featured alkaloids, heterocyclic nitrogenous 102 compounds and bacteriocin.

Figure 4.7: An example of intra- coding sequences diversification within 112 an anti-microbial gene cluster.

Figure 4.8: Potential examples of horizontal gene transfer of anti- 114 microbial gene clusters leading to species level evolutionary diversification.

Figure 4.9: The anti-microbial compounds reviewed in this study grouped 118 by the phylogeny of their microbial hosts.

Figure 5.1: Isolation of fungal endophytes from finger millet and screen 138 for anti-Fusarium activity from endophyte extracts.

Figure 5.2: Testing the pathogenicity of the putative endophyte WF4 on 142 finger millet.

Figure 5.3: Root colonization study using confocal scanning laser 144 microscopy.

Figure 5.4: HPLC chromatograms of extracts derived from endophyte 146 WF4.

Figure 5.5: Mass spectroscopy of peaks with antifungal activity detected 148 in extracts from endophyte WF4.

Figure 5.6: Structures of the purified anti-Fusarium compounds from 153 endophyte WF4.

Figure 5.7: The effects of the purified compounds on F. graminearum in 155 vitro using the agar disc diffusion assay and neutral red staining.

Figure 5.8: The effects of the purified compounds on F. graminearum in 157 vitro using Evans blue staining.

Figure 6.1: Characterization of the putative fungal endophytes previously 171 isolated from finger millet and the antifungal activity of their extracts.

Figure 6.2: Plant pathogenicity assay of each putative endophyte 177 following inoculation onto finger millet.

Figure 6.3: Test for the ability of the putative fungal endophytes to 179 colonize finger millet roots using confocal scanning laser microscopy.

Figure 6.4: Structures of the purified anti-Fusarium compounds from 182 fungal endophyte strains WF5, WF6 and WF7.

Figure 6.5: The effects of the purified endophyte-derived anti-fungal 186 compounds on F. graminearum in vitro using neutral red staining.

Figure 7.1 Isolation, Identification and Antifungal Activity of Endophytes 204

Figure 7.2 Confocal imaging of M6-Fusarium interactions in finger millet 207 roots.

Figure 7.3 Behavior and interactions of endophyte M6 and F. 210 graminearum in vitro on microscope slides.

Figure 7.4 Characterization of phenazine mutant ewpR-5D7::Tn5. 214

Figure 7.5 Characterization of fusaric acid resistance mutant ewfR- 218 7D5::Tn5.

Figure 7.6 Characterization of di-guanylate cyclase mutant ewgS- 222 10A8::Tn5.

Figure 7.7 Characterization of colicin V mutant ewvC-4B9::Tn5. 226

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Figure 8.1: Origin of endophytes used in this study and results of the in 248 vitro anti-Fusarium screen.

Figure 8.2: Taxonomic characterization of candidate anti-Fusarium 253 endophytes.

Figure 8.3: Electron microscope images of the anti-fungal endophyte 257 strains.

Figure 8.4: Microscopic in vitro interactions between each anti-fungal 259 endophyte and F. graminearum.

Figure 8.5: The effects of the candidate endophytes on F. graminearum in 261 vitro using the vitality stain, Evans blue.

Figure 8.6: Molecular and biochemical detection of the candidate anti- 264 fungal compound, fusaricidin, in Paenibacillus strains.

Figure 8.7: Greenhouse trial 1 to test for the ability of the candidate 267 endophytes to suppress Gibberella Ear Rot (GER) in a modern hybrid.

Figure 8.8: Greenhouse trial 2 to test for the ability of the candidate 269 endophytes to suppress Gibberella Ear Rot in a modern hybrid.

Figure 8.9: Test for the ability of the candidate endophytes to reduce DON 275 mycotoxin accumulation in maize grain during storage.

Figure 9.1: Origin of endophytes used in this study, activity against F. 292 gramineraum in maize and GFP-tagging of the endophytes to test wheat colonization.

Figure 9.2: Greenhouse trial 1 to test for the ability of the candidate 296 endophytes to suppress Fusarium Head Blight in spring wheat.

Figure 9.3: Greenhouse trial 2 to test for the ability of the candidate 298 endophytes to suppress Fusarium Head Blight in spring wheat.

Figure 9.4: Test for the ability of the candidate endophytes to reduce DON 301

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mycotoxin accumulation in wheat grain during storage.

Figure A5.1 Flow chart illustrating the bio-guided purification of the active 379 anti-Fusarium compounds from the extract of endophyte WF4 grown on rice culture.

Figure A5.2 Flow chart illustrating the bio-guided purification of the active 380 anti-Fusarium compounds from the extract of endophyte WF4 grown on millet culture.

Figure A5.3 Preliminary toxicity assays of the extract from endophyte 381 WF4.

Figure A7.1 Image of Finger Millet Seedlings Previously Seed-coated with 402 GFP-tagged M6 Showing No Pathogenic Symptoms, Consistent with the Strain Behaving as an Endophyte

Figure A7.2 Suppression of F. graminearum (Fg) by M6 and its effect on 403 grain yield in greenhouse trials.

Figure A7.3 Assay for production of indole-3-acetic acid (IAA, auxin) by 404 wild type strain M6.

Figure A7.4 Tn5 mutagenesis-mediated discovery, validation and 405 complementation of genes required for the anti-Fusarium activity of strain M6.

Figure A7.5 Model to illustrate the interaction between strain M6, the host 407 plant and F. graminearum pathogen.

Figure A7.6 Assays for production of butanediol and chitinase by strain 409 M6.

Figure A8.1 Test for the ability of the candidate endophytes to detoxify 422 DON in vitro.

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LIST OF ABBREVIATIONS

C-di-GMP: Bis-(3’,5’) cyclic diguanylic acid F. graminearum: Fusarium graminearum FHB: Fusarium Head Blight GER: Gibberella Ear Rot HPLC: High performance liquid chromatography IAA: Indole-3-acetic acid LB: Luria-Bertani medium LC-MS: Liquid chromatography-mass spectroscopy MS medium: Murashige and Skoog medium NMR: Nuclear magnetic resonance PCR: Polymerase chain reaction PDA: Potato dextrose agar QRT-PCR: Quantitative real time polymerase chain reaction Rpm: Revolutions per minute SCLM: Scanning confocal laser microscopy SEM: Scanning electron microscope TEM: Transmission electron microscope Tn: Transposon

Chapter 1: Thesis outline and general Introduction

1.1 Thesis outline The thesis includes 9 chapters, in addition to the General discussion, References and Appendices.

Chapter 1: General introduction

Chapters 2-4: Comprehensive literature review for the research-related background. In Chapter 2 (Mousa and Raizada, 2016), I discuss various strategies and mechanisms of natural disease control in cereal grains with a focus on biological control. In Chapter 3 (Mousa and Raizada, 2013), I review diverse chemical classes of antimicrobial secondary metabolites purified from endophytic microbes. I discuss emerging themes from a multidisciplinary perspective. In Chapter 4 (Mousa and Raizada, 2015), I review biosynthetic gene clusters that encode antimicrobial secondary metabolites in plant-associated microbes including, but not limited to, endophytes. I discuss various evolutionary events that led to cluster diversification including horizontal gene transfer among kingdoms.

Chapter 5 (Mousa et al., 2015b) and Chapter 6: In Chapter 5, I report the isolation of fungal endophytes from the ancient Afro-Indian crop, finger millet, for the first time and screening of their extracts for anti-Fusarium activity. Focusing on the most potent extract, derived from a Phoma sp., I use bio-guided purification followed by spectroscopic structural elucidation to isolate and characterize four diverse natural products underlying the antifungal activity. In Chapter 6, I characterize three anti-Fusarium compounds produced by three other fungal endophytes of finger millet. I study the endophytic behavior of finger millet fungi and demonstrate some insights into the mode of action of each purified molecule.

Chapter 7: In Chapter 7, I describe the isolation of bacterial endophytes from finger millet and screen them for anti- F. graminearum activity. Focusing on the most potent endophyte, strain M6, I study the in planta interactions, discover genes and natural products required for the antifungal activity, and test its ability to suppress F. graminearum in maize and wheat and to decrease mycotoxin contamination in grains after poor storage conditions. I describe the ability of M6 to create an unusual Root Hair Endophyte Stacking (RHESt) structure to physically hinder the entrance of the pathogen, which is then killed inside the RHESt. I discover genes and molecules that are required for the killing machinery of M6. Discovery of RHESt illustrates the value of exploring the microbiome of orphan crops of subsistence farmers for promising biological control.

Chapter 8 (Mousa et al., 2015a) and Chapter 9: In Chapter 8, I describe the screening of a library of ~200 bacterial endophytes previously isolated from diverse maize genotypes by a former PhD student in the Raizada lab, Dr. David Johnston-Monje, for antifungal activity against F. graminearum. Candidate endophytes were then tested for their ability to suppress F. graminearum in maize (Chapter 8) and wheat (Chapter 9). I report that the most potent endophytes were isolated from wild maize species. I study their in planta colonization and suggest a possible mode of action based on biochemical, molecular and microscopy experiments. This study highlights the value of exploring the microbiomes of wild relatives of modern crops, grown without fungicides, for potential biological antagonists.

1.2 General Introduction

1.2.1 Endophytes are important plant endo-symbionts

Endophytes are microbes that inhabit the internal tissues of plants without causing disease (Johnston-Monje and Raizada, 2011; Mousa and Raizada, 2013). Endophyte- plant interactions may develop a range of relationships which can involve symbiosis, mutualism and commensalism (Johnston-Monje and M.N., 2011; Ryan et al., 2008). It is worth noting that many endophytic bacteria are capable of living outside their host plant, even reported to be soil-borne microorganisms, an observation that has important implications for horizontal and lateral transmission (McInroy, 1994). Endophytes are often found more abundantly in roots than shoots. They are suggested to penetrate plant tissue by secreting enzymes such as pectinase and cellulase (Hurek et al., 1994), and can colonize a host plant by several routes: 1) penetration from the soil into the root, 2) entrance through stomates to the leaves (Sharrock et al., 1991), and 3) transmission through vectors such as insects (Ashbolt and Inkerman, 1990). Following entry, an endophyte may reside in a specific host tissue or intracellular space, perhaps regulated by interactions with the host and other microbes (Ryan et al., 2008).

Endophytes perform vital tasks for their host plants including growth promotion, nutrient acquisition, and tolerance to biotic and abiotic stress (McInroy, 1994). Endophytic strains of Pseudomonas, Enterobacter and Staphylococcus were reported to promote the growth of plants by producing growth regulators such as auxins and cytokinins (Yoav et al., 2004). With respect to nutrient acquisition, endophytes have been shown to assist in biological nitrogen fixation (James, 2000). Endophytes are also reported to help plants tolerate environmental stress such as flooding. For example, some endophytic bacteria produce 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which breaks down the ACC precursor of ethylene - a signal molecule which is otherwise induced during flooding/anoxic conditions and inhibits root growth (Glick et al., 1998). Endophytes help defend their hosts against phytopathogens such as fungi, bacteria or even insects and nematodes (Ryan et al., 2008). Different anti-pathogenic mechanisms

3 have been demonstrated or hypothesized including: 1) competition between endophytes and pathogens for nutrients or space, 2) induction of systemic resistance (ISR), and 3) secretion of anti-microbial natural products. In Chapter 2, various mechanisms of microbial antagonism will be discussed in detail (Mousa and Raizada, 2016).

1.2.2 Antimicrobial secondary metabolites from plant endophytes

Antimicrobial natural products from fungal endophytes are better characterized than those from bacterial endophytes. In the case of bacteria, many of the pathogen targets that have been studied are human.

1.2.2.1 Antimicrobial secondary metabolites produced by fungal endophytes

Antimicrobial natural products produced by fungal endophytes belong to diverse chemical classes including terpenoids, alkaloids, phenylpropanoids, aliphatic compounds, polyketides and peptides. In Chapter 3, all classes of antimicrobial compounds produced by endophytic fungi will be reviewed (Mousa and Raizada, 2013).

1.2.2.2 Antimicrobial secondary metabolites produced by bacterial endophytes

As noted above, anti-microbial molecules produced by bacterial endophytes have been less-studied than those produced from fungi. Some examples of antimicrobial secondary metabolites produced by bacterial endophytes include the following:

1.2.2.2.1 Ecomycins - These are peptide compounds purified from Pseudomonas viridiﬂava, an endophyte that colonizes Lactuca sativa (lettuce). Ecomycins showed broad spectrum antifungal activity against human pathogens including Candida albicans and Cryptococcus neoformans, and plant pathogens including Rhizoctonia solani, Fusarium oxysporum and Sclerotinia sclerotiorum (Miller et al., 1998).

1.2.2.2.2 Coronamycins - These peptide compounds were purified from a Streptomyces sp. that colonizes Monstera sp. vine in Peru. Coronamycins showed antifungal activity against several plant pathogenic fungi including Pythium ultimum, Aphanomyces cochlioides and Phytophthora cinnamomi, and human pathogens including Streptococcus pneumonia (a bacterium) and C. neoformans (a fungus) (Ezra et al., 2004).

1.2.2.2.3 Munumbicins - These peptide compounds were obtained from a Streptomyces sp., an endophyte of the medicinal plant Kennedia nigriscans, native to Australia. Munumbicins showed activity against human pathogens including C. neoformans, C. albicans, A. fumigates, S. aureus, Enterococcus faecalis, as well as the plant pathogenic fungi P. ultimum, R. solani, P. cinnamomi, Geotrichum candidum, S. sclerotiorum and P. syringae (Castillo et al., 2002).

1.2.2.2.4 Fusaricidins A-D – These are small antifungal peptides originally purified from the culture methanolic extract of Paenibacillus polymyxa (not an endophyte) (Beatty and Jensen, 2002). Later, I identified fusaricidin molecules in the culture filtrates of three endophytic P. polymyxa strains with potent activity against the fungal pathogen Fusarium graminearum. More details are given in Chapter 8 (Mousa et al., 2015b). In addition, fusaricidins were previously reported to inhibit the growth of various fungal pathogens including Leptosphaeria maculans, A. niger, A. oryzae, F. oxysporum and Penicillium thomii (Kajimura, 1996; Kajimura and Kaneda, 1997; Beatty and Jensen, 2002).

1.2.2.2.5 Oocydin A- This is a chlorinated macrocyclic lactone produced by Serratia marcescens, not an endophyte but an epiphyte living on the aquatic plant species Rhyncholacis penicillata (Strobel et al., 1999). Oocydin A showed antifungal activity against several crop pathogens including P. cinnamomi, P. parasitica, P. citrophora and P. ultimum.

1.2.2.2.6 Lipoproteins (LPs) - This large group of compounds includes surfactin, iturin and fengycin purified from Bacillus sp. and syringomycin, syringopeptin, syringofactins, arthrofactin, viscosin, orfamide, massetolides, putisolvins and entolysins purified from Pseudomonas. These lipoproteins have been reported to cause lysis and growth inhibition of several pathogens including viruses, mycoplasmas, bacteria, fungi and oomycetes (Raaijmakers et al., 2010).

1.2.2.2.7 Diacetylphloroglucinol (DAPG) - This group of phenolic polyketides is produced exclusively by Pseudomonas sp. (mainly fluorescent Pseudomonads), and is one of the primary compound classes responsible for the biocontrol activity of Pseudomonas sp. against different plant pathogens, including soil-borne pathogens (Yang and Cao, 2012) such as P. capsici, Colletotrichum gloeosporioides and F. oxysporum (Chung 2008).

1.2.2.2.8 Chitinase – These are enzymes that break down chitin, a polysaccharide consisting of repeating N-acetyl-D-glucose-2-amine units linked by β-1,4 glycosidic bonds. Chitinases are produced from various bacterial strains including Pseudomonas, Streptomyces, Bacillus and Burkholderia. Chitinases were found to antagonize different fungal pathogens by degrading the fungal cell wall, an activity that is correlated to the antipathogenic activity of the bacterial strains that employ this mechanism (Quecine et al., 2008). Chitinases produced from fluorescent Pseudomonas strains isolated from the sugarcane rhizosphere showed antifungal activity against C. falcatum, the causative agent of red rot disease in sugarcane (Viswanathan and Samiyappan, 2001). Actinoplanes missouriensis was reported to produce high levels of chitinase which degrades the hyphae of Plectosporium tabacinum, the causal agent of lupin root rot in Egypt, through plasmolysis and cell-wall lysis (El-Tarabily, 2003).

1.2.3 Biosynthetic genes that encode antimicrobial natural products in plant endophytes

Genes that encode structural enzymes required for the biosynthesis of antimicrobial compounds and other regulatory genes have also been identified. In Chapter 4, I have reviewed examples of genes responsible for the antimicrobial activity in several plant associated microbes, not only endophytes (Mousa and Raizada, 2015).

1.2.4 The fungal pathogen, Fusarium graminearum

The genus Fusarium includes widespread pathogens of cereal crops, including F. verticillioides in tropical maize which is associated with the production of carcinogenic mycotoxins, and F. graminearum (Figure 1.1 A), the causal agent of Gibberella ear rot in maize and Fusarium head blight in wheat (Sutton, 1982). In grain, F. graminearum produces sesquiterpene trichothecene mycotoxins including deoxynivalenol (DON) (Figure 1.1 B). DON is a mycotoxin that inhibits DNA and protein synthesis, resulting in various toxicity effects in both humans and animals. DON is an important virulence factor which enhances the ecological fitness and survival of Fusarium during the saprophytic growth phase through exerting its toxic effect on soil inhabitants (Audenaert et al., 2013). Moreover, DON plays a vital role in pathogen host interactions, in particular during the necrotic growth phase through induction of peroxides synthesis and subsequent cell death (Walter et al., 2010; Audenaert et al., 2013). The detailed role of DON in fungal infection and disease development have been extensively reviewed (Munkvold, 2003b; Voss, 2010; Kazan et al., 2012; Audenaert et al., 2013; Hassan et al., 2015). In addition to DON, F. graminearum produces fusaric acid (Figure 1.1 C), a mycotoxin/antibiotic that interferes with survival and metabolism of other microbes (Notz et al., 2002; van Rij et al., 2005).

Figure 1.1: The fungal pathogen, Fusarium graminearum and its mycotoxins. (A) Picture of F. graminearum stained with calcofluor fluorescent stain and viewed by confocal laser scanning microscopy. (B) Structure of deoxynivalenol mycotoxin (DON). (C) Structure of fusaric acid mycotoxin.

F. graminearum overwinters in crop debris, and is then transferred by rain and wind to infect maize at silking and wheat at flowering (Sutton, 1982). In maize, F. graminearum spores germinate and extend down the silk where infection of the developing seeds occurs (Miller et al., 2007). In wheat, spores germinate on the surface of pollen receptive spikelets then enter through natural opening such as stomata (Bushnell et al., 2003). Current disease management strategies to combat F. graminearum have achieved low to moderate success and rely on breeding for resistant genotypes, optimizing cultural practices or use of fungicides (Munkvold, 2003a; Reid et al., 2009). There is growing interest in developing bio-based strategies to combat plant pathogens and fungi to overcome negative impacts associated with the use of synthetic pesticides including pesticide resistance, soil and water pollution, toxicity from crop residues following treatment, and long term effects on human health and ecosystems.

Bacterial species from several genera including Bacillus, Burkholderia, Agrobacterium, Pseudomonas, Serratia, Arthrobacter, Azotobacter, Stenotrophomonas, Streptomyces, Collimonas and Pantoea have been reported as biological control agents (Raaijmakers, 2012). Among the bacteria used as biocontrol agents, Bacillus and Pseudomonas have received considerable attention and are already used commercially, with more than 10 registered products of Bacillus now available (Pérez-García et al., 2011). In the screening of biocontrol agents to control F. graminearum, Paenibacillus polymyxa strains were reported to be effective (He et al., 2009; Feng et al., 2011). The combination of an antibiotic-producing Pseudomonas sp. and two Bacillus sp. was found to be efficient against maize root pathogens including F. graminearum, F. moniliforme and Macrophomina phaseolina (Pal et al., 2001). Pantoea agglomerans and B. megaterium have also been used as biocontrol agents against Fusarium sp. (Luz, 2006). However, exploration of ancient and wild plants for endophytes with antifungal activity, in particular against F. graminearum, appears to be rare.

1.2.5 Ancient and wild relatives of crops as sources of endophytes

The ancient, marginalized crops of subsistence farmers, as well as the ancient landraces and wild relatives of staple crops, are thought to be more resistant to pests and

9 pathogens than their modern, mainstream counterparts (Rosenthal et al., 1997; Wang et al., 2005; Dávila-Flores et al., 2013; Lange et al., 2014). I hypothesized that this disease resistance-state might be due to endophytic microbes. To test this hypothesis, endophytes isolated from the ancient cereal, finger millet, as well as diverse maize genotypes including wild relatives and ancient landraces, were screened for their antifungal activity against F. graminearum.

1.2.5.1 The ancient Afro-Indian crop, finger millet

Finger millet (Eleusine coracana) is an Afro-Indian cereal grown by subsistence farmers (Vietmeyer, 1996; Goron and Raizada, 2015) (Figure 1.2). Finger millet was domesticated in Ethiopia and Western Uganda around 5000 BC then reached India by 3000 BC (Hilu and Wet, 1976). Finger millet is well known to be resistant to many pathogens (Munimbazi and Bullerman, 1996; Goron and Raizada, 2015) but there have been no reports of endophytes isolated from finger millet, including those that might contribute to its disease resistance. Fungal and bacterial endophytes were isolated in this study from finger millet for the first time [Chapter 5 (Mousa et al., 2015a), Chapter 6 and 7].

Figure 1.2: Finger millet fields. (A-B) Pictures of finger millet fields in Nepal, inset within (A) shows individual finger millet heads (source: Malinda Thilakarathna, University of Guelph, 2015).

Figure 1.3: Finger millet farmers. (A-B) Nepalese finger millet farmers (source: Manish Raizada, University of Guelph, 2011 and 2013, respectively). (C) African finger millet farmers (Source: Creative Commons, Peter Lewis, UK Department of International Development).

1.2.5.2 Wild relatives of modern maize

The primary ancestor of modern maize is the wild teosinte, Parviglumis (Zea mays ssp. parviglumis) (Figure 1.4) which remains extant in its native region (Matsuoka et al., 2002). There are other species of wild teosintes including the perennial Zea diploperennis (Figure 1.5) (Iltis and Doebley, 1980), grown in Mexico and Central America. Maize was bred and propagated throughout the Americas by local farmers, resulting in many ancient and traditional landraces. Modern scientists have since expanded upon the original germplasm by breeding inbreds and hybrids (Matsuoka et al., 2002; Smith et al., 2004). Wild maize has been reported to be more resistant to some pests than their modern relatives (Wang et al., 2005; Lange et al., 2014), a phenomena that might be attributed to loss of original endophytes during migration, breeding and evolution (Johnston-Monje and Raizada, 2011). The source of maize endophytes used in this study is a library of bacterial endophytes constructed by a former student in the Raizada lab, Dr. Johnston- Monje. These endophytes were isolated from diverse maize genotypes including wild, traditional and modern varieties (Johnston-Monje and Raizada, 2011; Johnston-Monje et al., 2014).

Figure 1.4: Illustration of Zea mays ssp. parviglumis

(Source: Creative Commons, Lisa Smith, University of Guelph, Canada).

Figure 1.5: Illustration of Zea diploperennis

(Source: Creative Commons, Lisa Smith, University of Guelph, Canada).

1.3 Hypothesis

Ancient crops and their wild relatives, which grow without added synthetic fungicides, may contain endophytes that help their host plants to combat fungal pathogens including F. graminearum. The mechanism of antifungal activity may involve the biosynthesis of antifungal natural products.

1.4 Objectives

1.4.1 To identify endophytes that can combat F. graminearum from the ancient cereal, finger millet, and from diverse maize genotypes, including wild relatives and ancient landraces.

1.4.2 To characterize anti-Fusarium natural products produced by the candidate endophytes.

1.4.3 To identify and characterize gene(s) encoding the observed antifungal activities, focusing on one potent bacterial endophyte.

Chapter 2: Natural disease control in cereal grains

Authors’ contributions

Walaa K Mousa wrote the manuscript.

Dr. Manish N. Raizada edited the manuscript.

Publication status: Published as book chapter.

Mousa W.K., and Raizada M.N. (2016) Natural Disease Control in Cereal Grains. In: Wrigley, C., Corke, H., and Seetharaman, K., Faubion, J., (eds.) Encyclopedia of Food Grains, 2nd Edition, pp. 257-263. Oxford: Academic Press.

2.1 Abstract

Grain diseases impact global food security and human health. The indiscriminate use of pesticides is a hazard to both humans and the environment. Biocontrol, the use of living organisms to suppress crop disease, is an alternative strategy of disease management. This chapter reviews the different mechanisms for biocontrol including competition, antibiosis, induced host resistance, soil suppression, hyperparasitism and transgenic plants. Examples are provided for each mechanism. The chapter concludes by discussing how biocontrol strategies can be successful under real world conditions.

2.2 Introduction

The expected dramatic increase in human population and shortages in food supply make it a necessity to explore new strategies to improve crop yields and effectively manage crop diseases with more natural, environmental friendly strategies. Given the nutritional and economic importance of grains, microbial diseases are a real danger to global food security. Approximately 10% of global food production is lost due to plant pathogens, contributing to more than 800 million people not having enough food (Strange and Scott, 2005). Plant pathogens may be difficult to identify and pathogens may evolve, which cause further challenges. Phytopathogens may result in a catastrophe as illustrated by the Southern Corn Leaf Blight epidemic of 1970–1971 in the United States caused by the fungus Cochliobolus heterostrophus (Ullstrup, 1972). Another example was the Great Bengal Famine of 1943 in India, caused by the fungus Cochliobolus miyabeanus, which resulted in the deaths of two million people due to their dependence on rice as the staple food (Padmanabhan, 1973). Today, the rice blast fungus, Pyricularia oryzae, causes 10%–30% crop losses annually (Talbot, 2003). Moreover, the host range of this fungus extends to include other cereals such as wheat (Igarashi et al., 1986), as well as finger millet (Eleusine coracana) where the infection may result in complete crop loss (Ekwamu, 1991).

Grain infection caused by toxin-producing fungal species such as Aspergillus, Fusarium, Penicillium, Alternaria and to a lesser extent Pithomyces, Phomopsis and Acremonium, is serious, because these mycotoxins may be consumed by animals including humans. For example, the rice and maize (corn) pathogenic fungus Fusarium moniliforme, secretes the mycotoxin fumonisin B1 which has been linked to esophageal cancer (Gelderblom et al., 1991), equine leukoencephalomalacia and porcine pulmonary oedema (Placinta et al., 1999). Fusarium graminearum, the disease causing agent of head blight in barley and wheat, and ear rot in maize, synthesizes cancer-causing trichothecenes such as deoxynivalenol (DON) (Sutton, 1982). Aspergillus flavus, the causal agent of kernel rot in maize, produces aflatoxin on preharvest maize and during storage (Payne and Widstrom, 1992). Together, aflatoxin and DON result in $1.5 billion

19 in losses annually across different crops in the United States alone (Robens and Cardwell, 2003). There is extensive contamination of grains in the United States and Canada with trichothecenes, with a higher incidence reported in maize than wheat (Scott, 1997). Fusarium mycotoxins have been reported worldwide, particularly fumonisins on maize (Shephard et al., 1995).

2.3 Overview of Disease Management Strategies

Crop diseases are managed by chemical, physical, cultural and/or natural control methods including biological control. Chemical management involves the use of synthetic pesticides (fungicides). However, these chemicals may lack specificity and affect the beneficial soil microbiota; their indiscriminate use may result in negative impacts on soil ecosystems. Moreover, some chemical pesticides may remain in the soil and in crop tissues, potentially causing harmful effects to end users, either animals or humans. Physical methods to control crop disease include burning of infected crops, barriers that prevent insect vectors from gaining access to crops, and segregation of infected seeds from healthy seeds by grain weight (infected grains have less weight). Cultural methods include crop rotation (when a pathogen has a specific crop host), field sanitation (e.g. burning of infected crop residues), disinfecting of field tools and machinery, site selection, and use of seeds certified to be free of pathogens. Natural control strategies are numerous and include the selection and breeding for disease resistant crop cultivars. For example, naturally resistant Chinese genotypes of wheat have been shown to have lower Fusarium-derived mycotoxin levels compared to more susceptible Canadian cultivars (Wong et al., 1995). An additional natural control strategy involves the use of intercropping “companion crops” that attract or repel insect vectors of disease. A final major type of natural control is biological control or biocontrol which is defined as “the use of living organisms to suppress the population density or impact of a speciﬁc pest organism, making it less abundant or less damaging than it would otherwise be” (Eilenberg, 2006). Biocontrol involves the use of soil and plant associated beneficial microbes, including bacteria and fungi. The increasing public interest in organic products

20 strongly enhances the idea of biocontrol, since organic agriculture does not allow synthetic chemicals but does permit spraying with beneficial microbes.

2.4 Introduction to Soil and Plant Associated Microbes

The plant is an attractive host to a variety of beneficial soil microbes (bacteria and fungi). These microbes include those that live in the: (1) rhizosphere (area surrounding the root and influenced by root secretions); (2) mycorhizosphere (area surrounding root- associated mycorrhizal fungi); and (3) endosphere (internal plant tissues; these microbes are termed endophytes). There may be cross-talk between the plant host, beneficial microbes and pathogens. On the plant side, roots produce secretions such as sugars, amino acids and organic acids that stimulate the microbial population including both antagonists and pathogens. The roots may also produce pathogen-stimulating factors, allelochemicals and repellents which have their peak effects during vegetative development, with these effects decreasing with increased distance from the root system. On the microbial side, plant growth promoting rhizobacteria (PGPR), plant growth promoting fungi, nitrogen fixing rhizobia and mycorrhizal fungi, play roles in nutrient cycling and also in biocontrol.

2.5 Overview of Biocontrol Mechanisms

Mechanistically, beneficial soil and plant associated microbes may control pathogens indirectly or directly. Indirect anti-pathogenesis occurs when microbes enhance plant health by making soil nutrients more available (e.g. solubilize rock phosphate) or fix nitrogen (convert atmospheric N2 gas into NH3 fertilizer). Direct anti- pathogenesis occurs when microbes combat pathogens by competition, antibiosis (production of anti-pathogen substances), induced systemic resistance (ISR: stimulation of plant defences), soil suppression (enhanced populations of soil microbiota to suppress disease), hyperparasitism (use of microbes as parasites of the pathogen) and the use of microbial derived genes with anti-pathogenesis activity inserted into transgenic crops (i.e.

Genetically Modified Organisms, GMOs). This review will focus on the direct mechanisms of biocontrol of grain disease. Diverse mechanisms of biocontrol are discussed below:

2.5.1 Competition

Competition-based biocontrol involves the use of fast growing, antagonist organisms to outcompete with soil borne pathogens that share the same ecological niche, where nutrients and/or space are limited. The success of the biocontrol agent is determined by its ecological fitness among a soil microbial community that is already in equilibrium. Competition-based biocontrol is also affected by the physical/chemical properties of the soil. For example, the biocontrol fungal agent Trichoderma has its activity enhanced in moist soil and diminished in higher pH soils. The use of non-target fungicides also diminishes the competition ability of any fungal biocontrol agent.

An example of a biocontrol agent that competes for nutrients is the bacterium, Pseudomonas fluorescens, that secretes siderophores to chelate iron, decreasing its availability for many soil borne pathogens including the bacteria, Erwinia carotovora (Haas and Défago, 2005). Another example of competition based biocontrol is the use of non-antagonist soil bacteria including Pseudomonas sp, Brevundimonas sp., Pedobacter sp. and Luteibacter sp., which when combined, were shown to reduce the biomass (ergosterol content) of the fungal pathogens, Rhizoctonia solani and Fusarium culmorum, and the saprophytic fungus, Trichoderma harzianum (de Boer et al., 2007). The authors argued that the combined biocontrol species competed for nutrients or triggered the production of antimicrobial metabolites (de Boer et al., 2007). Interestingly, non pathogenic strains of Fusarium oxysporum have the ability to control F. oxysporum strains, the causal agents of wilt, a disease that affect many crops including grains (Harvás et al., 1997); the mechanism of action involves competition for carbon if the biocontrol inoculum is applied in excess to the pathogen (Couteaudier and Alabouvette, 1990).

Antagonists can also compete for space by occupying infection sites, thus preventing the pathogen from gaining access to the plant host. For example, the fungus Phialophora graminicoa effectively controls the fungal pathogen, Gaeumannomyces graminis var. tritici, by occupying the infection sites of wheat root cortical tissue, resulting in reduced pathogen invasion (Deacon, 1976). The protective F. oxysporum strain was isolated from the stems of healthy plants or from wilt disease suppressive soil (see below) where it was present at an exceptionally high density (Ogawa and Komada, 1984). However, when the inoculum is low, F. oxysporum also retains some of its biocontrol activity; one possibility is that it may induce host resistance as an avirulent form of the pathogen (explained below). More recently, competition on root hairs and branches has been proposed to hinder the pathogen attachment and penetration (Bolwerk et al., 2005).

The addition of animal manures is widely employed particularly in organic agriculture to provide soil with nutrition and also to enhance soil beneficial microbiota, which then compete against pathogens. For example, animal manures can combat many plant pathogens including Gaeumannomyces graminis, Fusarium sp., Phymatotrichum omnivorum and Sclerotinia sclerotiorum (Conn and Lazarovits, 1999).

2.5.2Antibiosis

Antibiosis is a major mechanism of biological control, in which the antagonist produces substance(s) which could be an antibiotic, lytic enzyme (degrades plant cell wall), volatile substance or toxin that effectively target and destroy the pathogen. Below, examples are provided for each mechanism:

2.5.2.1 Antibiosis by production of antibiotics

2.5.2.1.1 Antibiotics from bacteria

The phenolic polyketide 2,4-diacetylphloroglucinol (DAPG) is produced exclusively by Pseudomonas bacterial species (mainly fluorescent Pseudomonas) and is one of the primary compounds responsible for the biocontrol activity of Pseudomonas sp. against different plant pathogens. For example, Pseudomonas brassicacearum was isolated as an endophyte from the roots of Artemisia sp. (Jinju area, Korea) and was shown to control the fungal plant pathogens Phytophthora capsici, Colletotrichum gloeosporioides and Fusarium oxysporum mainly by secretion of DAPG and 2,4,6-trihydroxyacetophenone (Chung 2008). Pseudomonas fluorescens strain HC1-07, isolated from the wheat phyllosphere suppresses wheat take-all disease caused by the fungus G. graminis var.tritici as well as root rot of wheat caused by Rhizoctonia solani (Yang et al., 2014a). The mechanism of action involves production of a cyclic lipopeptide (CLP). Extracted CLP inhibited the growth of G. graminis var.tritici and R. solani in vitro (Yang et al., 2014a).

The fungus, Chaetomium globosum, isolated from decaying organic matter and soil, effectively controls seedling blight of corn caused by Fusarium roseum and F. graminearum through the production of cochliodinol antibiotics (Chang and T, 1968).

Ecomycins, which are peptide compounds purified from the bacteria Pseudomonas viridiﬂava, an endophyte that colonizes Lactuca sativa (lettuce), showed broad spectrum antifungal activity against some fungal pathogens of plants, including Rhizoctonia solani, Fusarium oxysporum and Sclerotinia sclerotiorum (Miller et al., 1998).

Munumbicins, peptides obtained from a Streptomyces sp., an endophyte of the medicinal plant Kennedia nigriscans, Australia, showed activity against some plant pathogenic fungi such as Pythium ultimum, Rhizoctonia solani, Phytophthora cinnamomi, Geotrichum candidum and Sclerotinia sclerotiorum (Castillo et al., 2002).

Fusaricidins, A-D peptides originally purified from the methanolic culture extract of Paenibacillus polymyxa (Beatty and Jensen, 2002), have been isolated from different Paenibacillus strains and have been reported to combat several important fungal pathogens of plants including Aspergillus niger, Aspergillus oryzae, Fusarium oxysporum and Penicillium thomii (Kajimura and Kaneda, 1997; Kajimura, 1996).

Oocydin A, a chlorinated macrocyclic lactone produced by the bacteria, Serratia marcescens, an epiphyte (plant surface dwelling) living on the aquatic plant species, Rhyncholacis penicillata (Strobel et al., 1999a), showed antifungal activity against several crop pathogens including Phytophthora cinnamomi, Phytophthora parasitica, Phytophthora citrophora and Pythium ultimum.

Myxobacteria can efficiently combat the plant fungal pathogens, Phytophthora capsici, Pythium ultimum, Rhizoctonia sp., Sclerotinia minor and to a lesser extent Fusarium oxysporum, through the production of a group of macrocyclic lactone, lactam rings and linear cyclic peptide antibiotics (Bull et al., 2002).

2.5.2.1.2 Antibiotics from fungi

Rice blast disease, caused by Magnaporthe oryzae, is one of the most devastating diseases worldwide. Spore germination of M. oryzae was shown to be strongly inhibited by helvolic acid, a terpenoid compound purified from the yeast, Pichia guilliermondii, isolated from the medicinal plant, Paris polyphylla (Zhao et al., 2010). M. oryzae is also affected by cryptocin, isolated from the fungal endophyte, Cryptosporiopsis quercina, which colonizes the inner stem bark tissue of Tripterygium wilfordii, (Li et al., 2000). F.culmorum and F. graminearum could be inhibited by pestalachloride A isolated from Pestalotiopsis adusta, a fungal endophyte that inhabits the stem tissue of a Chinese tree (Li et al., 2008a).

The fungal pathogen, Helminthosporium sativum, the causal agent of seedling blight and root rot of cereals could be inhibited by colletotric acid, a tridepside compound purified from Colletotrichum gloeosporioides, a fungus that colonizes the stems of Artemisia mongolica (Zou et al., 2000).

Phomenone, an antifungal sesquiterpene (eremophilane class) purified from Xylaria sp., a fungal endophyte isolated from Piper aduncum, was demonstrated to combat the wheat pathogen, Cladosporium cladosporioides, as well as C. sphaerospermum (common indoor mold) (Silva et al., 2010).

Pyrrocidines, a group of polyketide-amino acid-derived antibiotics, were originally cultured from an endophyte of maize kernels, Acremonium zeae; this fungus has a protective effect on pre-harvest kernels against fungal pathogens, perhaps by effectively competing for the same host habitat (Wicklow et al., 2005). Pyrrocidines showed antifungal activity on agar disc experiments against mycotoxin-producing Fusarium verticillioides and Aspergillus flavus (Wicklow et al., 2005). The study reviewed previous experiments which suggested that pyrrocidine A reduces growth of several Gram-positive bacteria. The authors claimed that A. zeae can cause stalk rot and hence may be selected against by pathologists and breeders, perhaps making commercial hybrid maize more susceptible to fungal pathogens. In a follow up study, pyrrocidine A showed biocontrol activity against stalk and ear rot pathogens of maize, including Fusarium graminearum, Nigrospora oryzae, Stenocarpella maydis and Rhizoctonia zeae (Wicklow and Poling, 2009). The authors proposed that pyrrocidine A may protect disease-susceptible seedlings against pathogens, after colonization of seedlings by the endophyte. Pyrrocidine A also showed anti-pathogen effects against the seed-rot saprophytes, Aspergillus flavus and Eupenicillium ochrosalmoneum, and as well as against the biological agent of fungal leaf spot disease, Curvularia lunata, and the bacteria Clavibacter michiganense, the disease- causing agent of Goss’s wilt (Wicklow and Poling, 2009).

2.5.2.2 Antibiosis by production of lytic enzymes and volatiles

The enzyme chitinase was found to antagonize different fungal pathogens by degrading chitin present in the fungal cell wall. Chitinase secretion is correlated with the antipathogenic activities of many bacterial strains including Pseudomonas, Streptomyces, Bacillus and Burkholderia (Quecine et al., 2008). Chitinases produced from fluorescent Pseudomonas strains isolated from the sugarcane rhizosphere showed antifungal activity against the fungus Colletotrichum falcatum, the causative agent of red rot disease in sugarcane (Viswanathan and Samiyappan, 2001). Actinoplanes missouriensis, a Gram positive bacterium, was reported to produce high levels of chitinase which was shown to degrade the hyphae of Plectosporium tabacinum, the causal agent of lupin root rot in Egypt, through plasmolysis and cell-wall lysis (El-Tarabily, 2003). The bacteria, Bacillus cereus, isolated from a soil that suppresses take-all decline disease, was found to lyse the hyphae of the corresponding fungal pathogen Gaeumannomyces graminis var. tritici (Campbell and Faull, 1979).

2.5.2.3 Antibiosis by volatile compounds Muscodor albus, endophytic fungus obtained from Cinnamomum zeylanicum (Sri Lankan cinnamon), produces a mixture of volatile compounds including acids, alcohols, ester, lipids and ketones (Strobel et al., 2001). Among these volatiles, 1-butanol, 3- methyl-, acetate ester, is the most potent and targets a range of pathogens including the smut fungus, Ustilago hordei (a pathogen of barley), Fusarium oxysporum (pathogen of wheat and maize), Rhizoctonia solani and Pythium ultimum (Strobel et al., 2001).

2.5.3 Induced host resistance

In addition to biocontrol agents that directly suppress crop pathogens (discussed above), biocontrol agents may also induce natural defence mechanisms in host plants, a process which is termed priming or induced host resistance (Goellner and Conrath, 2008). Plant host resistance can be induced either locally or systemically (throughout the plant)

27 by biotic or abiotic inducers resulting in disease control without direct contact between the antagonist and the pathogen. This form of biocontrol usually results in a 20-85% reduction in disease symptoms rather than complete control. Induced host resistance form of biocontrol is also dependent on the genotype of the host and the level of induction in addition to environmental conditions such as crop nutrient health (Walters et al., 2013). Induced host resistance can involve stimulation of plant signalling hormones that induce plant defence genes (salicylic acid, ethylene, jasmonic acid) ( Ryals et al., 1996; Pieterse et al., 1998). Host disease resistance can be mediated by plant recognition of surface components of some biocontrol species such as the flagella (flagellin protein) and lipopolysaccharides, which mimic the surface features of pathogens (Van Wees et al., 2008).

Arbuscular mycorrhizal fungi (AMF) and other root colonizing fungi are well known as biotic inducers of systemic disease resistance in host plants. Examples of such root colonizers are: (1) the AMF Glomus intraradices, which induces pathogenic resistance genes in rice upon root colonization to combat the fungal blast pathogen, Magnaporthe oryzae (CAMPOS‐SORIANO et al., 2012); (2) the AMF Glomus mosseae which induces systemic resistance in maize against Rhizoctonia solani, the causal agent of corn sheath blight (Song et al., 2011); and (3) the endophytic fungus, Piriformospora indica, which induces host resistance against the basidiomycete fungus Blumeria graminis, the disease causing agent of powdery mildew in barley (Molitor et al., 2011). With respect to abiotic inducers, the commercial fungicide probenazole was used to induce resistance in maize against the fungus Bipolaris maydis, the disease causing agent of southern leaf blight (1.8 kg active ingredient per ha) (Yang et al., 2011); the application of this elicitor did not significantly affect the vegetative growth of the plant under greenhouse conditions. Acetoin (3-hydroxy-2-butanone) is a volatile compound produced by some microbial species such as Bacillus subtilis and thought to play a role in plant growth promotion, with potential to induce host resistance (Ryu et al., 2004). For example, acetoin has been shown to decrease disease severity caused by Erwinia carotovora on Arabidopsis seedlings when the seedlings were exposed to a mixture of

28 volatiles (including acetoin) produced by Bacillus subtilis and B. amyloliquefaciens when compared to untreated seeds (Ryu et al., 2004). Moreover, exposure to a racemic mixture of 2,3-butanediol also activated induced host resistance (Ryu et al., 2004).

2.5.4 Disease suppressive soil

Soil that suppresses crop disease due to the specific structure of its microbial community is known as disease suppressive soil. Suppressive soil is an attractive method of biocontrol, because it has the potential to be sustainable over many seasons under favourable conditions. Suppressive soil is divided into two categories, general and specific:

2.5.4.1 General soil suppression

General suppressive soils are those that have a high total microbial biomass, resulting in low levels of protection against multiple pathogens. This strategy is dependent on the quality and quantity of soil organic matter (Hoitink and Boehm, 1999) (composts, green manures and cover crops) that provides supplemental nutrients to enhance populations of beneficial microbes intended to antagonize associated crop pathogens primarily by occupying plant infection sites.

2.5.4.2 Specific soil suppression

In contrast to a general suppressive soil, specific suppressive soil is one that has a high concentration of one or more specific microbial species and results in high levels of protection against specific pathogens (Weller et al., 2002). Take-all disease of wheat, caused by Gaeumonomyces graminus var. tritici, is one of the most studied root diseases, and its control by specific soil suppression is used as model system for biocontrol research. This disease is mainly controlled by biological and culture strategies as there is no resistant cultivar nor effective fungicide available. The suppressive soil is developed

29 by continuous monocropping up to 5-7 years. The suppression is due to soil enhancement of Pseudomonas fluorescence that produces the antibiotics, phenazine carboxylic acid and 2,4 diacetyl phloroglucinol (James Cook, 2003).

Another example of specific soil suppression involves control of Fusarium wilt, a crop disease caused by Fusarium oxysporum (Mazzola, 2002). Here, an abiotic factor such as soil pH affects the microbial community and results in enhancement of non- pathogenic Fusarium sp. which induce host systemic resistance and outcompete the pathogen for access to nutrients and infection sites. This disease suppressive soil was also found to contain a high density of Pseudomonas fluorescence which induces host defence and releases siderophores (to decrease iron availability to other organisms). As previously mentioned, both Fusarium sp. and Pseudomonas act cooperatively rather than individually (Mazzola, 2002).

2.5.5 Hyperparasitism (parasite of the parasite)

Hyperparasitism involves specific recognition between the antagonist and the pathogen that terminates with the death of the pathogen. Hyperparasitism is most common in fungi that form sclerotia which are dense masses of pathogenic hyphae that remain in crop residues to promote infection in the next crop season. For example, the soil fungus Coniothyrium minitans is a potential microparasite of Sclerotinia sclerotiorum, a pathogen of legumes (e.g. beans); the biocontrol agent colonizes and penetrates the sclerotia resulting in reduced inoculation of the pathogen in the soil (Trutmann et al., 1980). As another example, Trichoderma is a common parasite of Rhizoctonia solani, the causative agent of bare patch disease of cereals and sheath blight of rice. Trichoderma can specifically recognize and effectively penetrate the hyphal cell wall of the parasite by release of cell wall degrading enzymes (Chet and Baker, 1981).

2.5.6 Transgenic plants (GMOs, genetically modified organisms)

Isolation of genes from microbial antagonists followed by their introduction into transgenic plants is a bio-based method of disease and pest control. This technology is controversial, has poor public acceptance and is restricted by government regulations worldwide. A few examples are provided here, focusing on Fusarium-derived mycotoxins including fumonisins.

The most common transgene for crop pest control on the market today involves transgenic maize plants that express the Cry genes [CryIA(b), CryIA(c), or Cry9C], isolated from the bacterium, Bacillus thuringiensis, which encodes the Bt proteins that are toxic to certain insects (Munkvold et al., 1999). This technology is noted here because these transgenic plants were found to contain lower concentrations of the mycotoxin fumonisin than non-transgenic maize (Munkvold et al., 1999), perhaps by reducing the number of entry sites of Fusarium pathogen associated with insect damage.

Recently a patented method was developed to isolate a fumonisin-degrading enzyme by fermentation of the fungi Exophiala spinifera or Rhinocladiella atrovirens on fumonisin containing media (Duvick, 1998). The gene encoding this degrading enzyme was isolated and introduced into plants (Duvick, 1998).

Transgenic plants have been reported that are claimed to detoxify the trichothecene mycotoxins of Fusarium (Hohn, 2002). These transgenic plants contain TR1101, a gene encoding trichothecene-3-o-acetyl transferase cloned from F. sporotrichierides, which catalyzes the acetylation of the C3 hydroxyl position of many trichothecenes including DON (Hohn, 2002).

2.6 Microbes that Combine Multiple Mechanisms for Biocontrol

Some microbes can contribute to biocontrol by integrating multiple mechanisms. Examples of such microbes include mycorrhizal fungi and endophytes. Mycorrhizal fungi have the potential to combat many pathogens by different mechanisms: (1) induced host resistance as noted above, (2) physical blockage of the inoculation and penetration sites, (3) increased lignification of roots, (4) increased production of antimicrobial isoflavonoids, (5) hyperparasitism, and (6) improved nutrient availability including as a bridge to deliver phosphate solubilised by PGPR, resulting in better plant health (Barea et al., 2005).

As noted above, endophytes are microbes that live inside plants without causing disease (Ryan et al., 2008). Endophytes may be able to combine multiple mechanisms to combat plant pathogens including: (1) competition for plant nutrients; (2) induced host resistance; and (3) secretion of anti-pathogenic molecules (e.g. secondary metabolites, antifungal peptides or enzymes such as chitinase). For example, the fungal endophytes of corn, Trichoderma koningii and Alternaria alternata, were found to possess antifungal activity against Fusarium sp., the causal agent of seedling blight and root and stalk rot (Orole and Adejumo, 2009). Paenibacillus polymyxa, an endophytic bacterium that inhabits wheat (Huaibei City, Anhui Province, China), was identified as a strong antagonist against F. graminearum by inhibiting mycelium growth and spore germination (Feng et al., 2011).

2.7 Examples of commercial biocontrol products

Among the most famous examples of biocontrol products currently on the market include Bacillus sp. and Pseudomonas sp. which have received considerable attention, with more than 10 registered products of Bacillus now available (Pérez-García et al., 2011). Pseudomonas species exert direct antifungal activity against various phytopathogens by the production of various secondary metabolites including:

32 siderophores, hydrogen cyanide, 2,4-DAPG, phenazines, 2,5-dialkylresorcinol, quinolones, gluconic acid, in addition to lipoproteins (Raaijmakers, 2012).

2.8 Discussion

Biocontrol faces some challenges including government regulation, public acceptance, the high costs of commercial production (e.g. fermentation) in addition to safety concerns for ecosystems, animals and humans. In practice, the key challenge is how to enhance the ecological fitness of the biocontrol agent within a complex ecosystem (field) under real world environmental conditions. The introduction of an antagonist in a well established soil is usually ineffective because the biocontrol agent may be lost due to competition with pre-existing soil microbiota and environmental unpredictability (temperature, moisture, seasonal variation, synthetic chemicals). Overcoming this challenge will require a better understanding of the complex factors that impact the interactions between host, pathogen and biocontrol agent under varied environmental conditions. Promising areas of future research are:

2.8.1 To target a pathogen at its most susceptible life cycle stage. For example, a promising direction is to target biocontrol strategies to the saprophytic stage of the pathogen (when a pathogen lives on dead host tissue) and to the dormancy stage of the pathogen (the stage of the pathogen life cycle outside of its host plant when the pathogen has less energy and hence lower resistance). Hyperparasites are most effective at these stages, because the pathogen is most unshielded. In contrast to hyperparasites, competitive antagonists may be most effective during pathogen spore germination when the pathogen has consumed large amounts of stored energy. In situations where the pathogen only survives in a living host (obligate pathogen), then host-based strategies such as induced resistance and transgenic plants as well as endophytes might be most effective.

2.8.2 To target a pathogen with the appropriate biocontrol based on its mechanism, timing and location of action. For example, endophytes and antibiosis secreting biocontrol agents are most appropriate when they share the same host tissue/cell type and developmental stage with the target pathogen. For soil borne pathogens, soil suppression is an ideal strategy because it can reduce the reservoir of the pathogen through competition for nutrients and/or compete for common host entry sites such as on roots. For fast growing pathogens, the competitive antagonist should be added prior to the rapid multiplication stage of the pathogen.

2.8.3 To take into account the defence strategies of the pathogen. For example, when considering antibiosis-based biocontrol, it is important to consider whether the target pathogen can degrade the biocontrol derived antibiotics.

2.8.4 To consider environmental stress conditions. It is important to select biocontrol agents that can either tolerate anticipated unfavourable conditions such as dryness or promote stress resistance in the host. For example, Trichoderma biocontrol agents have been well documented to improve host tolerance to low nitrogen and water.

Chapter 3: The diversity of anti-microbial secondary metabolites produced by fungal endophytes: An interdisciplinary perspective

Authors’ contributions

Walaa K Mousa wrote the manuscript.

Dr. Manish N. Raizada edited the manuscript.

Publication status: Published.

Mousa, W.K. and Raizada, M.N. (2013). The diversity of anti-microbial secondary metabolites produced by fungal endophytes: An interdisciplinary perspective. Frontiers in Microbiology 4:65

3.1 Abstract

Endophytes are microbes that inhabit host plants without causing disease and are reported to be reservoirs of metabolites that combat microbes and other pathogens. Here I review diverse classes of secondary metabolites, focusing on anti-microbial compounds, synthesized by fungal endophytes including terpenoids, alkaloids, phenylpropanoids, aliphatic compounds, polyketides and peptides from the interdisciplinary perspectives of biochemistry, genetics, fungal biology, host plant biology, human and plant pathology. Several trends were apparent. First, host plants are often investigated for endophytes when there is prior indigenous knowledge concerning human medicinal uses (e.g. Chinese herbs). However, within their native ecosystems, and where investigated, endophytes were shown to produce compounds that target pathogens of the host plant. In a few examples, both fungal endophytes and their hosts were reported to produce the same compounds. Terpenoids and polyketides are the most purified anti-microbial secondary metabolites from endophytes, while flavonoids and lignans are rare. Examples are provided where fungal genes encoding anti-microbial compounds are clustered on chromosomes. As different genera of fungi can produce the same metabolite, genetic clustering may facilitate sharing of anti-microbial secondary metabolites between fungi. I discuss gaps in the literature and how more interdisciplinary research may lead to new opportunities to develop bio-based commercial products to combat global crop and human pathogens.

3.2 Introduction Plant pests and pathogens including viruses, bacteria, nematodes, insects and fungi reduce crop yields by 30-50% globally, contributing to malnutrition and poverty (Pimentel, 2009). There are 700 known plant viruses, such as cassava mosaic virus (CMV) which devastates the livelihoods of cassava farmers in Africa (Thresh and Cooter, 2005). Amongst bacterial pathogens, blights caused by Xanthomonas species cause 350 different plant diseases including rice blight disease (X. oryzae) (Leyns et al., 1984). Nematodes are considered significant problems in tropical and subtropical regions, but are often undiagnosed because they exert their damage on plant roots (Shurtleff and Averre, 2000). Nematodes such as those belonging to the genera Paratrichodorus and Trichodorus are also vectors of plant pathogenic viruses (Boutsika et al., 2004). Approximately 9,000 species of insects and mites damage crops, causing an estimated 14% loss in global crop yields, requiring $13 billion in pesticide controls in the U.S. alone (Pimentel, 2009). Fungi are considered to be particularly serious plant pathogens because they can also potentially produce mycotoxins that are then consumed by humans and animals. For example, the maize and rice pathogenic fungus Fusarium moniliforme produces fumonisin B1 which is associated with esophageal cancer (Gelderblom et al., 1991). The maize pathogenic fungus Aspergillus flavus, which causes kernel rot, produces aflatoxin on preharvest corn and in storage (Payne and Widstrom, 1992). Fusarium graminearum, the causal agent of head blight in wheat and ear rot in maize, produces carcinogenic trichothecenes including deoxynivalenol (DON) (Sutton, 1982). Combined, aflatoxin and DON cause $1.5 billion in losses each year across different crops in the U.S. alone (Robens and Cardwell, 2003). Other serious fungal pathogens of crops include: Magnaporthe grisea and Pyricularia oryzae, which cause rice blast diseases in Asia; Puccinia sp., the causal agents of rusts in barley, maize, and wheat; Phytophthora infestans, an oomycete fungus which infects the Solanaceae family and led to the potato blight in Ireland in the 1840’s; and Rhizoctonia solani, the causal agent of damping-off in diverse crops and stem cranker in potato (Strange and Scott, 2005).

Synthetic pesticides including fungicides are widely used in pathogen management but there are increasing demands to develop environmentally friendly, bio-based products. Bio-based strategies include the use of biocontrol agents such as plant growth promoting bacteria (Compant et al., 2005), and fungi such as Trichoderma viridi, which has been used to control Rhizoctonia stem canker and black scurf of potato (Beagle- Ristaino and Papavizas, 1985). A potential opportunity to control crop pathogens is the use of endophytes and their derived secondary metabolites. Endophytes are microorganisms which inhabit plants in the tissues beneath the epidermal cell layers but cause no apparent harm to their hosts (Stone, 2000). In fact, endophytes can confer diverse beneficial traits to host plants (Johnston-Monje and M.N., 2011; Johnston-Monje and Raizada, 2011). All plants that have been investigated for endophytes possess them (Strobel and Daisy, 2003), and since there are more than 300,000 species of land plants, these microbes represent a large reservoir of biological resources including bioactive compounds with potential applications for medicine, industry and agriculture, including pathogen control.

The objective of this paper is to review the diversity of anti-microbial compounds synthesized by fungal endophytes including terpenoids, alkaloids, phenylpropanoids, aliphatic compounds, polyketides and peptides. Bioactive compounds in general synthesized by fungi were reviewed from different perspectives (Strobel and Daisy, 2003; Aly et al., 2010). I review compounds from the interdisciplinary perspectives of biochemistry, genetics, fungal biology, host plant biology human and plant pathology. I conclude by discussing common themes.

3.3 Diverse classes of fungal antimicrobial secondary metabolites

3.3.1 Terpenoid compounds

Sesquiterpenes, diterpenoids and triterpenoids are the major terpenoids that have been isolated from endophytes.

3.3.1.1 Sesquiterpenes:

The structures of sesquiterpene derivatives described in this review are shown (Figure 3.1).

Trichodermin (1) - Trichodermin was characterized from Trichoderma harzianum, an endophytic fungus living in Ilex cornuta, an evergreen holly shrub from East Asia (Chen et al., 2007). Trichodermin is a member of the 12,13-epoxytrichothecene mycotoxin family which has been used as a template for chemical synthesis of pharmaceutical compounds and plant growth regulators (Cutler and LeFiles, 1978). Trichodermin has been reported to protect against the Solanaceous plant pathogens Alternaria solani and Rhizoctonia solani in vitro (Chen et al., 2007). However, trichodermin has been shown to have inhibitory effects on plant growth including wheat coleoptiles (Triticum aestivum), tobacco (Nicotiana tabacum), beans (Phaseolus vulgaris) and corn (Zea mays) (Cutler and LeFiles, 1978). Mechanistically, trichodermin has been shown to be a very potent inhibitor of eukaryotic protein synthesis, specifically by inhibiting peptide-bond formation at the initiation stage of translation (Carter et al., 1976) and by inhibiting peptidyl transferase activity required for translational elongation and/or termination (Wei et al., 1974).

Phomenone (2) - The antifungal eremophilane sesquiterpene, phomenone, is produced from Xylaria sp., an endophytic fungus associated with Piper aduncum, a tree of the pepper family from the New World. Phomenone has been claimed to have antifungal activity against Cladosporium cladosporioides (wheat pathogen) and C. sphaerospermum (common indoor mold) though supporting evidence was not presented (Silva et al., 2010). Phomenone is also a phytotoxin and is assumed to be a causal agent of wilted leaves in tomato; citing unpublished data, the authors suggested that phomenone induces electrolyte loss and dysfunction of cell membrane permeability (Capasso et al., 1984). Phomenone is structurally similar to the sesquiterpene eremophilane ring system of the PR toxin produced by Penicillium roqueforti, which inhibits RNA polymerase and

39 protein synthesis at the initiation step as well as elongation (Moule et al., 1976). Phomenone has been used as a natural precursor for synthesis of anticancer ester drugs (Weerapreeyakul et al., 2007).

8α-acetoxyphomadecalin C (3) and phomadecalin E (4) – These eremophilane sesquiterpenes were obtained from the endophytic fungus, Microdiplodia sp. KS 75-1, from the stems of conifer trees (Pinus sp.). The compounds showed moderate antibiosis activities on agar assays against the pathogen Pseudomonas aeruginosa (ATCC 15442) (Hatakeyama et al., 2010), a standard strain used to evaluate bactericidal disinfectants. Phomadecalin C was also isolated from cultures of Phoma sp. (NRRL 25697), a fungus originally isolated from wood decay stromata (Che et al., 2002). Phomadecalin C was shown to be antagonistic to Bacillus subtilis (ATCC 6051) in standard disk assays (Che et al., 2002).

Cycloepoxylactone (5) and cycloepoxytriol B (6) – These compounds were purified from cultures of the endophytic fungus Phomopsis sp. (Valsaceae), isolated from the leaves of Laurus azorica (Lauraceae), a laurel tree from the Canary Island of Gomera. Cycloepoxylactone has been shown on agar plates to inhibit the growth of an anther smut fungus (Microbotryum violaceum) and a soil inoculant bacterium (Bacillus megaterium), while cycloepoxytriol B also inhibited growth of an alga (Chlorella fusca) (Hussain et al., 2009).

3,12-dihydroxycadalene (7) – This was one of five cadinane sesquiterpenes isolated from Phomopsis cassiae collected from Cassia spectabilis (Senna spectabilis), a tree from tropical America belonging to the legume family Fabaceae. This compound showed potent antifungal activity against the phytopathogenic fungi Cladosporium cladosporioids and C. sphaerospermum using a TLC-based assay (Silva et al., 2006).

1α-10α-epoxy-7α-hydroxyeremophil-11-en-12,8-β-olide (8) - This compound, structurally related to eremophilanolide sesquiterpenes, was obtained from Xylaria sp. BCC 21097, isolated from the palm Licuala spinosa (Isaka et al., 2010). The compound was active against Candida albicans (causative agent of human genital and oral infections) and exhibited activity against the malaria parasite Plasmodium falciparum using a microculture radioisotope assay in which failure of the parasite to uptake radiolabelled nucleic acid precursors indicated anti-parasitic activity. The authors hypothesized that this activity may be structurally related to the epoxide moiety (Isaka et al., 2010).

Heptelidic acid (9) and hydroheptelidic acid (10) - These compounds were isolated from Phyllosticta sp., an endophytic fungus inhabiting the needles of Abies balsamea (balsam fir tree) from New Brunswick, Canada (Calhoun et al., 1992). In this region, this tree species is affected by defoliating larvae of spruce budworm (Choristoneura fumiferana). The endophyte-derived compounds were shown to be toxic to these larvae (Calhoun et al., 1992) which have caused hundreds of millions of dollars in losses to the Canadian forestry sector (Chang et al., 2012).

5-hydroxy-2- (1-oxo-5-methyl-4-hexenyl)benzofuran (11) and 5-hydroxy-2-(1-hydroxy-5- methyl-4-hexenyl) benzofuran (12). These two new benzofuran-carrying normonoterpene derivatives were isolated from cultures of an unidentified endophytic fungus obtained from Gaultheria procumbens, a ground cover plant that grows between Canadian forest trees infected by spruce budworm, as noted above. These endophyte-derived compounds were found to be toxic to the cells and/or larvae of spruce budworm (Findlay et al., 1997).

Chokols (13) – These compounds were isolated from Epichloë typhina, an endophyte of Phleum pratense (perennial Timothy-grass). The compounds were found to be fungitoxic to the leaf spot disease pathogen Cladosporium phlei (Yoshihara et al., 1985).

Figure 3.1: Structures of sesquiterpene derivatives of fungal endophyte origin (1-13)

3.3.1.2 Diterpenes:

The structures of diterpenes and triterpenes derivatives discussed here are illustrated (Figure 3.2).

Paclitaxel (14) - The anti-cancer and antifungal drug paclitaxel (Taxol) was reported to be produced from the endophytic fungus Taxomyces andreanae originally isolated from the inner bark of the yew tree Taxus brevifolia in northwestern Montana (Stierle et al., 1993; Stierle et al., 1995). Paclitaxel is reported to be produced by ~20 different endophyte species inhabiting different plant species (Zhou et al., 2010b). Paclitaxel possesses a unique chemical structure composed of a taxane ring with a four-membered oxetane ring and a C-13 ester side chain. Paclitaxel acts by stabilizing microtubules and inhibiting spindle function leading to disruptions in normal cell division (Horwitz, 1994). Paclitaxel is derived from geranylgeranyl diphosphate (GGDP), a compound synthesized from dimethyl diphosphate and indole pyrophosphate, catalyzed by GGDP synthase (Eisenreich et al., 1996). Cyclization of GGDP to taxa-(4,5),(11,12)-diene is catalyzed by taxadiene synthase (TS) which represents the first step in paclitaxel biosynthesis (Hezari et al., 1995). We have independently confirmed the presence of a protein of the expected molecular weight (110 kDa) of TS in a paclitaxel-producing fungal endophyte using a plant anti-TS antibody (S. Soliman and M. Raizada, unpublished data). Interestingly, using selective chemical inhibitors and genetic studies, we recently showed that endophytic paclitaxel may be derived from both mevalonate and non-mevalonate pathways (Soliman et al., 2011), a surprising result since non-mevalonate pathway enzymes have only been shown to exist in bacteria and plastids but not fungi. A curiosity remains as to why both fungal endophytes and their host plants produce the same compound, apparently redundantly. Isolation of the genes responsible for fungal paclitaxel biosynthesis may help to reveal whether these two pathways evolved from convergent evolution or parallel evolution.

Periconicins A (15) and B (16) – These fusicoccane diterpenes were identified from the endophytic fungus Periconia sp. collected from the inner bark of Taxus cuspidata by

43 bioassay guided fractionation (Kim et al., 2004). Periconicin A was found to be a more potent antimicrobial agent than periconicin B against Bacillus subtilis, Klebsiella pneumoniae, and the opportunistic human pathogen Proteus vulgaris (ATCC 3851) using a microtiter broth dilution method (Kim et al., 2004). Periconicin A was also reported to have potent anti-fungal activity against the human pathogens Candida albicans, Trichophyton mentagrophytes (causative agent of cutaneous infections) and T. rubrum (causative agent of jock itch, athlete’s foot and ringworm) (Shin, 2005). Periconicins having the same carbon skeleton as fusicoccins, which are a group of plant growth regulators resembling the major plant hormone gibberellins (de Boer and Leeuwen, 2012). Though many anti-fungal compounds have adverse effects on plant growth, fusicoccins have the potential to remove seeds from dormancy, stimulate seed germination, open leaf stomata and promote plant growth by cell elongation, though these effects vary by crop species and dosage (Muromtsev et al., 1994; Shin, 2005; de Boer and Leeuwen, 2012).

Sordaricin (17) – This pimarane diterpene is the aglycon of sordarin and was purified from the fungi Xylaria sp. isolated from the leaves of Garcinia dulcis, a tropical fruit tree of southeast Asia (Pongcharoen et al., 2008). The compound exhibited moderate antifungal activity against Candida albicans ATCC90028 using an agar diffusion assay (Pongcharoen et al., 2008). C. albicans is an important pathogen of human immunocompromised patients. Sordarin was previously shown to inhibit fungal protein synthesis by selectively binding and inhibiting elongation factor 2 (EF-2) which catalyzes ribosomal translocation during translation (Justice et al., 1998). Replacement of the sugar moiety of sordarin with alkyl side chains increased the antifungal activity against yeast in a manner proportional to the lipophilicity of the alkyl side chain (Tse et al., 1998). Introduction of oxime moities to sordarin could increase antifungal activity against C. albicans and C. glabrata, perhaps by altering the spatial orientation of the lipophilic side chain (Serrano-Wu et al., 2002).

Diaporthein B (18) – This pimarane diterpene was purified from the culture broth of the fungus Diaporthe sp. BCC 6140, isolated from unidentified wood in Thailand, and it showed strong inhibition of growth of Mycobacterium tuberculosis using a metabolic indicator colorimetric assay (Alamar Blue) (Dettrakul et al., 2003). The bioassay suggested that the ketone group at position C-7 may be important for the antifungal activity (Dettrakul et al., 2003). This compound may hold promise against the global tuberculosis epidemic, which affects nearly 9 million new patients each year (WHO, 2012).

Guanacastepene (19) - This novel diterpenoid, produced by an unidentified fungus CR115 from a branch of the tree Daphnopsis americana growing in Costa Rica, was shown to have antibacterial activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium (Singh et al., 2000). As the possible mode of action, guanacastepene was reported to damage bacterial membranes (Singh et al., 2000).

Scoparasin B (20) - Structurally related to cytochalasins, this diterpenoid was isolated from the endophytic fungus Eutypella scoparia PSU-D44 inhabiting the leaves of Garcinia dulcis, a tropical fruit tree of Southeast Asia as noted earlier. The compound was shown to have antifungal activity against an important skin pathogen, Microsporum gypseum, using a hyphal-extension inhibition assay (Pongcharoen et al., 2006).

Compound JBIR-03 (21) and Asporyzin C (22) – These compounds, belonging to the tremorgenic mycotoxin indoloditerpenes, were identified from Aspergillus oryzae, an endophyte of the marine red algae Heterosiphonia japonica, a Pacific seaweed which has become invasive to Europe. JBIR-03 exhibited strong insecticidal activity against brine shrimp (Artemia salina), while asporyzin C exhibited potent activities against Escherichia coli (Qiao et al., 2010).

Diterpene CJ-14445 (23) – This compound was isolated from solid cultures of the endophytic fungus Botryosphaeria sp. MHF associated with the leaves of Maytenus hookeri, a medicinal plant containing the potent antitumor agent maytansine (Yuan et al., 2009). The compound inhibited growth of Candida albicans, Saccharomyces cervisiae, and Penicillium avellaneum using standard agar disk diffusion assays (Yuan et al., 2009).

3.3.1.3 Triterpenes:

Helvolic acid (24) – This nordammarane triterpenoid was isolated from the yeast Pichia guilliermondii Ppf9 (asexual form is Candida guilliermondii) that colonizes the Himalayan lily-family medicinal plant Paris polyphylla (Zhao et al., 2010). Helvolic acid was reported to exhibit strong antibacterial activity using broth dilution. It was also reported to inhibit spore germination of Magnaporthe oryzae, the causative agent of rice blast disease (Zhao et al., 2010), one of the most damaging diseases of rice. In Aspergillus fumigatus, genes encoding helvolic acid are clustered together in the sub- telomere chromosome region (Lodeiro et al., 2009), suggesting that the pathway may have been derived from horizontal gene transfer, possibly from bacteria. In A. fumigatus, a major human pathogen, evidence was presented that the helvolic acid gene cluster may be transcriptionally regulated by the major virulence-controlling transcription factor LaeA (Bok et al., 2005).

Figure 3.2: Structures of diterpene and triterpene derivatives of fungal endophyte origin (14-24).

3.3.1.4 Steroids:

Structure of steroids compounds discussed here are illustrated (Figure 3.3)

Penicisteroid A (25) – This steroid was isolated from the culture extracts of a fungus, Penicillium chrysogenum, cultured from an unidentified marine red algal species belonging to the genus Laurencia. Penicisteroid A showed potent antifungal activity against Aspergillus niger (plant black mould) and moderate activity against Alternaria brassicae (pathogen of Brassica plants such as cabbage) (Gao et al., 2011).

Steroids 3β,5α-dihydroxy-6β-acetoxy-ergosta-7,22-diene (26), 3β,5α-dihydroxy-6β- phenylacetyloxy-ergosta-7,22-diene (27), 3β-hydroxy-ergosta-5-ene (28) , 3-oxo-ergosta- 4,6,8(14),22-tetraene (29), 3β-hydroxy-5α,8α epidioxy-ergosta-6,22-diene (30) – These compounds were isolated from the liquid culture of a fungal endophyte Colletotrichum inhabiting the stems of Artemisia annua (Lu et al., 2000), a traditional Chinese medicinal herb. The authors reported that all the compounds except (30) have antifungal activity against several crop pathogens including Phytophthora capisici, Gaeumannomyces graminis, Rhizoctonia cerealis and Helminthosporium sativum. All of the compounds except (28) also showed antibacterial activity against Pseudomonas sp., Bacillus subtilis, Sarcina lutea (Micrococcus luteus, a human skin pathogen) and Staphylococcus aureus, and antifungal activity against Aspergillus niger and Candida albicans (Lu et al., 2000).

Figure 3.3: Structures of additional terpenoid derivatives of fungal endophyte origin (25-30).

3.3.2 Alkaloids The structures of the alkaloidal derivatives described in this review are summarized (Figure 3.4).

3.3.2.1 Amines and amides: Peramine (31) – This pyrrolopyrazine alkaloid was characterized from perennial ryegrass (Lolium perenne L.) produced by the endophytic fungus Acremonium lolii (Rowan, 1993) famous for its production of loline alkaloids (described later). Ryegrass infected with A. lolii or purified peramine were shown to be potent anti-feedants of Argentine stem weevil without negatively impacting mammals (Rowan, 1993). Peramine levels were highest in young leaves, and peramine has been identified in a number of grass genera (Rowan, 1993). In Arizona fescue infected with another fungal endophyte, Neotyphodium, peramine concentrations were observed to differ between plant genotypes inhabited by the same endophyte haplotype, suggesting that plant genotype plays a major role in regulating this secondary metabolite (Faeth et al., 2002).

Ergot alkaloids (32) – These compounds are produced within different species of the grass subfamily Pooideae by sexual Epichloe fungi and their asexual derivatives belonging to the genus Neotyphodium within the Clavicipitaceae family (Schardl, 2010). These metabolites can act as antifeedants and/or toxins against insects, nematodes and mammalian herbivores (Powell and Petroski, 1992; Wallwey and Li, 2011). Related ergot alkaloid-producing fungal parasites (especially Claviceps purpurea) of animal grass feed (e.g. tall fescue) have been shown to cause toxicity to livestock, in particular ergovaline (Powell and Petroski, 1992). The first committed step in ergovaline biosynthesis is the prenylation of L-tryptophan with dimethylallylpyrophosphate (DMAPP) to produce 4- dimethylallyltryptophan (4-DMAT), catalyzed by the enzyme DMAT synthase (Heinstein et al., 1971). In total, ergot alkaloid biosynthesis has been shown to involve 14 co-expressed genes which are arranged in a chromosomal cluster in C. purpurea (Wallwey and Li, 2011). Seven of these genes are conserved across different fungi and

50 are thought to be responsible for the biosynthesis of the tetracylic ergoline scaffold. Interestingly, in Epichloe festucae, the genes for ergovaline biosynthesis were only highly expressed during biotrophic growth of the fungus within the plant not when the mycelia were cultured separately, suggesting that the plant was needed to induce expression of the fungal gene cluster (Wallwey and Li, 2011). For more details about ergot alkaloids, please refer to excellent recent reviews on the subject (Tudzynski et al., 2001; Schardl, 2010; Wallwey and Li, 2011).

Phomopsichalasin (33) – This compound is a unique cytochalasan derivative with a novel ring system involving an isoindolone moiety fused to a 13-membered tricyclic system. Phomopsichalasin was isolated from an endophytic Phomopsis sp. originating from the twigs of the willow shrub, Salix gracilostyla (Horn et al., 1995). Cytochalasans are well known fungal metabolites that can bind actin (Binder and Tamm, 1973) and have been shown to have anti-bacterial, anti-fungal, anti-viral and anti-inflammatory activities (Pendse and Mujumdar, 1986). Phomopsichalasin was shown to be antibacterial against Bacillus subtilis, Staphylococcus aureus and Salmonella gallinarum (poultry pathogen), and antifungal against the human pathogenic yeast Candida tropicalis (Horn et al., 1995).

Phomoenamide (34) – This compound was detected in cultures of an endophytic Phomopsis sp. fungus obtained from the leaves of Garcinia dulcis, an Indonesian tropical fruit tree. The compound has been shown to have antimicrobial activity against Mycobacterium tuberculosis using the Alamar Blue Assay (Rukachaisirikul et al., 2008).

Cryptocin (35) – This tetramic acid analogue was purified from the endophytic fungus Cryptosporiopsis quercina which inhabits the inner bark of the stems of Tripterygium wilfordii, a Chinese medicinal plant used to treat rheumatoid arthritis. Cryptocin has antifungal activity against a wide range of plant pathogens including Pyricularia oryzae, the

51 fungus causing rice blast disease (Li et al., 2000), one of the most devastating crop diseases worldwide.

Pestalachloride A (36) – This compound was purified from Pestalotiopsis adusta, an endophytic fungus that inhabits the stem of an unknown Chinese tree; the compound showed antifungal activity against the plant pathogens, Fusarium culmorum, Gibberella zeae (anamorph Fusarium graminearum) and Verticillium albo-atrum (Li et al., 2008a). The authors classified this compound as a new chlorinated benzophenone alkaloid belonging to the amine and amide subclass. The authors further noted that pestalachloride A differs significantly from other known alkaloids by having a somewhat rare 2,4- dichloro-5-methoxy-3-methylphenol moiety attached to the isoindolin-1-one core.

3.3.2.2 Indole derivative

Loline alkaloid (37) - This indole derivative, a saturated 1-aminopyrrolizidine with an oxygen bridge, was detected in the grass Festuca pratensis (Lolium pratense) originating from its fungal endophyte, Neotyphodium uncinatum (Blankenship et al., 2001). Loline has broad-spectrum anti-insect and anti-aphid activity resulting in increased resistance of the host plant against insect herbivores (Wilkinson et al., 2000). A fascinating four- species ecological interaction involving loline was reported, in which the loline- producing endophytic fungus (Neotyphodium uncinatum) inhabiting its host grass protects a non-host plant (Rhinanthus serotinus, a parasite of the host grass of the endophyte) against aphids (Aulacorthum solani) (Lehtonen et al., 2005). In N. uncinatum, two homologous gene clusters encoding loline were identified, named LOL-1 and LOL- 2, with LOL-1 containing nine genes within a 25-kb chromosomal segment; as the genes between the two clusters were generally in the same order and orientation, these clusters likely represent relatively recent duplications (Spiering et al., 2005). Based on the identification of these genes, a biosynthetic route for loline was proposed involving the precursors proline and homoserine (Spiering et al., 2005).

Neotyphodium is the asexual form, but the sexual derivative, Epichloe, can also produce loline. Epichloe fungi can be pathogenic to host plant inflorescences and are horizontally transmitted by re-infection, whereas Neotyphodium are mutualistic and are transmitted through spores vertically on healthy inflorescences (Schardl, 2010). Expression of the loline biosynthetic genes has recently been used to better understand the pathogenic versus mutualistic forms. In contrast to plants infected by pathogenic Epichloe fungus, the fungal loline biosynthetic genes were upregulated in inflorescences of healthy plants inhabited by the mutualistic (Neotyphodium), suggestive of evolutionary selection on the endophyte for increased expression of genes encoding this beneficial insecticide (Zhang et al., 2010).

Figure 3.4: Structures of alkaloidal derivatives of fungal endophyte origin (31-37).

3.3.3 Phenolic compounds The structures of these phenolic compounds are described below and summarized (Figure 3.5) (Simple structures are not shown).

3.3.3.1 Phenols and phenolic acids: 2-Methoxy-4-hydroxy-6-methoxymethylbenzaldehyde (38) – This phenolic compound was shown to be antifungal against the cucumber phytopathogen Cladosporium cucumerinum using an antifungal TLC assay. The compound is produced by Pezicula strain 553, a fungal endophyte colonizing an unknown tree (Schulz et al., 1995).

P-hydroxybenzoic acid (39), p-hydroxyphenylacetic acid (40), tyrosol (41), p-coumaric acids (42) – These antifungal phenolic acids were purified from the stromata of Epichloe typhina, which can be a symptomless endophyte, but can also act as a pathogen against its host Phleum pratense (European Timothy-grass) (Koshino et al., 1988).

Colletotric acid (43) – This tridepside compound was characterized from the liquid culture of Colletotrichum gloeosporioides, a fungus that colonizes the stems of Artemisia mongolica, an Asian plant which shows resistance to insects and pathogens. The compound was shown to have antimicrobial activity against the bacteria Bacillus subtilis, Staphylococcus aureus and Sarcina lutea (Micrococcus luteus), and the fungus Helminthosporium sativum (Zou et al., 2000), the latter being a seedling blight and root rot pathogen of cereals.

Cytonic acids A (44) and B (45) – Three novel isomeric tridepsides (p-tridepside isomers) were obtained from Cytonaema sp, an endophytic fungus of Quercus sp. (oak tree). These compounds showed inhibitory activity against the opportunistic human pathogen cytomegalovirus by inhibiting a protease required for normal assembly of the viral nucleocapsid (Guo et al., 2000).

Altenusin (46) - This biphenyl fungal metabolite was isolated from Alternaria sp. (UFMGCB55), an endophyte of the Asteraceae family plant Trixis vauthieri, collected from a natural preserve in Minas Gerais, Brazil (Cota et al., 2008). This plant was investigated because it was known to contain compounds active against the human protozoan parasites Trypanosoma and Leishmania, which infect millions of people worldwide. Altenusin was shown to inhibit trypanothione reductase (TR), an enzyme required to protect these parasites against oxidative stress, though the compound itself did not diminish parasite viability perhaps due to an inability to traverse intracellular compartments (Cota et al., 2008). Altenusin was reported to have antifungal activity against clinical isolates of Paracoccidioides brasiliensis, which causes human Paracoccidioidomycosis, perhaps by inhibiting cell wall synthesis or assembly (Johann et al., 2012).

3.3.3.2 Isocoumarin derivatives: (R)-Mellein (47) - This isocoumarin was purified from Pezicula livida (strain 1156), an endophytic fungus isolated from the European beech tree Fagus sylvatica growing in Lower Saxony, Germany. In plate assays, the compound was inhibitory against the bacteria Bacillus megaterium and Escherichia coli, the fungi Ustilago violacea and Eurotium repens, and the alga Chlorella fusca (Schulz et al., 1995). Mellein and 4- hydroxymellein were also shown to be synthesized by Aspergillus ochraceus and show structural similarity to the dihydroisocoumarin moiety of ochratoxin A, one of the most abundant mycotoxins found in food (Moore et al., 1972). Mullein and 3-hydroxylated derivatives were also isolated from Botryosphaeria obtusa, a grapevine pathogen; in grape these compounds are considered potential molecular markers for the presence of pathogenic fungi (Djoukeng et al., 2009).

3.3.3.3 Flavonoids:

Tricin (48) and related flavone glycosides (49) – These flavonoids were isolated from Big Bluegrass (Poa ampla), a perennial grass native to Western North America, infected with

Neotyphodium typhnium, a symbiotic fungus (Ju et al., 1998). However, the compound was not reported from pure fungal cultures. These flavonoids were found to be toxic to the larvae of Culex pipiens (Ju et al., 1998), the common house mosquito which can also act as a vector for West Nile Virus in North America. The researchers initially investigated Big Bluegrass for endophytes producing anti-insecticide after observing that endophyte-free plants were more susceptible to spider mites.

3.3.3.4 Lignans:

Podophyllotoxin (50) – This aryl tetralin lignan, first described in 1880, is today an important anti-cancer drug originally isolated from Podophyllum plant species in both the Himalayas and North America where indigenous peoples used the plant for medicinal purposes (Stähelin and von Wartburg, 1991). Podophyllum has also been shown to have anti-viral and insecticidal properties (Sudo et al., 1998; Gao et al., 2004). More recently, podophyllotoxin was also purified from endophytes inhabiting Podophyllum sp. including the fungus Phialocephala fortinii (Eyberger et al., 2006). Podophyllotoxin is thus another example of a secondary metabolite produced by both an endophyte and its host. Podophyllotoxin was also obtained from Fusarium oxysporum, an endophyte of the medicinal plant Juniperus recurva which also originates from the Himalayan mountains (Kour et al., 2008). Podophyllotoxin production was also reported from Aspergillus fumigatus which is an endophyte of Juniperus communis (Kusari et al., 2009). With respect to its anti-cancer activity, podophyllotoxin and its derivatives have been shown to prevent mitosis in late S/early G2 phase by binding to and inhibiting the enzyme (topoisomerase II) required to unwind the double helix of DNA (Canel et al., 2000). The anti-viral activity of Podophyllotoxin appears to be due to its ability to disrupt viral replication and inhibit reverse transcriptase (Canel et al., 2000).

Figure 3.5: Structures of phenylpropanoid derivatives of fungal endophyte origin (43-50).

3.3.4 Aliphatic compounds

The structures of the aliphatic derivatives described in this review are illustrated (Figure 3.6).

Brefeldin A (51) – Brefeldin A has become an important research chemical used by cell biologists: Brefeldin A blocks the transport of proteins from the endoplasmic reticulum to the Golgi apparatus resulting in inhibition of secretion (Misumi et al., 1986). An early report showed that this compound could be isolated from cultures of the endophyte Eupenicellium brefeldianum (Harri et al., 1963). During the last 50 years, there have been additional reports of endophytes of different hosts producing breveldin A, including Paecilomyces sp. and Aspergillus clavatus, inhabitants of the conifer trees Taxus mairei and Torreya grandis, respectively, from southeast China (Wang et al., 2007). Brefeldin A has been shown to have anti-bacterial, anti-viral, anti-nematode and anti-fungal activities (Betina, 1992) including against the fungi Aspergillus niger, Candida albicans and Trichophyton rubrum (Wang et al., 2007).

Pestalofones C (52) and E (53) - These cyclo hexanone derivatives were isolated from cultures of Pestalotiopsis fici, an endophytic fungus that colonized the branches of an unidentified tree in Hangzhou, China. The compound showed antifungal activity against Aspergillus fumigates (Liu et al., 2009). A. fumigates causes invasive lung diseases that may cause mortality especially in immune-compromised people.

Gamahonolide A (54) and B (55) - These compounds were characterized from stromata of Epichloe typhina growing on Phleum pretense (Timothy grass). Gamahonolide A showed antifungal activity against Cladosporium herbarum, the fungal plant pathogen and common inhalant allergen of humans, using antifungal- guided isolation (Hiroyuki et al., 1992).

Figure 3.6: Structures of aliphatic derivatives of fungal endophyte origin (51-55).

3.3.5 Polyketides The structures of the polyketide derivatives noted below are summarized (Figure 3.7).

6-O-methylalaternin (56) and Altersolanol A (57) - These tetrahydroanthraquinones were isolated from fungal cultures of Ampelomyces sp. (Leptosphaeriaceae), isolated from the medicinal plant Urospermum picroides (Asteraceae), collected in Egypt (Aly et al., 2008b). Ampelomyces were among the first fungi used as biocontrol agents of plant parasitic fungi (Yarwood, 1932). Both quinone compounds exhibited antibacterial activity against Enterococcus faecalis, Staphylococcus aureus and S. epidermidis (Aly et al., 2008b).

Altersolanol A along with other derivatives were also isolated from the fungus Alternaria solani responsible for black spot disease of Lycopersicon esculentum (tomato) (Okamura et al., 1993). In the latter study, the compound was found to inhibit growth of the bacteria Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Escherichia coli and Pseudomonus aeruginosa and the fungi, Candida albicans and Candida utilis (Okamura et al., 1993). With respect to its antibacterial properties, Altersolanol A was reported to interfere with the respiratory chains of bacterial membranes by acting as an electron acceptor (Haraguchi et al., 1992).

Palmarumycin CP17 (58) and palmarumycin CP18 (59) – These new antiparasitic natural products with pentacyclic spiroketal structures were isolated from an Edenia sp. (Pleosporaceae) fungus, obtained from mature leaves of Petrea volubilis (Verbenaceae), a tropical woody vine collected in Coiba National Park, Panama ( art ne -Luis et al., 2008). The compounds have an unusual structural feature involving two or three oxygen atoms which act as bridges between two original naphthalene subunits (Zhou et al., 2010a). These metabolites were shown to significantly inhibit growth of the amastigote form of the protozoan parasite Leishmania donovani ( art ne -Luis et al., 2008), a genus considered to be the second largest global parasitic killer of humans after malaria. The

61 palmarumycins were also shown to have antineoplastic effects in mammalian cells by inhibiting the G2/M transition of the cell cycle through an unknown mechanism (Lazo et al., 2001).

Rugulosin (60) – This bis-anthraquinoid pigment was isolated using extracts of Hormonema dematioides, an endophytic fungus of Canadian balsam fir trees, using bioassay-guided fractionation for inhibition of growth of spruce budworm (Calhoun et al., 1992). Conifer needles infected with the endophyte were associated with reduced weight gain of spruce budworm larvae when used as feed (Miller et al., 2002). Rugulosin has also been reported to be produced by various other fungal species including Penicillium (Ueno et al., 1980). Rugulosin is cytotoxic to both prokaryotes and eukaryotes and causes fatty degeneration, liver cell necrosis and to a lesser extent hepatocarcinogenesis to mice and rats (Ueno et al., 1980). For these reasons, this compound is an important mycotoxin in yellow rice consumed in Asia (Chu, 1977).

Nodulisporins - Nodulisporium sp. fungal endophytes (Xylariaceae) isolated from the endangered plant Juniperus cedrus (Canary Island Juniper, a gymnosperm) yielded nodulosporins A-C (61-63) which exhibited antifungal activity against Microbotryum violaceum (Dai et al., 2006). Interestingly, the same endophyte was isolated from an Angiosperm shrub, Erica arborea, from the Canary Island of Gomera from which related compounds were isolated called nodulisporins D- F (64-66) (Dai et al., 2009). Nodulisporins D- F showed antibacterial activity against Bacillus megaterium, antifungal activity against Microbotryum violaceum (anther smut fungus) and anti-algal activity against Chlorella fusca, using agar diffusion assays.

Pyrrocidines A (67) and B (68) - These polyketide-amino acid-derived antibiotics were isolated from an endophyte of maize kernels, Acremonium zeae, a fungus which protects pre-harvest kernels against fungal pathogens, perhaps by competing for the same host niche in a temperature-dependent manner (Wicklow et al., 2005). Pyrrocidines displayed

62 significant antifungal activity on agar disc assays against mycotoxin-producing Aspergillus flavus and Fusarium verticillioides (Wicklow et al., 2005). The authors reviewed previous studies which reported that pyrrocidine A inhibits growth of several Gram-positive bacteria. The study noted that A. zeae has sometimes been implicated as causing stalk rot and hence is selected against by breeders and pathologists, perhaps making commercial maize more susceptible to pathogens. In a subsequent study, pyrrocidine A showed potent activity against important ear rot and stalk rot pathogens of maize, including Nigrospora oryzae, Fusarium graminearum, Rhizoctonia zeae and Stenocarpella (Diplodia) maydis (Wicklow and Poling, 2009). The authors suggested that pyrrocidine A may protect vulnerable seedlings, in particular against pathogens, following colonization of the seedling by the endophyte from the seed. Pyrrocidine A also showed anti-pathogen activity against the seed-rot saprophytes Eupenicillium ochrosalmoneum and A. flavus as well as against the causal agent of fungal leaf spot disease, Curvularia lunata, and the bacteria Clavibacter michiganense subsp. nebraskense, the causative agent of Goss’s wilt (Wicklow and Poling, 2009). Other non- disease causing protective fungal endophytes were not as sensitive to pyrrocidines suggestive of evolutionary selection for fungal endophyte compatibility (Wicklow and Poling, 2009). However, pyrrocidin A showed antibiosis activity against two bacterial endophytes of maize used as biological control agents, Bacillus mojaviense and Pseudomonas fluorescens (Wicklow and Poling, 2009).

Isofusidienol A to D (69-72) – These chromone-3-oxepine-polyketides were isolated from the fungus Chalara sp. (strain 6661), an endophyte of Artemisia vulgaris, an herb known as Mugwort which grows along the Baltic Sea coast. These compounds exhibited antifungal activity against the pathogenic yeast Candida albicans and antibacterial activity against Bacillus subtilis, Staphylococcus aureus and Escherichia coli of which isofusidienol A was the most potent using agar disc assays (Lösgen et al., 2008).

Chaetoglobosins A (73) and C (74) – These chlorinated azaphilone derivatives were characterized from cultures of the fungal endophyte Chaetomium globosum isolated from the leaves of Ginkgo biloba. These compounds exhibited significant toxicity towards brine shrimp larvae and antifungal activity against the industrial microbe Mucor miehei (Qin et al., 2009) a fungus used for the production of enzymes used in the cheese industry.

Chaetomugilin A (75) and D (76) - These azaphilone derivatives were also isolated from the fungal endophyte C. globosum as noted above. The compounds exhibited inhibitory activity against brine shrimp larvae (Qin et al., 2009).

Pestalotheol C (77) - This compound was isolated from the endophytic fungus Pestalotiopsis theae which inhabits the branches of an unidentified tree in Hainan Province, China. In its pathogenic form, this fungus causes Tea Gray Blight disease. The compound showed inhibitory activity against HIV replication based on ELISA assays (Li et al., 2008b).

CR377 (78) - This pentaketide was obtained from a Fusarium sp., an endophytic fungus living inside the stems of Selaginella pallescens (a resurrection plant given its ability to recover from severe dehydration), collected from the Guanacaste Conservation Area of Costa Rica. The compound exhibited antifungal activity against Candida albicans using an agar diffusion assay with an inhibition zone similar to the fungicide nystatin (Brady and Clardy, 2000).

Xanalteric acids I (79) and II (80) - These compounds were purified from an Alternaria sp. fungus, an endophyte isolated from the leaves of the Chinese mangrove plant Sonneratia alba. This plant is known as Mangrove Apple, an edible salt-tolerant plant

64 eaten by humans and camels in Africa and the Pacific, but also used as a traditional herb against skin or intestinal parasites. These compounds exhibited weak antibiotic activity against Staphylococcus aureus (Kjer et al., 2009).

Pestalachloride B (81) – This compound was isolated from Pestalotiopsis adusta, an endophytic fungus isolated from the stem of an unknown tree in China; the compound displayed significant antifungal activity against three important plant pathogens, Fusarium culmorum, Gibberella zeae (anamorph Fusarium graminearum) and Verticillium albo-atrum (Li et al., 2008a).

Figure 3.7: Structures of polyketide derivatives of fungal endophyte origin (56-66).

Figure 3.7: Continued (67-81).

3.3.6 Peptides The structures of the peptide derivatives discussed in this review are illustrated (Figure 3.8).

Leucinostatin A (82) - This compound was isolated from cultures of Acremonium sp., an endophytic fungus that colonizes Taxus baccata (European yew), an evergreen conifer tree. The compound was shown to have anti-cancer activities and could act as a fungicide against the oomycete Pythium ultimum (Strobel et al., 1997), an important plant pathogen that causes damping-off and root rot diseases.

Echinocandin A (83) - This lipopetide was purified from cultures of the endophytic fungi Cryptosporiopsis sp. and Pezicula sp., inhabitants of Pinus sylvestris (Scots pine) and Fagus sylvatica (European beech), respectively. The compound showed antifungal activity against Candida albicans and Saccharomyces cerevisiae (Noble et al., 1991). Mechanistically, echinocandins were found to inhibit the synthesis of cell wall glucans by inhibiting glucan synthase leading to cell lysis (Chapman et al., 2008).

Cryptocandin (84) – This lipopeptide, related chemically to echinocandin, was isolated from the fungus Cryptosporiopsis quercina, an endophyte of the Chinese medicinal plant Tripterigeum wilfordii (Strobel et al., 1999b). Cryptocandin was shown to have antifungal activity against multiple human pathogens including Candida albicans and Histoplasma capsulatum (causal agent of the lung disease Histoplasmosis), in addition to Trichophyton rubrum and T. mentagrophytes (Strobel et al., 1999b) – the latter two fungi cause skin and nail diseases in humans. The compound was also shown to inhibit the growth of phytopathogenic fungi including Sclerotinia sclerotiorum, the fungus that causes white mold disease which affects over 400 plant species, and Botrytis cinerea, a necrotic fungus that primarily affects grapes (Strobel et al., 1999b).

Figure 3.8: Structures of peptides derivatives of fungal endophyte origin (82-84)

3.4 Conclusions and future directions The objective of this paper was to review the diversity of secondary metabolites with anti-microbial activities produced by endophytic fungi, from the interdisciplinary perspectives of biochemistry, genetics, fungal biology, host plant biology, human and plant pathology. This review covered ~80 compounds with diverse activities against plant and human pathogens, produced from a wide taxonomic diversity of endophytes inhabiting a range of plant species. Several major themes are apparent from the literature. With respect to biochemistry, fungal endophytes are able to produce almost all chemical classes of secondary metabolites, with terpenoids and polyketides being apparently the most common, and flavonoids and lignans being the rarest. Where endophytes have been investigated in depth biochemically such as Neotyphodium typhnium, many compounds have been identified, suggesting that significant numbers of secondary metabolites remain to be discovered from less explored or unexplored endophytes. With respect to the genetic studies reviewed here, genes of fungal endophytes encoding anti-microbial secondary metabolites were observed to be clustered on chromosomes (e.g. loline alkaloids, ergot alkaloids, helvolic acid). Combined with the observation that the same secondary metabolite can be produced by different genera of endophytic fungi (e.g. paclitaxel, brefeldin A, echinocandin), this genetic clustering may have facilitated horizontal gene transfer of secondary metabolic pathways between fungal species during evolution. With respect to fungal biology, a complexity of this field of study is that fungi can exist in both sexual and asexual forms and can sometimes switch between endophytic and pathogenic lifestyles (e.g. Neotyphodium typhnium/Epichloe typhina), with each type potentially producing different classes of secondary metabolites. Finally, with respect to the biology of the host plants, angiosperms, gymnosperms and lower plants were all found to be inhabited by fungal endophytes that could combat specific pathogens. However, treatment of crops with fungicides may be reducing natural fungicides by killing protective fungal endophytes of the host (e.g. pyrrocidine A produced from Acremonium zeae). Often host plants that were investigated for endophyte-derived anti- microbial compounds were Chinese herbal medicinal plants or plants associated with indigenous knowledge concerning their anti-microbial properties (e.g. cryptocandin). Surprisingly, in some cases, both fungal endophytes and their host plants were reported to

70 produce the same complex secondary metabolites, apparently redundantly (e.g. podophyllotoxin, paclitaxel). I conclude that fungal endophytes are potentially vital sources for natural products for agriculture, medicine and industry, with significant potential to combat global crop and human pathogens which are becoming increasingly resistant to drugs and pesticides.

Despite the apparent progress, significant gaps remain in this field of research from an interdisciplinary perspective. Biochemically, many biosynthetic pathways and enzymes remain unidentified. How the endophyte and the host plant coordinate metabolic biosynthesis remains unexplored. For example, when both the plant and its endophyte produce the same secondary metabolite (e.g. paclitaxel), does the biosynthesis of this metabolite occur independently or is their signalling across organisms (e.g. feedback inhibition)? Furthermore, very little is known about the intracellular location of biosynthesis or mode of secretion of these anti-microbial compounds. Genetically, very few genes encoding the relevant biosynthetic enzymes have been isolated, and there is limited research as to how the expression of these genes is regulated at the molecular level, with only a few exceptions (e.g. Ergot alkaloids). Concerning fungal biology, factors which trigger the endophyte to change from mutualism to parasitism, along with associated changes in secondary metabolite production, remain poorly investigated. Concerning host plant biology, the literature appears to be biased for sampling endophytes from leaf, stem and seed tissues compared to flowers, fruits and roots. With respect to understanding the activities of these endophytes, there is limited information about structure-function relationships, specifically identifying moieties which can enhance and/or reduce the toxicity of the compound. Moreover, reducing the general toxicity of the compound may have positive effects on human health and natural ecosystems. Often anti-pathogen data is from in vitro studies only; however results from the natural environment may be different. Studies on anti-microbial activities of endophytes are often limited to a few model species including human pathogens with limited reports of plant viruses as targets. A better understanding of the contextual ecology of the host plant may be critical since identifying pathogens which inhabit the

71 host may provide clues as to the specific anti-pathogenic targets of the endophyte under investigation (Acremonium zeae produces pyrrocidines A to combat Aspergillus flavus). In conclusion, a more comprehensive understanding of the biochemistry, genetics and biology of endophyte and host, may lead to new opportunities for developing bio-based commercial products to combat global crop and human pathogens.

Chapter 4: Biodiversity of genes encoding anti-microbial traits within plant associated microbes

Authors’ contributions

Walaa K Mousa wrote the manuscript.

Dr. Manish N. Raizada edited the manuscript.

Publication status: published.

Mousa WK and Raizada MN (2015). Biodiversity of genes encoding anti-microbial traits within plant associated microbes. Frontiers in Plant Science 6: 231

4.1 Abstract

The plant is an attractive versatile home for diverse associated microbes. A subset of these microbes produce a diversity of anti-microbial natural products including polyketides, non-ribosomal peptides, terpenoids, heterocylic nitrogenous compounds, volatile compounds, bacteriocins and lytic enzymes. In recent years, detailed molecular analysis has led to a better understanding of the underlying genetic mechanisms. New genomic and bioinformatic tools have permitted comparisons of orthologous genes between species, leading to predictions of the associated evolutionary mechanisms responsible for diversification at the genetic and corresponding biochemical levels. The purpose of this review is to describe the biodiversity of biosynthetic genes of plant- associated bacteria and fungi that encode selected examples of antimicrobial natural products. For each compound, the target pathogen and biochemical mode of action are described, in order to draw attention to the complexity of these phenomena. I review recent information of the underlying molecular diversity and draw lessons through comparative genomic analysis of the orthologous genes. I conclude by discussing emerging themes and gaps, discuss the metabolic pathways in the context of the phylogeny and ecology of their microbial hosts, and discuss potential evolutionary mechanisms that led to the diversification of biosynthetic gene clusters.

4.2 Introduction The plant is an attractive versatile home for diverse microbes that can colonize internal plant tissues (endophytes), live on the surface (epiphytes) or in the soil surrounding the root system (rhizosphere microbiota) (Barea et al., 2005; Johnston-Monje and Raizada, 2011). Plant associated microbes have the potential to be used as biocontrol, the use of living organisms to suppress crop disease (Eilenberg, 2006) through various mechanisms including the production of antibiotics (Compant et al., 2005). Diverse classes of antimicrobial secondary metabolites of microbial origin have been reported (Mousa and Raizada, 2013), including polyketides, non-ribosomal peptides, terpenoids, heterocylic nitrogenous compounds, volatile compounds, bacteriocins as well as lytic enzymes. Polyketides and non-ribosomal peptides constitute the majority of microbial derived natural products (Cane, 1997). Interestingly, the tremendous structural diversity of antimicrobial secondary metabolites originated via limited metabolic pathways utilizing few primary metabolites as precursors (Keller et al., 2005). Underlying the diversification of antimicrobial metabolites must have been a corresponding genetic diversification of ancestral genes driven by co-evolutionary pressures (Vining, 1992).

The revolution in genomics, genome mining tools and bioinformatics offers a new opportunity to connect biochemical diversity to the underlying genetic diversity and to analyze the evolutionary events leading to biodiversity (Zotchev et al., 2012; Scheffler et al., 2013; Deane and Mitchell, 2014).

The scope of this review is to describe the biodiversity of biosynthetic genes of plant-associated microbes (bacteria and fungi) that encode selected examples of antimicrobial secondary metabolites and lytic enzymes. For each example, the target pathogen(s) and mode of action are described where known, in order to highlight the diversity of biochemical targets. Out of necessity, the review focuses on compounds for which in depth molecular analysis has been conducted. I review data pertaining to the underlying molecular diversity and highlight comparative genomic data of the

75 orthologous genes. The review concludes with a discussion of common themes and gaps in the literature, and discusses the role of evolution in the diversification of biosynthetic gene clusters.

4.3 Biosynthetic genes encode diverse chemical classes of anti-microbial compounds The compounds described in this review, the underlying genes, microbes and pathogenic targets are summarized (Tables 4.1 and 4.2).

Table 4.1: Summary of anti-microbial compounds belonging to genetic pathways that show evidence of horizontal gene transfer (HGT).

Compound Class Genes* Producing Pathogen target Significance

microbes (examples) to plant /human (examples) 2,4 -DAFG Phenolic phlD and Pseudomonas Gaeumannomyces Disease suppressive

polyketide phlACB fluorescens1 graminis var. tritici soils of crops

operon Agrocin 84 Nucleotide agn Agrobacterium Agrobacterium sp. Biocontrol of crown 2 analogue operon radiobacter gall

1 Chitinases Lytic See text P. fluorescens Colletotrichum Suppression of crop (family 18) enzymes Actinoplanes falcatum diseases such as lupin missouriensis Plectosporium root rot

Streptomyces sp. tabacinum

Chitinases Lytic See text Green and Various fungal Suppression of crop (family 19) enzymes purple bacteria pathogens diseases

Actinobacteria

Helvolic acid Fusidane See text Aspergillus Magnaporthe oryzae Rice blast disease triterpene fumigates Loline Indole LOL-1 Neotyphodium Insects Protects its host plants

alkaloid and LOL- uncinatum* from insects

2 Epichloe Mupirocin Polyketide mup P. fluorescens1 Staphylococci and Clinical use for human

operon Streptococci

Haemophilus influenzae Neisseria gonorrheae

*Indicates that the genes noted have been studied in this species. A superscript beside the species refers to the recent genome sequence data as indicated below;1P. fluorescens (Martínez-García et al., 2015), 2Agrobacterium radiobacter (Zhang et al., 2014), 3B. cereus (Takeno et al., 2012)

Table 4:1: Continued.

Compound Class Genes* Producing Pathogen target Significance microbes (examples) to plant /human (examples) Paclitaxel Diterpene See text Penicillium Heterobasidion Anticancer aurantiogriseum annosum Phaeolus schweinitzii Perenniporia subacida Phenazines Hetero- phzADEFG P.fluorescens*1 Rhizoctonia solani Combat soil borne cyclic operon P. aeruginosa Gaeumannomyces pathogens compounds Burkholderia graminis var. cepacia tritici

Pantoea Pythium sp. agglomerans Fusarium

(Erwinia oxysporum herbicola) Pyoluteorin Phenolic pltABCDE P. fluorescens1 P. ultimum Damping-off polyketide FG operon disease in cotton Pyrrolnitrin Chlorinated prnABCD P. fluorescens*1 Trichophyton sp. Treatment of skin phenyl- operon Br. pyrrocinia Botrytis cinerea mycoses pyrrole Myxococcus Rhizoctonia solani fulvus G.graminis var. tri Pantoea tic agglomerans Serratia sp.

Zwittermicin Polyketide/ zmaA- B. cereus*3 Phytophthora Contro root-rot in A nonribosom zmaV B. thuringiensis medicaginis alfalfa and

al peptide and kabR, chickpeas hybrid kabA – kabD *Indicates that the genes noted have been studied in this species. A superscript beside the species refers to the recent genome sequence data as indicated below;1P. fluorescens (Martínez-García et al., 2015), 2Agrobacterium radiobacter (Zhang et al., 2014), 3B. cereus (Takeno et al., 2012).

Table 4.2: Summary of anti-microbial compounds belonging to diverse genetic pathways, grouped by different evolutionary levels of diversification.

Compound Class Genes* Example of producing Best studied Significance microbe pathogen target to plant /human Genome level diversification Difficidin Polyketide PKS3 B. amyloliquefaciens*1 Broad spectrum Crop and B. subtilis antibacterial human pathogens Loline Indole LOL-1 Neotyphodium Insects Protects its alkaloid and LOL- uncinatum* host plants 2 Epichloe from insects Intra gene cluster diversification Agrocin 84 Nucleotide agn  Agrobacterium  Agrobacterium Biocontrol of analogue operon radiobacter2 sp. crown gall Polymyxins Non- pmxA-E  Paenibacillus  Klebsiella Limited A, B, D, E ribosomal operon polymyxa3 pneumonia clinical (colistin) and lipopeptide  P. aeruginosa application M (mattacin)  Acinetobacter due to sp. toxicity Trichodermi Terpenoids tri cluster  Trichoderma  Rhizoctonia Crop n and arundinaceum* solani pathogens Harzianum  T. brevicompactum*  Alternaria A solani

Intra CDS diversification Chitinases Lytic See text  P. fluorescens4  Colletotrichum Suppression (family 18) enzymes  Actinoplanes falcatum of crop missouriensis  Plectosporium diseases such  Streptomyces sp. tabacinum as lupin root  Bacillus sp. rot *Indicates that the genes noted have been studied in this species.A superscript beside the species refers to the recent genome sequence data as indicated: 1B. amyloliquefaciens (Kim et al., 2015), 2Agrobacterium radiobacter (Zhang et al., 2014), 3P. polymyxa (Xie et al., 2014), 4P. fluorescens (Martínez-García et al., 2015), 5B. subtilis (Barbe et al., 2009; Belda et al., 2013).

Table 4.2: Continued.

Compound Class Genes* Example of Best studied Significance

producing pathogen target to plant microbe /human

Intra CDS diversification

Ergots Alkaloid See text Epichloe Nematodes, Protect the Neotyphodium insects, and host plant Aspergillus sp. mammalian

A. fumigatus herbivores Penicillium sp.

Claviceps purpurea Fusaricidins Non- fusGFEDCB P. polymyxa3 Aspergillus sp. Control black

ribosomal A operon Fusarium root rot in peptides oxysporum canola

Iturins Non- ituDABC B. subtilis*5 Rhizoctonia Control

bacillomycin ribosomal operon B. solani Gibberella bacillopeptin cyclolipo- amyloliquefacien Fusarium sp. ear rot 1 iturins peptides s* mycosubtilin

Complex dynamic interaction

Jadomycin Polyketide jad operon Streptomyces Methicillin- Clinical use venezuelae resistant S. aureus

Others Bacilysin Non- ywfB-G B. subtilis*5 Candida Clinical use

ribosomal operon and B. pumilus albicans peptide ywfH HCN Volatile hcnABC P. aeruginosa* Thielaviopsis Black root rot

compound operon P. fluorescens basicola of tobacco Phomenone Sesquiterp- See text Xylaria sp. Cladosporium Targets

ene cladosporioides fungal wheat

pathogen * *Indicates that the genes noted have been studied in this species.A superscript beside the species refers to the recent genome sequence data as indicated: 1B. amyloliquefaciens (Kim et al., 2015), 2Agrobacterium radiobacter (Zhang et al., 2014), 3P. polymyxa (Xie et al., 2014), 4P. fluorescens (Martínez-García et al., 2015), 5B. subtilis (Barbe et al., 2009; Belda et al., 2013).

4.3.1 Polyketides The structures of Polyketides described in this review are shown (Figure 4.1)

Diacetylphloroglucinol: 2,4-diacetylphloroglucinol (2,4-DAPG) is a well-studied fluorescent polyketide metabolite produced by many strains of fluorescent Pseudomonas sp. that contributes to disease-suppressive soils of crops (McSpadden Gardener and Thomashow, 2000; Mavrodi et al., 2001). 2,4-DAPG is synthesized by the condensation of three molecules of acetyl coenzyme A and one molecule of malonyl coenzyme A to produce the precursor monoacetylphloroglucinol (MAPG) (Shanahan et al., 1992). In P. fluorescens strain Q2-87, four coding sequences (CDS) within the phl operon are responsible for biosynthesis of 2,4-DAPG: a single CDS (phlD) encoding a type III polyketide synthase is responsible for the production of phloroglucinol from the condensation of three acetyl-CoAs, and then three CDS (phlACB) encoding acetyltransferases are sufficient to convert phloroglucinol to 2,4-DAPG via MAPG (Bangera and Thomashow, 1999; Yang and Cao, 2012). It was suggested that the peptides encoded by phlACB may exist as a multi-enzyme complex (Bangera and Thomashow, 1999). phlD has been the subject of interest, because it has homology to chalcone and stilbene synthases from plants, which suggests horizontal gene transfer between plants and their rhizosphere microbial populations (Bangera and Thomashow, 1999). Whereas phlACB coding sequences are highly conserved between eubacteria and archaebacteria (Picard et al., 2000), a considerable degree of polymorphism was reported for phlD (Mavrodi et al., 2001). phlA transcription is negatively regulated by the product of phlF (Delany et al., 2000) which also appears to mediate repression by fusaric acid (Delany et al., 2000), a metabolite of pathogenic fungi of plants, that has previously been implicated in repression of biosynthesis of the anti-fungal compound, phenazine (see above) (van Rij et al., 2005). These observations demonstrate the ongoing arms race between plants, their fungal pathogens and associated anti-fungal antagonists, leading to gene diversification.

Mupirocin: The polyketide mupirocin or pseudomonic acid is one of the major antibacterial metabolites produced by Pseudomonas fluorescens (Fuller et al., 1971) and

81 is widely used as a clinical antibiotic (Gurney and Thomas, 2011). Mupirocin can inhibit the growth of methicillin resistant Staphylococci, Streptococci, Haemophilus influenzae and Neisseria gonorrheae (Sutherland et al., 1985). In terms of the mode of action, mupirocin inhibits isoleucyl-tRNA synthetase, and hence prevents incorporation of isoleucine into newly synthesized proteins, thus terminating protein synthesis (Hughes and Mellows, 1980). Biochemically, mupirocin has a unique chemical structure that contains a C9 saturated fatty acid (9-hydroxynonanoic acid) linked to C17 monic acid A (a heptaketide) by an ester linkage (Whatling and Thomas, 1995). Mupirocin is derived from acetate units incorporated into monic acid A and 9 - hydroxynonanoic acid via polyketide synthesis (Whatling and Thomas, 1995). At the molecular level, the mupirocin biosynthetic gene cluster (mup operon) in P. fluorescens is complex, and includes 6 Type I polyketide synthases that are multifunctional as well as 29 proteins of single function within a 65 kb region, which are incorporated into 6 larger coding sequences (modules mmpA-F) (El-Sayed et al., 2003; Gurney and Thomas, 2011).The gene cluster is non- standard as the CDS are not in the same order as the biosynthetic steps (El-Sayed et al., 2003; Gurney and Thomas, 2011). The acyltransferase (AT) domains of the polyketide synthases (PKS) are not present in each genetic module but are instead encoded by a separate CDS (from the mmpC module) and this classifies these polyketide synthases as in-trans AT PKSs (El-Sayed et al., 2003). With respect to gene regulation, two putative regulatory genes, mupR and mupI, were identified within the cluster that are involved in quorum sensing dependent regulation (El-Sayed et al., 2001).

An interesting feature of this system in P. fluorescens is that self-resistance to mupirocin is also encoded by a CDS (mupM) within the biosynthetic gene cluster (El- Sayed et al., 2003). mupM encodes a resistant Ile t-RNA synthetase (IleS) due to polymorphisms within the binding site of mupirocin (El-Sayed et al., 2003; Gurney and Thomas, 2011). A second resistant IleS was cloned from P. fluorescens NCIMB 10586 outside of the mup gene cluster which showed 28% similarity to the mupM product (Yanagisawa, 1994). Human pathogens that have high level mupirocin-resistance are associated with an additional gene that encode a novel IleS with similarity to eukaryotic

82 counterparts; this resistance gene is associated with transposable elements and is carried on plasmids, facilitating its rapid spread (Eltringham, 1997; Gurney and Thomas, 2011).

There is also genetic evidence that the entire mup gene cluster in Pseudomonas arose by horizontal gene transfer; specifically the genes encoding tRNAVal and tRNAAsp were found upstream of the mupA promoter region leading to speculation that the mup cluster arose from homologous recombination between chromosomal tRNA genes and possibly a plasmid containing the mup cluster (El-Sayed et al., 2003). The inclusion of a resistant IleS (mupM) within the mup biosynthetic cluster might have facilitated such horizontal gene transfer, as otherwise uptake of the mupirocin gene cluster would have been immediately suicidal.

Difficidin: a polyketide with an interesting geometry that involves four double bonds in the Z configuration (Chen et al., 2006). Difficidin is produced by various Bacillus species such as B. subtilis and B. amyloliquefaciens FZB 42 with broad antibacterial activity against human and crop pathogens (Zimmerman et al., 1987; Chen et al., 2006; Chen et al., 2009). A large gene cluster (pks3) encoding difficidin (and oxydifficidin) was characterized in B. amyloliquefaciens (Chen et al., 2006). This compound is included in this review, because pks3 is adjacent to other polyketide synthesis gene clusters, pks1 and pks2, that encode bacillaene and macrolactin, respectively (Chen et al., 2006; Schneider et al., 2007). All three gene clusters share sequence homology, a similar order of CDS and are located close to another on the chromosome, leading Chen et al. (2006) to hypothesize that they emerged from homologous recombination from a common ancestral gene cluster resulting in gene duplication. This system provides insights into the diversification of polyketides.

Pyoluteorin (PLt): a phenolic polyketide with bactericidal, herbicidal and fungicidal properties. Plt can suppress damping-off disease in cotton caused by the fungus, Pythium ultimum (Howell, 1980). Both pyoluteorin and phenazine (see below) may act

83 synergistically to suppress such soil-borne fungal diseases in plants, as some studies have suggested that the two biosynthetic pathways interact with one another (Ge et al., 2007; Lu et al., 2009). The biosynthesis of Plt involves condensation of proline with three acetate equivalents through chlorination and oxidation. The carbon skeleton is built up by the action of a single multienzyme complex (Nowak-Thompson et al., 1999). In Pseudomonas fluorescens Pf-5, a 24 kb segment contains the PLt biosynthetic operon (pltABCDEFG). PLt biosynthesis is catalyzed by type I polyketide synthases (pltB, pltC), an acyl-CoA dehydrogenase (pltE), an acyl-CoA synthetase (pltF), a thioesterase (pltG), and halogenases (pltA, pltD, pltM) with pltM located adjacent to the gene cluster (Nowak-Thompson et al., 1999). A significant delay in the expression of the pyoluteorin biosynthetic operon was reported in the cucumber spermosphere compared to cotton, which correlated to the timing of infection with the fungal root pathogen Pythium ultimum (Kraus and Loper, 1995). The authors suggest that such temporal differences may be responsible for differential disease suppression in diverse plant hosts.

The plt biosynthetic operon has been shown to be regulated by a LysR family transcriptional activator, encoded by pltR (Nowak-Thompson et al., 1999). Interestingly, pltR is tightly linked and transcribed divergently to the biosynthetic gene cluster (Nowak- Thompson et al., 1999). In earlier studies involving the biosynthetic operon of phenazine, its LysR transcriptional regulator gene (phzR) was also shown to be tightly linked to its corresponding biosynthesis gene cluster (Pierson Iii et al., 1998). As both phenazine and pyoluteorin combat soil-borne fungal diseases in plants, we speculate that strong evolutionary pressures in the rhizosphere may have promoted horizontal gene transfer of the biosynthetic operons to new rhizosphere microbial hosts; the activator-cluster gene module would facilitate activation of the biosynthetic CDS following such gene transfer.

Jadomycin: Jadomycin is a member of angucycline antibiotics produced by Streptomyces species such as S. venezuelae. Jadomycin production is induced under stress conditions such as phage infection or heat shock (Jakeman et al., 2009). The jadomycin (jad) biosynthetic gene cluster in S. venezuelae is closely related to type II

84 polyketide synthase genes (Han et al., 1994) with a complex biosynthetic gene cluster (Zou et al., 2014). Jadomycin is of interest here because upstream of the jad operon are sets of negative regulatory genes including jadR1R2R3 and jadW123 (Yang et al., 1995; Zou et al., 2014). jadW123 encodes enzymes for the biosynthesis of gamma- butyrolactones (GBL), whereas JadR2 is a pseudoreceptor for GBL which upon its binding activates JadR1 and JadR3 that subsequently act as positive and negative transcriptional regulators of the jad biosynthetic operon, respectively (Zou et al., 2014). GBLs are becoming well known as regulators of secondary metabolism in gram positive bacteria, analogous to the related acyl homoserine lactone compounds which mediate quorum sensing (QS) in gram negative bacteria (Nodwell, 2014). Quorum sensing is a method of communication between bacterial populations that activates genes based on high cell density through the signal molecule N-acyl-homoserine lactone (AHL) (Whitehead et al., 2001). Whereas QS signaling molecules are thought to be synthesized and sensed by the same species (Nodwell, 2014), the GBL/jad system is interesting, because recent data suggests that GBL can signal across different Streptomyces species to activate different polyketide biosynthetic pathways (Nodwell, 2014; Zou et al., 2014). Biologically, it has been shown that different Streptomyces species, which are soil microbes, can live on the same grain of soil alongside a diversity of bacteria (Keller and Surette, 2006; Vetsigian et al., 2011), suggesting there may have been evolutionary selection for inter-species coordination for antibiotic production (Nodwell, 2014), resulting in enhanced genetic complexity associated with the jad locus.

Figure 4.1: Structures of polyketide compounds featured in this review.

4.3.2 Non-ribosomal peptides The structures of non-ribosomal peptides described in this review are shown (Figure 4.2).

Zwittermicin A: a polyketide/nonribosomal peptide hybrid antibiotic produced by B. cereus and B. thuringiensis (Raffel et al., 1996) with activity against oomycetes such as Phytophthora medicaginis and some other pathogenic fungi (Silo-Suh et al., 1998). Zwittermicin A has a unique structure that includes glycolyl moieties, D amino acid and ethanolamine in addition to the unusual terminal amide produced from the ureidoalanine (nonproteinogenic amino acid) (Kevany et al., 2009). Zwittermicin A is thought to be biosynthesized as part of a larger metabolite that is processed twice to form zwittermicin A and two other metabolites (Kevany et al., 2009). The complete biosynthetic operon encoding zwittermicin A includes 27 open reading frames (CDS, zmaA and zmaV) that extend over 62.5 kb of the Bacillus cereus UW85 genome, in addition to five individual genes (kabR and kabA – kabD) (Kevany et al., 2009). In this study, support was gained for the hypothesis that the skeleton of zwittermicin A is catalyzed by a megasynthase enzyme involving multiple nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS); the megasynthase has multiple modules containing distinct domains that catalyze the different steps in the pathway (Emmert et al., 2004; Kevany et al., 2009). Evidence suggested that the CDS included 5 NRPS modules (Kevany et al., 2009). It is noteworthy that a similar gene cluster was characterized on a plasmid in B. cereus AH1134, suggesting that the pathway can be transferred horizontally (Kevany et al., 2009). Consistent with the mobility of this operon, an orthologous 72-kb region encoding for zwittermicin A in Bacillus thuringiensis, was shown to be flanked by putative transposase genes on both edges, suggesting that it may be a mobile element that was gained by B. cereus through horizontal gene transfer. Since zwittermicin A has been reported to enhance the activity of protein toxins that attack insects (Broderick et al., 2000), it was hypothesized that transfer of this operon into B. thuringiensis permitted the microbe to gain insecticide-promoting factors to combat insects during co-evolution (Luo et al., 2011).

Fusaricidins A-D: guanidinylated ß-hydroxy fatty acids attached to a cyclic hexapeptide including four D-amino acids (Kajimura and Kaneda, 1997; Schwarzer et al., 2003). These antibiotics are produced by Paenibacillus polymyxa strains and exhibit antifungal activity against diverse plant pathogens including, Aspergillus niger, Aspergillus oryzae, Fusarium oxysporum and Penicillium thomii (Kajimura, 1996; Kajimura and Kaneda, 1997) as well as Leptosphaeria maculans, the causal agent of black root rot in canola (Beatty and Jensen, 2002). The amino acid chains of fusaricidins are linked together and modified by a non- ribosomal peptide synthetase (NRPS). The multi-domain NRPS consists of up to 15,000 amino acids and is therefore considered among the longest proteins in nature (Schwarzer et al., 2003). NRPS incorporation is not limited to the 21 standard amino acids translated by the ribosome, and this promiscuity contributes to the great structural diversity and biological activity of non-ribosomal peptides (Li and Jensen, 2008).

In P. polymyxa E68, the fusaricidin biosynthetic gene cluster (fusGFEDCBA) has been characterized in which the NRPS gene, the largest CDS in the cluster, was observed to encode a six-module peptide (Choi et al., 2008; Li and Jensen, 2008; Li et al., 2013). The biosynthetic cluster includes other genes responsible for biosynthesis of the lipid moiety but does not contain transporter genes (Li and Jensen, 2008). In P. polymyxa, a promoter for the fus operon was identified and shown to be bound by a transcriptional repressor (AbrB) which previous studies implicated as a regulator of sporulation; this is of interest since fusaricidin was observed to be synthesized during sporulation, thus coordinating the microbe’s secondary metabolism with its life cycle (Li et al., 2013).

Allelic diversity is typically thought to be responsible for producing chemical diversity. However, an interesting feature of the fus cluster is that a diversity of fusaricidins, differing in their incorporated amino acids (Tyr, Val, Ile, allo-Ile, Phe), can be produced by a single allele of fusA; the underlying mechanism is that the NRPS A-domain, responsible for recognition of amino acids, has relaxed substrate specificity (Figure 4.3) (Han et al., 2012).

Polymyxins: a family of non-ribosomal lipopeptide antibiotics composed of ten amino acids, a polycationic heptapeptide ring and a fatty acid derivative at the N terminus (Storm et al., 1977). They are produced by Gram positive bacteria and target Gram negative species, by altering the structure of the cell membrane. The polymyxin family includes polymyxins A, B, D, E (colistin) and M (mattacin) (Shaheen et al., 2011). Polymyxin B exhibits potent antibacterial activity against Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter sp. (Gales et al., 2006). However, polymyxins exhibit a remarkable degree of neurotoxicity and nephrotoxicity which limit their clinical use (Li et al., 2006).

In Paenibacillus polymyxa PKB1 (the same strain that controls plant fungi by producing fusaricidins, see above), a 40.8 kb polymyxin biosynthetic gene cluster was shown to encode five coding sequences, pmxA-E. Three CDS (pmxA, B, E) encode subunits of non-ribosomal peptide synthetase (NRPS), each responsible for the modular incorporation of amino acids, while two genes (pmxC, D) encode a permease belonging to the ABC-type transporter family (Shaheen et al., 2011). In both P. polymyxa PKB1 and P. polymyxa E681, the arrangement of the NRPS coding sequences in the pmx cluster does not match the amino acid sequence in the produced polymyxin, which is unusual for NRPS-encoded peptides (Choi et al., 2009; Shaheen et al., 2011).

With respect to the diversity of polymyxins, polymyxins differ in the amino acid composition of residues 3, 6 and 7, in the D versus L stereochemistry of the incorporated amino acids, as well as in the lipid moiety (Choi et al., 2009; Shaheen et al., 2011). In P. polymyxa SC2 and P. polymyxa PKB1, an allelic variant was uncovered within NRPS domain 3 using bioinformatic analysis of the genome which correlated with incorporation of the D rather than L form of 2,4-diaminobutyrate in amino acid position 3, explaining the mechanism for the production of two subtypes of polymyxin B (Shaheen et al., 2011). With respect to the diversity of residues 6 and 7, P. polymyxa E681 and P. polymyxa PKB1 produce polymyxins that differ in these amino acids, producing polymyxin A and

B, respectively (Shaheen et al., 2011). Bioinformatic analysis revealed that the DNA sequences of the pmx gene clusters were 92% conserved at the nucleotide level, but differed considerably in the domains corresponding to modules 6 and 7 (Shaheen et al., 2011). These two sets of observations led the authors to suggest that the diversity of polymyxins arises from mixing and matching of alleles of the NRPS modular domains, hence combinatorial chemistry, rather than relaxed substrate specificity as seen in other secondary metabolites such as fusaricidins (see above).

Another interesting feature of the pmx gene clusters is that the polymyxin transporters might also transport fusaricidin, since the fus biosynthetic cluster lacks any transporter genes (see above), and as both antibiotics are cationic lipopeptides (Shaheen et al., 2011). The authors found support for this hypothesis, as deletion mutations in pmxC and D genes also reduced the antifungal activity of fusaricidin against Leptosphaeria maculans although the two biosynthetic gene clusters are not linked. It is worth noting that there is no evidence yet of genes responsible for lipidation of the peptide residue in the characterized polymyxin clusters, suggesting that this function might be encoded elsewhere in the genome (Shaheen et al., 2011).

Iturins: a family of non-ribosomal cyclolipopeptides consisting of seven α-amino acid residues and one ß-amino acid, the latter noted as a unique feature compared to other lipopeptide antibiotics (Constantinescu, 2001; Leclère et al., 2005; Hamdache et al., 2013). The iturin family includes compounds such as bacillomycins D, F and L, bacillopeptins, iturins A, C, E and E, and mycosubtilins (Hamdache et al., 2013). Iturins are produced by different strains of B. subtilis and B. amyloliquefaciens, and exhibit potent antifungal activity against major phytopathogens including R. solani, Fusarium oxysporum and F. graminearum, the latter responsible for Fusarium head blight in wheat (Gueldner et al., 1988; Constantinescu, 2001; Tsuge et al., 2001; Dunlap et al., 2013). The mechanism of action involves disruption of the target fungal plasma membrane (Thimon et al., 1995). In both B. subtilis RB14 and B. amyloliquefaciens AS43.3, the

90 iturin A biosynthetic operons were shown to contain four coding sequences (ituDABC) coding for: a putative malonyl coenzyme A transacylase, a protein with three functions (fatty acid synthetase, amino acid transferase, and peptide synthetase), and two peptide synthetases, respectively (Tsuge et al., 2001; Dunlap et al., 2013).

Regarding diversification within the chemical family, iturin A from B. subtilis RB14 has a similar structure as mycosubtilin that is produced by B. subtilis ATCC 6633 but with inverted amino acids at the 6th and 7th positions (Tsuge et al., 2001). By comparative analysis of orthologous CDS between these two strains (ituC and mycC, respectively), it was suggested that the NRPS amino acid adenylation domain may have been intragenically swapped during evolution, which would also imply a horizontal gene transfer event (Tsuge et al., 2001). Comparative genome analysis between at least three sequenced itu clusters may reveal further information concerning the diversification of the iturin family (Tsuge et al., 2001; Blom et al., 2012; Dunlap et al., 2013).

Bacilysin: a non-ribosomally produced dipeptide composed of an L-alanine residue at the N terminus and a non-proteinogenic amino acid, L-anticapsin, at the C terminus (Walker and Abraham, 1970; Stein, 2005). Compared to the more elaborate non-ribosomal peptides noted above, bacilysin is noteworthy because it is amongst the simplest peptides in nature, adding to the structural diversity of observed non-ribosomal peptides. Bacilysin is produced by Bacillus species such as B. pumilus, B. amyloliquefaciens and B. subtilis (Leoffler et al., 1986; Phister et al., 2004) and shown to have antimicrobial activity against various bacteria and fungi such as Candida albicans (Kenig and Abraham, 1976). Mechanistically, bacilysin is a prodrug that is activated by the action of a peptidase enzyme that releases the active moiety, anticapsin (Rajavel et al., 2009). Anticapsin inhibits bacterial peptidoglycan or fungal protein biosynthesis through blockage of glucosamine synthetase, resulting in cell lysis (Kenig et al., 1976). Biosynthesis of bacilysin originates from the prephenate aromatic amino acid pathway (Hilton et al., 1988; Parker and Walsh, 2012).

In B. subtilis the biosynthesis of bacilysin is encoded by the operon, bacABCDE (ywfB-G), in addition to a monocistronic gene (ywfH) (Inaoka et al., 2003). bacABC is likely responsible for the biosynthesis of anticapsin while bacDE (ywfEF) encodes a ligase and an efflux transporter protein for self protection, respectively (Rajavel et al., 2009; Steinborn et al., 2005). The bacilysin biosynthetic operon is positively regulated by quorum sensing pheromones, in particular PhrC (Ya gan et al., 2001; Köroğlu et al., 2011) and negatively regulated by ScoC, a transition state regulator (Inaoka et al., 2009). The transition state in bacteria is a period of decision making.

Figure 4.2: Structures of non-ribosomal peptide compounds featured in this review.

Figure 4.3: Diagram illustrating how a diversity of fusaricidins produced from a single allele of fusA which encodes the non-ribosomal peptide synthase (NRPS) A-domain. (A) Most enzymes have stringent substrate specificity. (B) By contrast, the NRPS A-domain can recognize and incorporate different amino acids to create diverse fusaricidins, and hence it is an example of an enzyme with relaxed substrate specificity (Han et al., 2012).

4.3.3 Terpenoids

The structures of terpenoids described in this review are summarized (Figure 4.4).

Trichodermin and Harzianum A: Trichothecene mycotoxins are produced by some fungal genera such as deoxynivalenol (DON) from Fusarium, and harzianum and trichodermin from Trichoderma arundinaceum and T. brevicompactum, respectively (Cardoza et al., 2011). Trichodermin was reported to have antifungal activity against the fungal pathogens Rhizoctonia solani and Alternaria solani (Chen et al., 2007) as well as other fungal genera (Tijerino et al., 2011). Trichodermin inhibits protein synthesis in eukaryotes by inhibiting peptidyl transferase that catalyzes translational elongation and/or termination (Wei et al., 1974) and by inhibiting peptide-bond formation at the initiation stage of translation (Carter et al., 1976).

Figure 4.4: Structures of terpenoid compounds featured in this review.

Comparative analysis has been conducted on the CDS responsible for trichothecene biosynthesis in Fusarium and Trichoderma. In Fusarium, trichothecenes are encoded by a gene cluster called the TRI cluster; this cluster also encodes regulatory and transport proteins (Proctor et al., 2009). In Trichoderma, an orthologous TRI cluster was discovered in which 7 CDS were conserved with Fusarium, but the two clusters showed interesting evolutionary divergence (Cardoza et al., 2011) which may be informative for understanding the genetics underlying other anti-fungal metabolites. In Fusarium, the TRI cluster includes tri5 that encodes trichodiene synthase, the first committed step in trichothecene biosynthesis, which catalyzes the cyclization of farnesyl pyrophosphate to form trichodiene (Hohn and Beremand, 1989). In Fusarium, tri5 is located within the TRI cluster, but surprisingly it is not associated with the orthologous cluster in Trichoderma. Three additional CDS responsible for trichothecene biosynthesis in Fusarium (tri7, tri8, tri13) are missing from the Trichoderma cluster, along with an CDS of unknown function (tri9) (Cardoza et al., 2011). Interestingly, two of the apparently conserved biosynthetic CDS (tri4 and tri11, based on sequence homology) were demonstrated to have diverged functionally between Trichoderma and Fusarium based on heterologous expression analysis: in Trichoderma, tri4 catalyzes three out of four oxygenation reactions carried out by its corresponding Fusarium ortholog; tri11 catalyzes distinctive hydroxylation reactions in Fusarium (C-15) and Trichoderma (C-4). Amongst the conserved CDS between Fusarium and Trichoderma, head-to-tail versus head-to-head rearrangements are observed (e.g. tri3, tri4) (Cardoza et al., 2011). These results demonstrate multiple evolutionary events (rearrangement, functional diversification, gene loss, gene gain) within one biosynthetic gene cluster (Figure 4.5).

Figure 4.5: Comparative analysis of the trichothecene biosynthetic gene clusters. (A) Trichoderma arundinaceum, (B) T. brevicompactum, (C) Fusarium sporotrichioides, and (D) F. graminearum. The illustration suggests that the ancestral gene cluster underwent multiple evolutionary events including rearrangements (blue arrows), gene gain or loss within the same genus (green arrows) and gene gain or loss between genera (orange and green arrows) (adapted from Cardoza et al., 2011).

Phomenone: a sesquiterpene synthesized by various fungi including Xylaria sp., an endophytic fungus isolated from Piper aduncum, and reported to have antifungal activity against the pathogen Cladosporium cladosporioides (Silva et al., 2010). Phomenone is structurally similar to the PR toxin metabolite of Penicillium roqueforti which functions by inhibiting RNA polymerase and thus inhibits protein synthesis at the initiation and elongation steps (Moule et al., 1976). A biosynthetic precursor for phomenone A is aristolochene (Proctor and Hohn, 1993). In P. roqueforti NRRL 849, a gene required for aristolochene (aril) biosynthesis was characterized and shown to encode a sesquiterpene cyclase named aristolochene synthase (AS) (Proctor and Hohn, 1993). Expression of aril occurs in stationary phase cultures and is regulated transcriptionally (Proctor and Hohn, 1993).

Paclitaxel (Taxol): the diterpene paclitaxel (Taxol) is reported to be produced by at least 20 diverse fungal endophyte genera inhabiting various plant species (Zhou et al., 2010b). Taxol was reported to be produced by some fungal endophytes that inhabit conifer wood and its ecological function was suggested to be a fungicide against host pathogens (Soliman et al., 2013). Taxol acts by stabilizing microtubules and inhibiting spindle function leading to disruptions in normal cell division (Horwitz, 1994). However, Taxol was originally purified from Taxus trees (Wani et al., 1971) and shown to be encoded by plant nuclear genes, apparently redundantly. As the number of plant genera that produce Taxol is very few, it is interesting to speculate whether the biosynthetic genes may have been transferred horizontally from fungi to plants.

The Taxol biosynthetic pathway in plants requires 19 enzymatic steps. The first committed step in biosynthesis of plant Taxol is cyclization of GGDP to taxa- (4,5),(11,12)-diene catalyzed by taxadiene synthase (TS) (Hezari et al., 1995). The 13 plant Taxol biosynthetic genes from Taxus were used in BLASTP searches to identify potential homologs in Penicillium aurantiogriseum NRRL 62431 (Yang et al., 2014b). Seven putative homologous genes were identified though the homology scores were as

99 low as 19%; these genes were claimed to encode: phenylalanine aminomutase (PAM), geranylgeranyl diphosphate synthase (GGPPS), taxane 5α-hydroxylase (T5OH), taxane 13α-hydroxylase (T13OH), taxane 7β-hydroxylase (T7OH), taxane2α-hydroxylase (T2OH) and taxane 10β-hydroxylase (T10OH). Another gene encoding an acyltransferase (PAU_P11263) was identified by using BLASTP against the GenBank database. However, no homologs were identified to plant TS; the authors claimed that the fungus might catalyze taxadiene synthesis by a unique enzymatic system (Yang et al., 2014b). Position-Specific Iterative BLAST showed one gene from the bacterial genus Mycobacterium with potential similarity to plant TS suggesting lateral gene transfer from plants to mycobacteria (Yang et al., 2014b).

In a parallel study to isolate fungal Taxol biosynthetic genes, a different approach was taken where PCR primers designed from the plant genes that encode Taxol were used as a primary screen against fungi (Xiong et al., 2013). The study identified putative homologs of fungal TS as well as BAPT (which encodes the critical C-13 phenylpropanoid side-chain CoA acyltransferase) with ~40% sequence identities to their plant counterparts. Despite this progress, other reports remain skeptical that fungi actually encode Taxol (Heinig et al., 2013).

Recent studies have demonstrated complex three-way interactions in Taxol biosynthesis between a Taxol-producing fungal endophyte, other endophytes and the host plant. Host endophytic fungi appear to elicit plant TS transcription or transcript accumulation. Specifically, taxadiene synthase (TS) transcript and the corresponding protein were reduced upon treating both young plantlets and old Taxus wood with fungicide (Soliman et al., 2013). In a parallel study, co-culture of the Taxol-producing endophyte Paraconiothyrium SSM001 with two presumptive fungal endophytes of the same yew tree host elicited paclitaxel accumulation from the endophyte, suggesting interspecies interactions between endophytes inhabiting the same host niche (Soliman and Raizada, 2013).

100

Helvolic acid: a fusidane triterpene produced by Aspergillus fumigatus (Lodeiro et al., 2009) and the yeast, Pichia guilliermondii Ppf9 (Zhao et al., 2010). Helvolic acid was reported to inhibit the spore germination of Magnaporthe oryzae, the causal agent of rice blast disease (Zhao et al., 2010). The biosynthetic genes for helvolic acid are clustered as nine genes coding for protostadienol synthase which catalyzes the precursor (17Z)- protosta-17(20),24-dien-3-ol, along with genes that encode squalene-hopene cyclase, four cytochrome P450 monooxygenases, short chain dehydrogenase, two transferases and 3- ketosteroid 1-dehydrogenase (Lodeiro et al., 2009). The authors reported that the P450 monooxygenases from different fungi shared substantial sequence identity across recent evolution, while the transferases duplicated and diversified into paralogous gene families (Lodeiro et al., 2009). This observation suggests that even within a single gene cluster, there may be different selection pressures on adjacent genes belonging to the same biosynthetic pathway. Interestingly, the helvolic acid biosynthetic gene cluster in A. fumigates is located in the sub-telomere chromosome region (Lodeiro et al., 2009) which is associated with high rates of evolutionary recombination and diversification. However, the gene cluster lacks introns which is a trait sometimes associated with subtelomeric regions, but this observation might also be evidence of horizontal gene transfer from bacteria (Lodeiro et al., 2009).

4.3.4 Alkaloids

The structures of alkaloids described in this review are summarized (Figure 4.6).

101

Figure 4.6: Structures of featured alkaloids, heterocyclic nitrogenous compounds and bacteriocin.

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Ergot: ergot alkaloids are produced from the sexual Epichloe fungi and their asexual derivatives Neotyphodium within the Clavicipitaceae family which inhabit Pooideae grasses (Schardl, 2010). Ergot alkaloids can interact with receptors of the central nervous system and exhibit toxic effect on nematodes, insects, and mammalian herbivores including livestock which graze these grasses (de Groot et al., 1998; Gröcer and Floss, 1998). In Europe in the Middle Ages, consumption of ergot-infected grain or grasses caused convulsions, paranoia and hallucinations in livestock and humans, known as St. Anthony’s Fire (Dotz, 1980). The diverse ergot alkaloids share a tetracyclic ergoline backbone derived from tryptophan and dimethylallyl diphosphate (Flieger et al., 1997). Gene clusters for ergot alkaloid biosynthesis have been identified in various Ascomycete species belonging to Aspergillus, Penicillium and Claviceps. Seven genes encode the ergoline scaffold including dimethylallyltryptophan synthase (DMATS) which catalyzes the first committed step. DMATS is responsible for the prenylation of L-tryptophan with dimethylallylpyrophosphate (DMAPP) to produce 4-dimethylallyltryptophan (4-DMAT) (Heinstein et al., 1971). Ergots have diversified into three classes, caused by diverse substituents attached to the carboxyl group of the tetracyclic ergoline backbone, in particular the presence of an amide group (creating ergoamides), a peptide-like amide moiety (creating ergopeptines) or the absence of these moieties (creating clavine alkaloids) (Wallwey and Li, 2011). These structural modifications are responsible for the differential physiological and pharmacological effects of the ergot family, that include treatment of postpartem hemorrhage, leukemia and Parkinson’s disease. The genetic basis for ergot diversification into these 3 major classes is associated with the presence or absence of nonribosomal peptide synthases (NRPS) which catalyse the biosynthesis of the peptide moieties on the ergoline backbone (Wallwey and Li, 2011). For example, four NRPS genes are present in Claviceps purpurea (which encodes ergopeptines) but absent in Aspergillus fumigatus (which produces clavine alkaloids). Inactivation of these genes suggests that two of the NRPS genes (lpsA and lpsB) are also responsible for synthesis of the ergoamides (Haarmann et al., 2008). Interestingly, further diversification of the peptide moiety within C. purpurea has been reported to be caused by fine-scale allelic diversification of the NRPS genes (Haarmann et al., 2005). There is additional evidence to suggest that diversification of the ergot alkaloid gene clusters is associated with DNA

103 transposons and retroelements, which were observed in the cluster encoding ergovaline, an ergot alkaloid from Epichloe festucae associated with livestock toxicity (Fleetwood et al., 2007). As an interesting note, the genes encoding ergovaline were highly expressed only during biotrophic growth of the fungus within the host grass plant not when the mycelia were cultured in vitro, suggesting that the host might have a regulatory role in the expression of the fungal gene cluster (Fleetwood et al., 2007).

Loline alkaloid: an indole alkaloid produced by Neotyphodium uncinatum fungus, the asexual mutualistic derivative of Epichloe, which is known to protect its host plants from insects (Blankenship et al., 2001; Schardl, 2010). The loline biosynthetic pathway was suggested to involve proline and homoserine (Spiering et al., 2005). In N. uncinatum, two homologous gene clusters encoding loline were identified, named LOL-1 and LOL-2 (Spiering et al., 2005). The cluster LOL-1 involves nine genes (lolF-1, lolC-1, lolD-1, lolO-1, lolA-1, lolU-1, lolP-1, lolT-1, lolE-1) within a 25-kb chromosomal segment, while the LOL-2 cluster contains the same homologs (except for lolF) ordered and oriented the same as in LOL-1. This evidence suggests that the loline clusters may represent a recent segmental duplication event (Spiering et al., 2005).

An interesting ecological situation exists in grasses infected with Epichloe fungi (sexual form of Neotyphodium): the fungus reduces the ability of these plants to propagate sexually (they choke the inflorescences), which, without compensatory mechanisms, would prevent vertical transmission of the fungus (Zhang et al., 2010). However, to compensate, the fungal stromata attract fly vectors which transmit the fungal spores to other plants, permitting horizontal transfer of the fungus. Loline accumulates in young tissues of the grasses, providing insect protection to these young hosts; however if loline was also to accumulate in the grass inflorescences, it would kill the fly vector of the fungus. Upon further investigation, this apparent paradox was resolved: in these grass inflorescences, transcription of the loline biosynthesis genes was dramatically downregulated compared to plants with healthy inflorescences (infected with the

104 symbiotic asexual Neotyphodium), permitting the fly vectors to survive (Zhang et al., 2010). These observations suggest strong selection pressure to evolve the regulatory elements of these genes.

4.3.5 Heterocyclic nitrogenous compounds

The structures of heterocyclic nitrogenous compounds described in this review are summarized (Figure 4.6).

Phenazines: a group of naturally occurring heterocyclic nitrogenous antibiotics produced exclusively by bacteria and widely reported in fluorescent Pseudomonas (Mavrodi et al., 2013). Phenazines are potent antifungal compounds that can combat soil borne pathogens (Ligon et al., 2000) such as Rhizoctonia solani, Gaeumannomyces graminis var. tritici , Pythium sp. (Gurusiddaiah et al., 1986) and Fusarium oxysporum (Anjaiah et al., 1998). Mechanisms of action include: (1) accumulation of toxic molecules such as hydrogen peroxide and superoxide due to the redox potential of phenazine (Hassan and Fridovich, 1980; Hassett et al., 1995); and (2) elicitation of induced host resistance (Audenaert et al., 2002). Ecologically, the evidence suggests that the plant rhizosphere promotes phenazine-producing bacteria to combat pathogens (Mavrodi et al., 2013).

Phenazine is derived from shikimic acid pathway, with amino-2-deoxyisochorismic acid (ADIC) as the branchpoint to phenazine (McDonald et al., 2001). ADIC is then converted to trans-2, 3-dihydro-3-hydroxy anthranilic acid which undergoes dimerization to form phenazine-1-carboxylic acid, the first derivative of the phenazines (McDonald et al., 2001). Phenazine biosynthesis in Pseudomonas fluorescens is encoded by a single or duplicated core of five genes, phzADEFG, that encode ketosteroid isomerase, isochorismatase, anthranilate synthase, trans-2,3-dihydro-3-hydroxyanthranilate isomerase , and pyridoxamine oxidase respectively (Mavrodi et al., 2013). In Pseudomonas, the core may include other genes such as phzB which was duplicated from

105 phzA, and phzC which encodes 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase that is responsible for diverting carbon from the shikimate pathway to phenazine (Pierson III and Pierson, 2010).

Comparisons between Pseudomomas species and other genera have revealed conservation yet diversity of the core phenazine biosynthetic genes. For example, the phenazine biosynthesis operon in Burkholderia cepacia maintains the five core enzymes observed in Pseudomonas as reviewed (Mavrodi et al., 2006). However, there is evidence to suggest that these genes spread to enteric bacteria and Burkholderia species via horizontal gene transfer, because these genes can be observed in plasmids and transposons (Mavrodi et al., 2010). For example, in Erwinia herbicola, a biosynthetic cluster of 16 genes (ehp) was isolated from a plasmid, of which 15 coded for D-alanyl griseoluteic acid (AGA) while ehpR was observed to encode for resistance to AGA (Giddens et al., 2002). Other differences in the core have also been observed between Pseudomonas species and others; for example in both Burkholderia cepacia and Erwinia herbicola, phzA is not duplicated (Mavrodi et al., 2006).

Structural diversity of phenazines in different species is achieved by specific genes that may be located within the cluster or elsewhere in the genome. For example, in P. chlororaphi, the phzH gene is located downstream of the phenazine operon, where it encodes an aminotransferase responsible for converting phenazine-1-carboxylic acid (PCA) to phenazine-1-carboxamide, the characteristic green pigment of P. chlororaphis (Chin-A-Woeng et al., 1998). In P. aureofaciens, phzO was identified as the gene that encodes an aromatic monooxygenase, responsible for catalyzing the hydroxylation of PCA to form the broad spectrum antifungal compound, 2-OH-PCA (Delaney, 2001). In P. aeruginosa (PAO1), two diversification genes were discovered: phzM was shown to be involved in the synthesis of pyocyanin while phzS gene encodes a monooxygenase that catalyzes the production of 1-hydroxy phenazine (Mavrodi et al., 2001).

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Pyrrolnitrin: a chlorinated phenylpyrrole antibiotic purified initially from Burkholderia pyrrocinia (Arima et al., 1964) then subsequently from other species including pseudomonads, Myxococcus fulvus, Enterobacter agglomerans and Serratia sp. (Chernin et al., 1996; Kirner et al., 1998; Hammer et al., 1999). Pyrrolnitrin was initially used for treatment of skin mycoses caused by Trichophyton fungus, then was developed as an effective fungicide for crops against Botrytis cinerea (Hammer et al., 1993), Rhizoctonia solani (El-Banna and Winkelmann, 1998) and Gaeumannomyces graminis var. tritici (Tazawa et al., 2000). In P. fluorescens, the pyrrolnitrin biosynthetic operon consists of four coding sequences (prnABCD) coding for tryptophan halogenase (prnA), a decarboxylase (prnB), monodechloroaminopyrrolnitrin halogenase (prnC), and an oxidase (prnD) (Hammer et al., 1997; Kirner et al., 1998). Comparative analysis indicates that the pyrrolnitrin biosynthetic operon is differentially conserved between different species with 59% similarity among diverse bacterial strains such as Pseudomonas, Myxococcus fulvus and Burkholderia cepacia, with lower similarity shown for prnA in M. fulvus (45%) (Hammer et al., 1999). Furthermore, RFLP-based polymorphisms within a 786 bp prnD fragment suggested that there may have been lateral gene transfer of the prn operon from Pseudomonas to Burkholderia pyrrocinia (Souza and Raaijmakers, 2003). Consistent with such mobility, transposase-encoding genes surrounding the prn biosynthetic operon were observed in Burkholderia pseudomallei (Costa et al., 2009).

4.3.6 Volatile compounds In this section, only the most well studied volatile compound, hydrogen cyanide, is discussed.

Hydrogen cyanide (HCN): a volatile secondary metabolite produced by P. aeruginosa, and diverse rhizosophere fluorescent pseudomonads, where they exhibit biocontrol activity against pathogenic fungi such as Thielaviopsis basicola, the fungal causal agent of black root rot of tobacco (Voisard et al., 1989; Voisard et al., 1994; Frapolli et al., 2012). Mechanistically, HCN functions by inhibiting important metalloenzymes such as

107 cytochrome c oxidase (Blumer and Haas, 2000) and/or by complexing metals in the soil (Brandl et al., 2008). HCN is biosynthesized from glycine (Castric, 1977) in an oxidative reaction catalyzed by HCN synthase, a membrane-bound flavoenzyme (Castric, 1994; Blumer and Haas, 2000). The biosynthesis of HCN occurs in the presence of an electron acceptor such as phenazine methosulfate (Wissing, 1974).

In P. aeruginosa PAO1, the HCN synthase biosynthetic operon hcnABC was characterized (Pessi and Haas, 2000). hcnA was reported to encode a protein similar to formate dehydrogenase while hcnB and hcnC encode products with similarity to amino acid oxidases (Laville et al., 1998; SVERCEL et al., 2007). In a phylogenetic analysis of 30 fluorescent pseudomonads, no evidence was found for horizontal gene transfer of the hcn gene cluster, but rather that the locus appears to be exclusively inherited vertically (Frapolli et al., 2012). Hydrogen cyanide has also been detected in Chromobacterium violaceum but the underlying genes have not been reported which might otherwise give new insights into hydrogen cyanide biosynthesis outside of the pseudomonads (Blom et al., 2011).

4.3.7 Bacteriocin: In this section, only the most well studied compound from this class is discussed. The structures of agrocin 84 described in this review is included (Figure 4.6).

Agrocin 84: a 6-N-phosphoramidate of an adenine nucleotide analogue (Roberts and Tate, 1977). This compound is produced by non-pathogenic strains of Agrobacterium radiobacter to biocontrol crown gall, a tumorous disease resulting from overproduction of auxin and cytokinin hormones stimulated by the Ti plasmid after it has transferred from A. radiobacter and integrated within host plant chromosomal DNA (Kerr, 1980; Wang et al., 1994). Recently, it was shown that agrocin 84 employs a novel mechanism to inhibit leucyl-tRNA synthetases and hence inhibit translation (Chopra et al., 2013),

108 though it was earlier suggested that agrocin 84 acts by inhibiting DNA synthesis (Das et al., 1978).

In Agrobacterium radiobacter K84, the biosynthesis and immunity to agrocin 84 is encoded by 17 coding sequences (the agn operon) located on a 44-kb conjugal plasmid, pAgK84, though the plasmid has 36 CDS in total (Kim et al., 2006). The two most interesting CDS are agnB2 and agnA which encode aminoacyl tRNA synthetase homologues. The agrocin 84 antibiotic is essentially a nucleotide attached to an amino acid-like moiety (methyl pentanamide), and its mode of action was proposed to involve competitive binding to the active site of leucyl-tRNA synthetases (Reader et al., 2005). agnB2 encodes a leucyl-tRNA synthetase homologue that confers self-immunity to agrocin 84 since it does not bind the antibiotic (Kim et al., 2006). Normally, a tRNA synthetase acts as a ligase that catalyzes the attachment of an amino acid to a tRNA which includes an anticodon; the catalysis results in a phosphoanhydride bond between the amino acid and ATP as the initial step in the aminoacylation of tRNA (Ibba and Söll, 2000). Surprisingly, agnA encodes a truncated homolog of an asparaginyl-tRNA synthetase which lacks the anticodon-binding domain, but maintains the catalytic domain. Thus, agnA appears to be a fascinating example of a gene that evolved from an ancient tRNA synthetase (for arginine), but now is a biosynthetic enzyme for an antibiotic that inhibits a paralogous enzyme (for leucine attachment) (Kim et al., 2006).

The agn operon may have an evolutionary history of horizontal gene transfer, as pAgK84 is inter and intra species transferable: Rhizobium that received the pAgK84 plasmid from Agrobacterium as trans-conjugates could synthesize agrocin 84 and received immunity as well (Farrand et al., 1985).

A final fascinating feature of the agn system is an apparent second form of ancient evolutionary pressure on the genes responsible for the biosynthesis of the antibiotic.

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Agrocin 84 is a chemical mimic of agrocinopines, a class of compounds that is a source of plant-derived nitrogen for the pathogens targeted by the antibiotic; the pathogens have their own Ti plasmids that encode for transporters that not only transport agrocinopines but also agrocin 84 (Ellis and Murphy, 1981; Hayman and Farrand, 1988; Kim and Farrand, 1997). Hence the agn biosynthetic genes evolved to create a chemical structure that not only mimics the tRNA synthetase substrate of the pathogen target, but also targets its nitrogen uptake machinery.

4.3.7 Enzymes In this section, only the most well studied anti-fungal enzyme, chitinase, is discussed.

Chitinase: enzymes that break down chitin, one of the fungal cell wall components composed of repeated units of N-acetyl-D-glucos-2-amine, linked by β-1,4 glycosidic bonds (Bhattacharya et al., 2007). Fungi and hence chitin are enriched in soil and thus soil microbes are abundant sources of chitinases (also to target insects) (Hjort et al., 2010). Examples of chitinase-producing microbes include: fluorescent Pseudomonas strains isolated from the sugarcane rhizosphere that can target Colletotrichum falcatum, the causative agent of red rot disease in this crop (Viswanathan and Samiyappan, 2001); Actinoplanes missouriensis that antagonizes Plectosporium tabacinum, the causal agent of lupin root rot in Egypt (El-Tarabily, 2003); and Stenotrophomonas maltophilia that suppresses summer patch disease in Kentucky bluegrass (Kobayashi et al., 2002). Chitinases are produced by diverse bacterial genera including Pseudomonas, Streptomyces, Bacillus and Burkholderia (Quecine et al., 2008). Chitinases are divided into two major categories, exochitinases and endochitinases. Of the four reported endochitinase family members (glycoside hydrolase families 18, 19, 23 and 48), primarily families 18 and 19 have been reported in bacteria, with only a single example of a family 23 chitinase (Prakash et al., 2010).

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In Stenotrophomonas maltophilia 34S1, the chitinase family 18 gene has one CDS that encodes for a protein with seven domains: a catalytic domain, a chitin binding domain, three putative binding domains, a fibronectin type III domain and a polycystic kidney disease domain (Kobayashi et al., 2002). Bacterial chitinase family 18 has been shown to display different types of diversity. First, sequence analysis has shown that the catalytic domain and substrate binding domain, which are separated by a linker, have evolved independently. As the domain sequences do not match the taxonomies of their hosts, it has been suggested that domain swapping has been an important generator of diversity in this family, combined with horizontal gene transfer (Figure 4.7) (Karlsson and Stenlid, 2008). Unusual examples of chitinase genes are those that contain multiple family 18 catalytic domains within the same peptide that appear to function independently of one another (Howard et al., 2004). Additional examples of family 18 biodiversity include genes that contain non-consensus sequences at the catalytic site, as well as a bacterial subgroup that consists solely of a catalytic domain (Karlsson and Stenlid, 2008).

Unlike family 18 chitinases that are widely distributed among the prokaryotes, family 19 chitinases are restricted to green non-sulfur and purple bacteria, as well as actinobacteria (Prakash et al., 2010). Based on sequence alignments of family 19 chitinases in prokaryotes and eukaryotes, strong evidence has emerged that this gene family in actinobacteria and purple bacteria was derived from flowering plants by horizontal gene transfer (HGT) (Prakash et al., 2010). Furthermore, HGT from plants to purple bacteria may have occurred as two independent events in the distant past, followed more recently by HGT to actinobacteria (Prakash et al., 2010). The core architecture and catalytic sites of bacterial and plant family 19 chitinases are nearly identical. The sequence analysis further suggests that there was subsequent HGT from purple bacteria and actinobacteria to nematodes and arthropods, respectively (Prakash et al., 2010).

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Figure 4.7: An example of intra- coding sequences diversification within an anti- microbial gene cluster: amongst the Family 18 chitinases is an example of a chitinase in which the catalytic domain has been duplicated (Howard et al., 2004).

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4.4 Discussion

The objective of this paper was to review the biodiversity of anti-microbial compounds, their mode(s) of action and underlying biosynthetic genes within plant associated microbes. This review covered diverse biosynthetic gene clusters that encode polyketides, non-ribosomal peptides, terpenoids, alkaloids, heterocyclic nitrogenous compounds, volatile compounds, bacteriocins and lytic enzymes. The reviewed evidence suggests that these biosynthetic genes have diversified at different orders, each based on distinct evolutionary mechanisms.

4.4.1 Levels of biosynthetic gene cluster diversifcation

4.4.1.1 Species level diversification. An emerging theme from the literature is that horizontal gene transfer (HGT) appears to have played a major role in the evolutionary diversification of plant-associated microbial species through inheritance of anti-microbial traits. There is evidence that HGT may have occurred from: bacteria to bacteria such as those that inhabit the rhizosphere (e.g. phenazine); from bacteria to fungi (e.g. helvolic acid); from bacteria to nematodes and arthropods (e.g. chitinase family 19); possibly from fungi to plants (e.g. Taxol); from plants to bacteria (e.g. phenazine and chitinase family 19); and even from higher eukaryotes to bacteria (e.g. IleS, pseudomonic acid resistance protein) (Figure 4.8). As noted in the literature, diverse factors might have facilitated these remarkable gene transfer events including: (1) the clustering of genes encoding the secondary metabolite; (2) homologous recombination between chromosomes and trans- conjugated plasmids (e.g. phenazine, zwittericin A); (3) the presence of mobile elements (DNA transposons and retroelements) flanking the biosynthetic operons (e.g. zwittermicin A, phenazine and ergovaline); and (4) the presence of genes that encode self-immunity to the antibiotic within the biosynthetic cluster as otherwise receiving the cluster would have caused immediate suicide (e.g. mupirocin). It is worth noting that some gene clusters show no evidence of HGT (e.g. hydrogen cyanide).

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Figure 4.8: Potential examples of horizontal gene transfer of anti-microbial gene clusters leading to species level evolutionary diversification.

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4.4.1.2 Genome level diversification. A second emerging theme from the literature is that a subset of associated plant- associated microbial genomes have diversified with respect to duplications of entire gene clusters responsible for the synthesis of antimicrobial compounds. For example, as noted above, in Neotyphodium uncinatum, there are two homologous gene clusters that encode loline, LOL-1 and LOL-2, the likely result of a segmental duplication event within this fungus. Another noted example is from Bacillus species in which three tandemly duplicated gene clusters, pks1, pks2 and pks3, encode the polyketides, bacillaene, macrolactin and difficidin, respectively, the likely result of a homologous recombination event.

4.4.1.3 Intra gene cluster diversification. A third interesting theme from the literature is that gene clusters encoding anti-microbial compounds have extensively diversified within, to permit biochemical diversification. The biosynthetic genes for these compounds are clustered in fungi or organized into operons in bacteria – in the latter, they are generally located on chromosomes but occasionally on plasmids (e.g. agrocin 84). Diversity within each cluster can include varying combinations of biosynthetic coding sequences, transporters for the respective compound, regulatory CDS and CDS that confer self-immunity (e.g. mupirocin, agrocin 84). The biosynthetic operons vary in how many CDS synthesize the core skeleton (e.g. synthetases) as well as in how many encode decoration enzymes (e.g. hydroxylases, acyltransferases). However, the decoration enzymes may be encoded outside the gene cluster (e.g. phenazine operon). Furthermore, the biosynthetic CDS may be organized into genetic modules (e.g. NRPS) that vary in number. Each gene cluster is also associated with distinct DNA regulatory elements, for example to receive signals such as from quorum sensing. For example, comparative analysis of the trichothecene biosynthetic gene clusters (TRI) in Fusarium and Trichoderma showed multiple evolutionary diversification events within a single biosynthetic gene cluster family (e.g. head-to-tail versus head-to-head rearrangements) (Figure 4.5). In another example, comparative analyses of the polymyxin operon showed mixing and matching of CDS, resulting in diversification of the compounds. Similarly, diversification of the phenazines likely arose through a diversity of biosynthetic

115 decoration enzymes (e.g. hydroxylases). Another intriguing observation is from the helvolic acid biosynthetic gene cluster, in which transferase CDS were shown to have duplicated and diversified into paralogous gene families. As noted above, an interesting feature of this gene cluster is that it located in the sub-telomere chromosome region which is associated with high rates of evolutionary recombination.

4.4.1.4 Intra CDS diversification. A final emerging theme from the literature is that diversification of anti-microbial traits in plant-associated microbes arose from allelic diversification. For example, intragenic swapping of domains was observed within the same genetic module (e.g. iturin A, mycosubtilins). As another example, whereas most chitinase genes possess a single catalytic domain, examples were noted where a single CDS encoded two catalytic domains (Figure 4.7). In general, the literature notes that domains within the same CDS can evolve independently (e.g. catalytic versus substrate binding domains of chitinase); combined with the existence of linker peptides between domains as sites of homologous recombination, these features can result in novel alleles following domain swapping (e.g. family18 chitinases). Whereas such allelic diversification plays a major role in the diversification of compound structures, caused for example by DNA mutations within the substrate binding domain, the literature demonstrates examples where biochemical diversity has arisen from relaxed substrate specificity of the biosynthetic enzymes (Figure 4.3). A representative example of the latter is the promiscuous fusaricidin NRPS in which the same recognition domain in different species can recognize and incorporate different amino acids, and furthermore it can recognize amino acids beyond the 21 standard amino acids translated by the ribosome, which results in significant structural diversity.

4.4.2 Dynamic evolutionary driven by selection pressures. It is interesting to speculate on the evolutionary selection pressures that have led to the diversification at the various biological levels noted above. At the most basic level, diversification was likely driven by a three-way co-evolution between the plant-associated microbe, its target pathogen

116 and the host plant. This co-evolution may have occurred within a specific plant tissue niche or within soil associated with the rhizosphere (e.g. phenazine and pyoluteorin to combat soil-borne pathogens). However, there is also evidence for four-way interactions, to also include additional microbes (e.g. Taxol, jadomycin) and insects (e.g. ergovaline). These complex interactions can be bi-directional (e.g. loline). Within the producing organism, there is evidence for selection pressure to coordinate biosynthesis of the anti- microbial compound with the life cycle of the microbe (e.g. fusaricidins). There may also have been selection for genetic efficiency (e.g. potential sharing of transporter genes between polymyxin and fusaricidin). These selection pressures have led to fascinating individual stories, including the evolution of mimicry to facilitate antibiosis (e.g. agrocin).

4.4.3 Ecological and evolutionary lessons

When the examples of anti-microbial pathways were grouped by the phylogeny of their host microbes, several trends were observed (Figure 4.9, Tables 4.1 and 4.2). Specifically: (1) some anti-microbial genes are apparently widely distributed among diverse taxonomic classes of bacteria (e.g. chitinases); (2) some metabolic pathways are widely distributed within one taxonomic class such as pyrrolnitrin that shows up in more than half of the presented species of Proteobacteria; (3) other anti-microbial pathways appear to be more restricted (eg. fusaricidin, polymyxin, jadomycin). These results may correlate to the evolutionary age of these genetic pathways, or may represent a bias based on how well the pathway has been studied. More widespread genome sequencing and/or the use of orthologous gene probes may help to inform the evolutionary origins of these anti-microbial pathways.

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Figure 4.9

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Figure 4.9: The anti-microbial compounds reviewed in this study grouped by the phylogeny of their microbial hosts. (A) for bacteria and (B) for fungi. The phylogenetic trees were generated using the interactive Tree of Life website (Letunic and Bork, 2011). The anti-microbial compounds produced by these species colour coded, panel of color coded as indicated (C). The scale bar represents the number of nucleotide substitutions per site.

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4.4.3.1 Bacterial pathway lessons: The selected examples of anti-microbial pathways from plant-associated bacteria found in the literature and presented in this review are distributed across Proteobacteria, Actinobacteria and Firmicutes (Figure 4.9). This may not be surprising as Proteobacteria and Actinobacteria are among the most widespread bacterial taxa associated with plants, perhaps because of their saprophytic capabilities (Bulgarelli et al., 2012).

Within these phyla, P. fluorescens (Proteobacteria) and B. subtilis (Firmicutes) were observed to produce a plethora of diverse antimicrobial compounds belonging to diverse chemical classes including polyketides, non-ribosomal peptides, heterocyclic nitrogenous compounds, volatiles and enzymes which reflect the diversity of the metabolic machineries of these species. As P. fluorescens and B. subtilis are both model systems, these results also support the above note of the bias within this literature.

Bacillus sp. and Pseudomonas sp. are ubiquitous microbes that can survive in diverse ecological niches (Compant et al., 2005). Both have elegant survival strategies that involve the production of antibiotics, surfactin, cyanide, biofilms, and induction of host resistance (Espinosa-Urgel, 2004; Dini-Andreote and van Elsas, 2013). These unique adaptations have led to their widespread study and use as biocontrol agents (Santoyo et al., 2012).

Genome analysis of P. fluorescens has provided insight into its ecological competency and evolutionary mechanisms. The versatile and rapid adaptability of P. fluorescens to diverse environmental clues may be attributed to over 200 characterized signal transduction proteins which enhance its sensing capability (Garbeva and de Boer, 2009; Humair et al., 2010). With respect to co-evolution, the P. fluorescens genome is exceptionally rich in repetitive extragenic palindromic (REP) elements, target sites for transposases and recombinases, with 1052 REP elements identified in P. fluorescens Pf-5

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(compared to 21 in P. aeruginosa PAO1 and 365 in P. syringae DC 3000) (Tobes and Pareja, 2005, 2006). REPs likely affected genome evolution either by gene gain, loss or rearrangement (Silby et al., 2011). The latest version of the genome sequence and annotation of P. fluorescens was recently released (Martínez-García et al., 2015).

B. subtilis is naturally competent genetically, with a cascade of competence- specific DNA-uptake proteins that bind and transport DNA, in addition to a dynamic recombination mechanism which transforms chromosomal or plasmid DNA via different pathway (Chen and Dubnau, 2004; Kidane et al., 2009). Additionally, the B. subtilis genome encodes integrative and conjugative element binding (ICEBs1) proteins responsible for excision, integration, transfer of DNA (Lee et al., 2007) that likely have facilitated HGT. Comparative genomic analysis of B. subtilis strains revealed 298 accessory segments that potentially originated from mobile elements including plasmids, transposons and phages. This implies extensive HGT events that lead to diversification of the arsenal of anti-microbial pathways within Bacillus (Zeglier, 2011). The complete genome sequence and genome annotation of B. subtilis is available (Belda et al., 2013).

4.4.3.2 Fungal pathway lessons: In contrast to bacteria, all the fungal examples presented in the review belong to a single classification - the Pezizomycotina (filamentous fungi), a subdivision of Ascomycota, the largest phylum of fungi (Blackwell, 2011) including representitatives from Eurotiomycetes and Sordariomycetes (Figure 4.9). Pezizomycotina has an ancient origin in the Cambrian period, ca 530 Mya (Prieto and Wedin, 2013).

Pezizomycotina species are the most ubiquitous fungi with extremely diverse lifestyles, suggesting a corresponding diversity of ecological strategies (Spatafora et al., 2006; Beimforde et al., 2014) reflected in their production of a range of anti-microbial metabolites. There may be at least two reasons for this metabolic diversity, HGT and

121 recombination. First, HGT from bacteria to fungi was previously reported in Ascomycota, of which 65% were observed in Pezizomycotina (Marcet-Houben and Gabaldón, 2010). Second, secondary metabolism gene clusters in Pezizomycotina show evidence of recent gene expansion (Arvas et al., 2007). Interestingly, most of these genes are located in the sub-telomere region (Rehmeyer et al., 2006) that is associated with a considerable high rate of recombination and correspondingly rapid evolution compared to other regions in the genome (Freitas-Junior et al., 2000), an example represented in this review by helvolic acid (Table 4.1).

4.5 Gaps and Future Perspectives Despite the apparent progress in understanding the genetic mechanisms underlying the diversity of anti-microbial compounds produced by plant-associated microbes, significant gaps and opportunities remain. The major challenge is that a vast majority of plant associated microbes are unculturable, a phenomena that, to a far extent, limits our understanding of species diversification and evolution. It is worth noting that considerable progress towards cultivation of unculturable microbes has been achieved (Pham and Kim, 2012; Stewart, 2012). The modified cultivation methods attempt to simulate the natural environment, and include community culturing, and the use of high- throughput microbioreactors and laser microdissection (Pham et al., 2012). Another challenge is that the literature appears to be biased for model organisms, with insufficient data from other organisms in the phylogenetic tree for comparative genomics and evolutionary studies. For example, despite our efforts to originally focus this review only on anti-microbial pathways from endophytes, it became clear that the number of associated genes from endophytes has largely been unexplored, compared to free living rhizosphere model species. Indeed, there remains a lack of detailed genetic analysis underlying many anti-microbial compounds across microbes (endophytic and non- endophytic) and a lack of information to connect allelic diversity with compound diversity.

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With respect to understanding the biosynthetic pathways of these metabolites, more information is needed as to the extent by which diverse anti-microbial pathways coordinate and share biosynthetic enzymes. An important question in metabolic biosynthesis is understanding how chemical substrates are channeled along metabolic pathways from one enzyme to the next; from this review, it appears that some anti- microbial pathways solve this problem by using mega-synthase enzymes (e.g. zwittermicin A), but for other pathways, investigation of enzyme-enzyme interactions will be informative. An interesting future area of study will be to investigate the subcellular location of biosynthetic and storage proteins, especially of self-toxic compounds that may need to be sequestered. To that end, there have been advances in studying compartmentalization and secondary metabolite trafficking machinery (Roze et al., 2011; Lim and Keller, 2014; Kistler and Broz, 2015), which offer strategies to move forward.

A significant challenge in this discipline is the study of anti-microbial compounds in their native ecological context, as most reports are based only on in vitro studies. In particular, because the target pathogen affects the host plant, more information is needed as to how the plant and the anti-pathogenic microbe coordinate and regulate one another. For example, in the jadomycin pathway, evidence suggests that the plant sensing of the pathogen stimulates the anti-pathogen pathway in the associated beneficial microbe. The potential complexity of plant-microbe interactions and associated signaling networks are well studied in model systems such as Rhizobium (Janczarek et al., 2015). Though Rhizobium is a symbiotic microbe of legume plants, these studies suggest that a wealth of information remains to be explored for other plant-associated microbes, in particular endophytes (Kusari et al., 2012).

Also within the ecological context, basic biochemical questions are raised such as whether the anti-microbial pathway is regulated by the target pathogen, for example feedback inhibition once the pathogen has been eliminated. To help understand the

123 genetic regulation of these anti-microbial pathways, analysis of gene expression with respect to the microbial life cycle would be a useful avenue of investigation, similar to the interesting findings from the fusaricidin pathway. An interesting study concerning aflatoxin, a polyketide mycotoxin, revealed strong evidence for the potential link between the fungal growth stage and polyketide biosynthesis (Zhou et al., 2000). Furthermore, intracellular tracking of aflatoxin biosynthetic enzymes in Aspergillus parasiticus showed significant accumulation in the vacuoles of specific cells but its absence in neighbouring ones (Hong et al., 2008). This surprising result led Roze et al. (2011) to hypothesize the possibility of special and temporal gene expression of the associated biosynthetic pathway, at different developmental resolutions ranging from a single cell to fungal colony.

A related major challenge is that there are many natural products that exist in the literature that were initially isolated as part of screens for new compounds from total extracts, and hence the ecological functions of these compounds, as well as their underlying genes, remains unknown. As anti-pathogenic metabolites may be self-toxic, the evolution of self-resistance is a particularly fascinating avenue of study, which this review demonstrates has been investigated for a limited number of pathways (e.g. mupirocin). Diverse self-resistance mechanisms have been reported in the microbial literature (Schäberle et al., 2011; Westman et al., 2013; Stegmann et al., 2015), suggesting that each plant-asssociated microbe with anti-microbial activity may employ unique self-protection strategies.

The recent advances in genome sequencing combined with gene editing tools will facilitate more in-depth analysis of orthologous biosynthetic genes in diverse species. Bioinformatic genome mining of biosynthetic gene clusters, combined with new advances in metabolomics, may also lead to the discovery of a diverse array of novel bioactive natural products. Moreover, merging these techniques with knowledge of microbial co-evolution and ecology (Vizcaino et al., 2014) along with advanced

124 microscopy and imaging techniques will open a new era of discovery to harvest the diversity of natural products to combat evolving pathogens.

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Chapter 5: An endophytic fungus isolated from finger millet (Eleusine coracana) produces anti-fungal natural products

Authors’ contributions

Walaa Kamel Mousa designed and performed all experiments and data analysis and wrote the manuscript.

Adrian Schwan contributed chemicals and helped in compounds structural elucidation.

Jeffrey Davidson provided technical assistance.

Philip Strange performed the fruit fly toxicity assay.

Huaizhi Liu provided technical assistance

Ting Zhou contributed chemicals and helped in the experimental design.

France-Isabelle Auzanneau contributed chemicals and helped in the experimental design.

Manish N. Raizada helped in the experimental design and edited the manuscript.

Publication status: published.

Mousa WK, Schwan A, Davidson J, Strange P, Liu H, Zhou T, Auzanneau F-I and Raizada MN (2015) An endophytic fungus isolated from finger millet (Eleusine coracana) produces anti-fungal natural products. Frontiers in Microbiology 6, 1157

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5.1 Abstract

Finger millet is an ancient African cereal crop, domesticated 7000 years ago in Ethiopia, reaching India at 3000 BC. Finger millet is reported to be resistant to various fungal pathogens including Fusarium sp. I hypothesized that finger millet may host beneficial endophytes (plant-colonizing microbes) that contribute to the antifungal activity. Here I report the first isolation of endophyte(s) from finger millet. Five distinct fungal species were isolated from roots and predicted taxonomically based on 18S rDNA sequencing. Extracts from three putative endophytes inhibited growth of F. graminearum and three other pathogenic Fusarium species. The most potent anti-Fusarium strain (WF4, predicted to be a Phoma sp.) was confirmed to behave as an endophyte using pathogenicity and confocal microscopy experiments. Bioassay-guided fractionation of the WF4 extract identified four anti-fungal compounds, viridicatol, tenuazonic acid, alternariol and alternariol monomethyl ether. All the purified compounds caused dramatic breakage of F. graminearum hyphae in vitro. These compounds have not previously been reported to have anti-Fusarium activity. None of the compounds, except for tenuazonic acid, have previously been reported to be produced by Phoma. I conclude that the ancient, disease-tolerant crop, finger millet, is a novel source of endophytic anti-fungal natural products. This paper suggests the value of the crops grown by subsistence farmers as sources of endophytes and their natural products. Application of these natural chemicals to solve real world problems will require further validation.

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5.2 Introduction

Finger millet [Eleusine coracana (L.) Gaertn.] is an important cereal crop widely grown by subsistence farmers in Africa and South Asia (Goron and Raizada, 2015). Finger millet is known in many ancient languages, referred to as wimbi (in Swahili), bule (Bantu), dagussa/tokuso (Amharic), mugimbi (Kikuyu), tailabon (Arabic) and ragi (in South Asian languages) (Board on Science and Technology, 1996). Finger millet was domesticated in Western Uganda and Ethiopia around 5000 BC then reached India by 3000 BC (Hilu and Wet, 1976). Finger millet is well known by local farmers as a crop that tolerates stress conditions and that resists diverse pathogens (Goron and Raizada, 2015). Though limited scientific research has been conducted on this crop, comparative analysis data from tropical Africa (Burundi) showed that whereas 92-94 identifiable fungal mould species were found in maize grain and 97-99 in sorghum, only 4 were found in finger millet grain (Munimbazi and Bullerman, 1996). Furthermore, whereas 295-327 non-identified mould colonies were found in maize grain, and 508-512 in sorghum, only 4 were found in finger millet grain (Munimbazi and Bullerman, 1996).

The genus Fusarium includes widespread pathogens of cereal crops, including F. verticillioides in tropical maize which is associated with the production of carcinogenic mycotoxins, and F. graminearum, the causal agent of Gibberella ear rot in maize and Fusarium head blight in wheat; the latter diseases are associated with the mycotoxin deoxynivalenol (DON) (Sutton, 1982). By contrast, in the above African study (Munimbazi and Bullerman, 1996), no Fusarium species were identified in finger millet grain, compared to 28-40 Fusarium species in maize grain, 11-25 in sorghum, 25-51 in common bean, 4-16 in peanut and 29-43 in mung bean. Other studies, however, have reported the presence of diverse Fusarium species in finger millet in India (Pall and Lakhani, 1991; Saleh et al., 2012) and Africa (Amata et al., 2010), including F. graminearum, but without apparent plant disease symptoms (Adipala, 1992; Penugonda et al., 2010; Ramana et al., 2012; Saleh et al., 2012). These reports suggest that finger millet may have tolerance to this family of pathogenic fungi – restricting either their presence and/or pathogenicity.

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The resistance of finger millet grain to mould has been attributed to abundant polyphenols (Chandrashekar and Satyanarayana, 2006; Siwela et al., 2010). However, an emerging body of literature suggests that microbes that reside in plants without themselves causing disease, defined as endophytes, may contribute to host resistance against fungal pathogens (Johnston-Monje and Raizada, 2011; Mousa and Raizada, 2013). The mechanisms involved in endophyte-mediated disease resistance include competition for nutrients and space (Haas and Defago, 2005), induction of host resistance genes (Waller et al., 2005), improvement of host nutrient status (Johnston-Monje and Raizada, 2011), and/or production of anti-pathogenic natural compounds (Mousa and Raizada, 2013). I hypothesized that endophytes might contribute to the resistance of finger millet to fungal pathogens including members of the genus Fusarium as reported by local farmers. Fusarium is an ancient fungal genus, dated to at least 8.8 millions of years ago, and their diversification appears to have co-occurred with that of the C4 grasses (which includes finger millet), certainly pre-dating finger millet domestication in Africa (O’Donnell et al., 2013). A diversity of F. verticillioides (synonym F. moniliforme) has been observed in finger millet in Africa and it has been suggested that the species evolved there (Saleh et al., 2012). Despite these observations, I could not find reports of endophytes isolated from finger millet.

The objective of this study was to isolate endophytes from finger millet, to investigate endophyte extracts for anti-fungal activity using F. graminearum, then to use bio-assay guided fractionation to purify the active anti-Fusarium compounds. I report, to the best of our knowledge, the first isolation of endophyte(s) from finger millet. I show that extracts from three putative fungal endophytes have anti-Fusarium activity. From the most potent strain, confirmed to be an endophyte, I report the structures of all anti- Fusarium compounds, and show the in vitro microscopic effects of these compounds against F. graminearum.

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5.3 Material and methods

5.3.1 Isolation of finger millet endophytes

Finger millet seeds were of commercial food grade, originating from Northern India. Plants were grown under semi-hydroponic conditions (i.e. all nutrients provided in solution; on clay Turface MVP, Profile Products, Buffalo Grove, Illinois, USA) in 22.5 L pails placed in the field (Arkell Field Station, Arkell, ON, Canada, GPS: 43˚39′ N, 80˚25′ W, and 375 m above sea level) during the summer of 2012 and irrigated with the following nutrient solution: urea (46% N content), superphosphate (16% P2O5), muriate of potash (60% K2O), Epsom salt (16% MgO, 13% S), and Plant-Prod Chelated Micronutrient Mix (3 g/L, Plant Products, Catalog #10047, Brampton, Canada) consisting of Fe (2.1 ppm), Mn (0.6 ppm), Zn (0.12 ppm), Cu (0.03 ppm), B (0.39 ppm) and Mo (0.018 ppm). Six tissue pool sets (3 sets of: 5 seeds and 5 root systems from pre- flowering plants) were surface sterilized as follows: samples were washed in 0.1% Triton X-100 detergent for 10 min with shaking; the detergent was decanted, 3% sodium hypochlorite was added (10 min twice for seeds; 20 min twice for roots), followed by rinsing with autoclaved, distilled water, washing with 95% ethanol for 10 min; and finally the samples were washed 5-6 times with autoclaved, distilled water. Effective surface sterilization was ensured by inoculating the last wash on PDA and LB plates at 25°C and 37°C, respectively; all washes showed no growth. Tissues were ground directly in LB liquid medium in a sterilized mortar and pestle, then 50 µl suspensions were plated onto 3 types of agar plates [LB, Potato Dextrose Agar (PDA) and Biolog Universal Growth media (Catalog #70101, Biolog, Inc, Hayward, CA, USA)]. Plates were incubated at 25˚C or 37˚C for 2-7 days. A total of 10 fungal colonies with unique morphologies were selected and purified by repeated culturing on fresh media.

5.3.2 Testing the pathogenicity of the isolated endophytes To test if the most potent putative fungal endophyte identified in this study (WF4) is a pathogen, finger millet seeds were surface sterilized (see above) and planted on sterile Phytagel based medium (7 seeds per tube). The medium was prepared using

130 distilled water as follows (per 2 L): 1 package Murashige and Skoog modified basal salt mixture (Catalog #M571, PhytoTechnology Laboratories, USA), 500 ml nicotinic acid (1 mg/ml), 1 ml pyridoxine HCl (0.5 mg/ml), 10 ml thiamine HCl (100 mg/L), 1 ml glycine

(2 mg/ml), 0.332 g CaCl2, 1 ml MgSO4 (18 g/100 ml), and 4 g Phytagel. The tubes were kept in the dark for three days then transferred to light shelves (25˚C, 16 h light). When plants were one week old, the candidate endophyte (or control) was applied onto the gel surface (as 11 mm diameter agar discs). The negative control consisted of seedlings that received no endophytes, while the positive control consisted of seedlings that received the fungal pathogen Alternaria alternate. Disease symptoms were assessed visually at 10 days after inoculation with each endophyte or the controls. There were three replicates (tubes) for each treatment.

5.3.3 Root colonization study

To test the ability of the putative candidate endophyte (WF4) to colonize the root of finger millet, confocal microscopy was used. Finger millet seeds were surface sterilized (see above) and planted on sterile Phytagel based medium in glass tubes, each with 4-5 seeds (for medium preparation and growth conditions, see above). The fungal endophyte was applied (as 100 μL of a 48 h old culture grown in potato dextrose broth) to finger millet seedlings (17 days after germination) and incubated at room temperature for 24 h. The control consisted of seedlings incubated with potato dextrose broth only. There were three replicate tubes for the fungal endophyte or the control. Thereafter, roots were stained with Calcofluor (catalogue #18909, Sigma Aldrich), following the manufacturer’s protocol and scanned with a Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems, Mannheim, Germany) at the Molecular and Cellular Imaging Facility, University of Guelph. The conditions for the scanning were as follows: excitation at 405 nm with an Argon laser (17%) and 488 nm with a green helium laser (25%); the emission ranges were 418-452 nm and 603-689 nm; pinhole [Au] = 1.0 airy; objective lens = 63x oil immersion; and frame average = 3 times.

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5.3.4 Molecular identification of endophytic fungi using 18S rDNA Genomic DNA from cultured fungal endophytes was extracted using a DNA extraction kit (Fungi/Yeast Genomic DNA Isolation Kit, Catalog #27300, Norgen Biotek Corp, Niagara-on-the-Lake, ON, Canada) and then quantified using a Nanodrop machine (Thermo Scientific, USA). The extracted DNA was used as template to PCR amplify 18S rDNA using universal, degenerate primers as previously described (Borneman & Hartin, 2000). The PCR master mix consisted of the following (per 20 μl): 50 ng/μl DNA, 2.5 μl Standard Taq Buffer (10X, New England Biolabs, USA), 0.5 μl of 25 m dNTP mix, 1 μl of 10 m primer nu-SSU-0817-5` with sequence 5’- TTAGCATGGAATAATRRAATAGGA-3’, 1 μl of 10 m primer nu-SSU-1536-3` with sequence ATTGCAATGCYCTATCCCCA, 0.25 μl of 50 m gCl2, 0.25 μl of Standard Taq (10U/μl, New England Biolabs, USA), and double distilled water up to 20 μl total. PCR amplification conditions were: 96°C for 3 min, followed by 35 amplification cycles (94°C for 30 sec, 48°C for 30 sec, 72°C for 90 sec), and a final extension at 72°C for 7 min, using a PTC200 DNA Thermal Cycler (MJ Scientific, USA). The PCR products were separated on a 1.5 % agarose gel at ≤5 V/cm; 700 bp sized bands were excised and eluted using a gel purification kit (Illustra GFX 96 PCR Purification kit, GE Healthcare, USA). The purified DNA was sequenced at the University of Guelph Genomic Facility. Five distinct fungal strains were identified based on 18S rDNA sequence comparisons using BLAST searches to Genbank (default parameters for BLASTN: Expect threshold 10, Match/Mismatch Scores 1,-2, Gap Costs: Linear).

5.3.5 Fermentation of fungal endophytes and antifungal screening To enable endophytic fungi to grow and secrete metabolites, for each endophyte, 50 g of commercial white rice (variety: Basmati long grain) was placed in 100 ml H20 in a 2 L flask, which was incubated overnight at room temperature, autoclaved, then inoculated with 5 ml of endophytic fungi inoculum; for each inoculum, mycelia were inoculated into potato dextrose broth medium and placed on a shaker at 100 rpm for ~3 days. Endophytic fungi were fermented on rice solid medium for 4 weeks in darkness at room temperature

132 without shaking. Each endophytic fungus was fermented in triplicate. For metabolite extraction: Ethyl acetate was added (3 x 1.5 L) and the samples were incubated overnight at room temperature, filtered [0.2 micron filter paper (Whatman, USA), cotton and/or cheesecloth], then concentrated on a rotary evaporator at 45˚C. The ethyl acetate extract was then washed with water to remove salts and sugars. The ethyl acetate fraction was then dried and subject to liquid fractionation between Methanol and n-Hexane to remove long chain hydrocarbons. ethanol condensates were stored at 4˚C and were used to screen for anti-fungal activity (at a concentration range of 6.8 to 7.4 mg/ml) and to purify candidate anti-fungal compounds. Methanol condensates were diluted in a minimal amount of methanol and used to screen endophytic fungi for inhibition of growth of Fusarium graminearum (obtained from Agriculture and Agrifood Canada, Guelph, ON) using the agar diffusion method: Briefly, F. graminearum was grown for 24-48 h (25˚C, 100 rpm) in potato dextrose broth medium, then mycelia were added to melted, cooled PDA medium (1 ml of fungus into 100 ml of medium), mixed and poured into Petri dishes (100 mm x 15 mm), then allowed to re-solidify. Wells (11 mm diameter) were created in this pathogen-embedded agar by puncturing with sterile glass tubes, into which the methanol endophyte extracts were applied (200 µl per well). The agar plates were incubated at 30˚C for 48 h in darkness. The radius of each one of inhibition was measured. The fungicides Amphotericin B (Catalog #A2942, Sigma Aldrich, USA) and Nystatin (Catalog #N6261, Sigma Aldrich, USA) were used as positive controls, and methanol was used as a negative control. Each endophyte was screened in three independent replicates.

5.3.6 Anti-fungal target spectrum of extracts Endophyte methanol extracts which tested positive for activity against F. graminearum were re-screened for activity against a diversity of other fungi (obtained from Dr. Ting Zhou, Agriculture and Agrifood Canada, Guelph, Canada) using the agar diffusion method (described above) to characterize the activity spectrum of each extract. The fungi tested included: Alternaria alternata, Alternaria arborescens, Aspergillus flavus, Aspergillus niger, Bionectria ochroleuca, Davidiella (Cladosporium) tassiana, Diplodia pinea, Diplodia seriata, Epicoccum nigrum, Fusarium lateritium, Fusarium

133 sporotrichioides, Fusarium avenaceum (Gibberella avenacea, two isolates), Nigrospora oryzae, Nigrospora sphaerica, Paraconiothyrium brasiliense, Penicillium camemberti, Penicillium commune, Penicillium expansum, Penicillium afellutanum, Penicillium olsonii, Rosellinia corticium, Torrubiella confragosa, Trichoderma hamatum and Trichoderma longibrachiatum.

5.3.7 General procedure to purify the active anti-F. graminearum compounds from WF4 To permit large scale purification of the active compound(s) from the most potent anti-fungal endophyte extract (from Phoma endophyte WF4), the fermentation procedure described above was scaled up to five 2 L flasks, each containing 50 g of commercial white rice (variety: Basmati long grain) or millet (Variety: Pearl millet) placed in 100 ml

H20. To purify the active compound from WF4, the dry methanol residues were separated by flash liquid chromatography and preparative high pressure liquid chromatography. For flash liquid chromatography, the column was filled with flash grade silica gel (SiliaFlash® P60 230-400,Catalog # R12030B, SiliCycle, USA) and saturated with the desired mobile phase just prior to sample loading. Three grams of the dry residue extract were dissolved in chloroform and after rotary evaporation, applied as a dried silica band on the top of the column. The mobile phase (step-wise elution using hexane-ethyl acetate then ethyl acetate-methanol mixture with increasing polarity) was then passed through the column with air pressure resulting in sample separation. For Preparative HPLC, the most appropriate solvent system was determined using analytical HPLC before running the HPLC separation (Waters prep LC 4000 system, USA). The mobile phase combination was purified water (Nanopure, USA) and acetonitrile, pumped in a gradient manner (starting at 99:1 and ends at 0:100). Each injection consisted of 250 mg of the fraction dissolved in 8 ml of the solvent system. The mobile phase was pumped through the column (Nova-Pak HR C18 Prep Column, 6 µm, 60 Å, 25 x 100 mm prepack cartridge, part # WAT038510, Serial No 0042143081sp, Waters Ltd, USA) at a rate of 5 ml/min. The eluted peaks were detected by the UV detector (2487 Dual λ Absorbance Detector, Waters Ltd, USA) and collected separately. Samples were then dried under inert nitrogen gas. Thin layer chromatography (TLC) was used to visualize the bands

134 using TLC glass silica gel (60 F254, Whatman, Germany). Chromatograms were developed using different solvents and detected under UV light (254 and 366 nm), and heated at 95°C after spraying with vanillin-H2SO4 reagent (0.5 g vanillin dissolved in a mixture of 85 ml methanol, 10 ml acetic acid and 5 mL H2SO4). The fractions were dried by rotary evaporator, re-dissolved in their solvents and screened for antifungal activity as described above. Purified fractions were screened in the anti-fungal disc diffusion assay (against Fusarium graminearum, described above) to identify the active fraction(s). The active fractions were further purified. The active anti-fungal compounds (20 µl of 5mg/ml) were rescreened against F. graminearum using the agar disc diffusion method, and then subjected to structural analysis as described below.

5.3.8 Structure elucidation of the anti-fungal compounds from WF4 The chemical structures of the anti-fungal compounds isolated from WF4 was elucidated primarily using one and two dimensional nuclear magnetic resonance (NMR) techniques in combination with mass spectrometry (MS) methods. Further spectroscopic methods, such as IR (infra-red) spectroscopy provided additional structural information. All NMR spectra were conducted at the Guelph NMR Facility using a 600 DPX spectrometer (Bruker, Germany) operating at 600 MHz. The spectra were recorded in

CDCl3 and DMSO-d6. Structural assignments were based on spectra resulting from the following NMR experiments: 1H, APT, 1H-1H COSY, 1H-13C direct correlation (HSQC), 1H-13C long range correlation (HMBC). IR spectroscopy was conducted using a Bruker Alpha IR Spectrometer instrument (Bruker, Germany) located in the Department of Chemistry, University of Guelph. MS was conducted in the mass spectroscopy facility of the Advanced Analysis Centre at the University of Guelph using the following acquisition parameters; Ion Source (ESI), Ion Polarity (Positive), Alternating Ion Polarity (off), capillary exit (Resolution, 140.0 V), Mass Range Mode (Enhanced), Scan Begin (70 m/ ), Scan End (1000 m/ ), Trap Drive (58.9), Accumulation Time (1348 μs), Averages (3 Spectra), Auto MS/MS (on).

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5.3.9 Microscopic mechanisms of action Microscope slides were used to view the in vitro interactions between pathogen and each purified compound. Each slide was coated with a thin layer of PDA, then 20 µl of each purified compound (5 mg/ml) was applied adjacent to 100 µl of F. graminearum (mycelia grown for 24-48 h in potato dextrose broth at 25˚C, with shaking at 100 rpm). For the positive control, 20 µl of PROLINE® 480 SC fungicide (Bayer CropScience, USA) was used (at a concentration of 48 g/L of the active ingredient Prothioconazole). Each slide was incubated at 25˚C for 24 h then stained with neutral red (Sigma Aldrich, Catalog #57993) or Evans blue (Sigma Aldrich, Catalog # E2129) by placing 100 µl of stain on the slide, followed by a 3-5 min incubation, then washing 3-4 times with deionized water. Pictures were taken using a light microscope (MZ8, Leica, Wetzlar, Germany for neutral red staining; and BX51, Olympus, Tokyo, Japan for Evans blue staining). There were 3-4 replicates for each slide.

5.3.9 Statistical analysis

All statistical analysis was performed using Prism Software version 5 (Graphpad Software, USA). All error bars shown represent the range of data points.

5.4 Results

5.4.1 Isolation and identification of fungal endophytes from finger millet

To isolate fungal endophytes from finger millet, non-sterilized seeds were germinated and grown in non-sterilized clay Turface in pales placed in the field (Figure 5.1A). Three replicate pools of seeds and intact root systems, each consisting of five samples (plants at pre-flowering stage), were surface sterilized. Sterilized tissues were ground in LB liquid medium and the extracts were plated onto LB agar, Potato Dextrose Agar (PDA) and Biolog Universal Growth media. A total of 10 fungal strains with diverse morphologies were isolated from roots; no fungal endophytes were isolated from seeds. 18S rDNA sequencing and BLAST searches predicted only five distinct fungal strains, which were named WF1 (GenBank # KF957636), WF3 (GenBank # KF957638),

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WF4 (GenBank # KF957639), WF6 (GenBank # KF957641) and WF7 (GenBank # KF957642) (Tables A5.1 and A5.2, Figure 5.1B-F). The fungal endophytes of finger millet most closely resembled the genera Aspergillus, Penicillium and Phoma based on 18S rDNA sequence identities (Tables A5.1 and A5.2).

5.4.2 In vitro screen for endophyte extracts with anti-fungal activity

Solid rice culture medium was used to ferment the putative endophytic fungi (Figure 1G), then their concentrated methanol extracts were used to test for activity against F. graminearum using the agar diffusion method (Figure 5.1H). Briefly, F. graminearum was embedded in agar; endophyte extracts were applied in parallel to standard fungicide controls (Nystatin, Amphotericin), and then any resulting zones of inhibition of F. graminearum growth were measured. Extracts from three of the five putative endophytes (WF4, WF6, WF7) consistently inhibited growth of F. graminearum, with the extract from WF4 causing the greatest inhibition, and WF7 causing the least inhibition (Figure 5.1I).

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Figure 5.1: Isolation of fungal endophytes from finger millet and screen for anti- Fusarium activity from endophyte extracts. (A) Picture of a finger millet plant. (B-F) Pictures of fungal endophytes isolated from finger millet roots, with the corresponding best BLAST match taxonomic identification in brackets based on 18S rDNA sequencing: (B) WF1 (Aspergillus niger), (C) WF3 (Penicillium griseofulvum), (D) WF4 (Phoma sp), (E) WF6 (Penicillium chrysogenum), (F) WF7 (Penicillium expansum). (G) An example of an endophyte fermented on rice medium. (H) Example of an endophyte extract showing suppression of F. graminearum hyphae (white) using the agar diffusion method, along with fungicide controls. (I) Quantification of the effects of different endophyte extracts on the growth of F. graminearum (diameter of inhibition zone, n=3).

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To determine the target spectrum of anti-fungal activity, the three positive extracts were then tested against 25 genetically diverse fungal strains (Table 5.1). Extracts from WF4- WF6 and WF7 were observed to inhibit the growth of six of the 25 fungi, of which three were pathogenic strains of Fusarium (Fusarium lateritium, Fusarium sporotrichioides, Fusarium avenaceum); these extracts also inhibited two other known fungal crop pathogens (Alternaria alternata, Aspergillus flavus) in addition to the soil fungus, Trichoderma longibrachiatum (Table 5.1). None of the endophytic extracts were observed to be specifically targeted to Fusarium species. The extract from WF4 was observed to have the widest target spectrum (7/26 fungal strains) and caused the greatest inhibition zone in all cases (Table 5.1).

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Table 5.1: Effect of endophyte extracts on the growth of a diversity of fungal species

Target fungal species Mean diameter of inhibition zone with each extract*

WF4 WF6 WF7 Nystatin Amphotericin B (5 µg/ml) (10 µg/ml) Alternaria alternata 3.0±0.2 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 Alternaria arborescens 0.0 0.0 0.0 0.0 0.0 Aspergillus flavus 4.0±0.2 3±0.2 3.0±0.0 0.0±0.0 1.0±0.2 Aspergillus niger 0.0 0.0 0.0 0.0 0.0 Bionectria ochroleuca 0.0 0.0 0.0 0.0 0.0 Davidiella tassiana 0.0 0.0 0.0 0.0 0.0 Diplodia pinea 0.0 0.0 0.0 0.0 0.0 Diplodia seriata 0.0 0.0 0.0 0.0 0.0 Epicoccum nigrum 0.0 0.0 0.0 0.0 0.0 Fusarium avenaceum 0.0 0.0 0.0 0.0 0.0 Fusarium graminearum 6.5±0.2 3.0±0.0 1.5±0.2 1.5±0.2 0.0±0.0 Fusarium lateritium 6.0±0.5 0.0±0.0 2.5±0.5 0.0±0.0 1.0±0.2 Fusarium sporotrichioides 4.0±0.2 2.0±0.2 3.0±0.2 1.0±0.2 1.0±0.0 Gibberella avenacea 4.5±0.3 3.5±0.3 0.0±0.0 0.0±0.0 0.0±0.0 Nigrospora oryzae 0.0 0.0 0.0 0.0 0.0 Nigrospora sphaerica 0.0 0.0 0.0 0.0 0.0 Paraconiothyrium 0.0 0.0 0.0 0.0 0.0 brasiliense Penicillium afellutanum 0.0 0.0 0.0 3.0±0.2 3.0±0.3 Penicillium camemberti 0.0 0.0 0.0 2.0±0.3 5.0±0.3 Penicillium commune 0.0 0.0 0.0 1.5±0.2 3.5±0.2 Penicillium expansum 0.0 0.0 0.0 2.0±0.2 4.5±0.3 Penicillium olsonii 0.0 0.0 0.0 0.0 0.0 Rosellinia corticium 0.0 0.0 0.0 0.0 0.0 Torrubiella confragosa 0.0 0.0 0.0 0.0 0.0 Trichoderma hamatum 0.0 0.0 0.0 0.0 0.0 Trichoderma longibrachiatum 8.5±0.3 5.5±0.2 7.0±0.5 2.0±0.2 3.5±0.3 *Data expressed as mean± standard error of the mean (SEM, Mann Whitney t-test), n=3 or 4 replicates.

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5.4.3 Toxicity assays of the WF4 extract

The general toxicity of the WF4 extract was then tested using three standard assays. First, maize leaf punches were immersed in different dilutions of each endophyte extract, which were then incubated in the dark (Figure A5.3A); the WF4 extract showed toxicity against maize leaf punches as indicated by lesions (Figure A5.3B). In the second assay, the effect of the endophyte extract on the germination of maize and wheat seeds was tested (Figure A5.3C-F); the extract did not significantly inhibit germination. Finally, the effect of the endophyte extracts on the development of fruit flies (Drosophila melanogaster) was monitored from egg hatching to larval instar stages, to encapsulation into pupa, and finally metamorphosis into adult flies (Figure A5.3G). The undiluted endophyte extract (or buffer control) was applied to six sets of eggs (each set = 30 eggs) and the onset and duration of each developmental transition was monitored over 23 days. The WF4 extract significantly inhibited egg hatching, did not delay the subsequent encapsulation of larva into pupa, but dramatically delayed the metamorphosis from pupa to adult flies (Figure A5.3H); however, the timing when each developmental stage peaked was significantly delayed and diminished compared to the control (P≤0.05, Mann Whitney t-test). Taking into account both the leaf and insect assays, I conclude that the WF4 extract shows toxicity against plants and insects.

5.4.4 Confirming the endophytic nature of WF4

An endophyte is defined as a microbe that is able to colonize the internal tissues of its plant host without causing disease (Wilson, 1995). To test these criteria, two experiments were undertaken for WF4, the focus of this study:

Pathogenicity: Finger millet seedlings were incubated with WF4 or with Alternaria alternata, a known pathogen of finger millet (Kumar, 2010). The seedlings incubated with A. alternata developed characteristic disease symptoms including reduction in plant length, black roots and leaf spots (Figure 5.2A), compared to the healthy buffer control (Figure 5.2B). In comparison, there were no disease symptoms observed in the seedlings challenged with WF4 (Figure 5.2C).

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Figure 5.2: Testing the pathogenicity of the putative endophyte WF4 on finger millet. (A) Representative pictures showing the effect of the known pathogen Alternaria alternata on finger millet seedlings (negative control). (B) Representative pictures showing the effect of buffer on finger millet seedlings (positive control). (C) Representative pictures showing the effect of the fungus WF4 on finger millet seedlings.

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Plant colonization: To test the ability of WF4 to colonize the roots of finger millet (from where it was originally isolated), confocal imaging was used. Seedlings that were inoculated with the buffer only (control) showed no observable fungal growth inside the tissues (Figure 5.3A-B). In comparison, WF4 could efficiently colonize the root tissues including the epidermis and sub-epidermal layers (Figures 5.3C-F).

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Figure 5.3: Root colonization study using confocal scanning laser microscopy. (A-B) Representative pictures of root tissues inoculated with the buffer control. (C- F) Representative pictures of root tissues inoculated with WF4. WF4 fluoresces purple-blue due to staining with Calcofluor. Plant tissues appear red due to autofluorescence. White arrows point to the fungus, WF4.

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5.4.5 Bio-assay guided purification and structure elucidation of the anti-fungal compounds from endophyte WF4

Since the extract from endophyte WF4 had the most potent and the widest antifungal target spectrum, it was subjected to bio-assay guided purification using F. graminearum to isolate the active anti-fungal compound(s). The fermentation and extraction procedures were scaled up (see Methods) to permit purification and structural elucidation using two different solid medium (rice and millet). Flow charts that illustrate the purification procedures are included (Figures A5.1 and A5.2). LC/MS analysis was conducted on the rice-derived extract (Figure 4A, Table S3) and millet-derived extract (Figure 5.4B, Table A5.4), and bioactive compounds eluted from the column were used as reference to track the retention time and relative abundance of these compounds relative to the total extract ingredients. The retention times (min) of each of the purified compounds were as follows: Compound 1: 14.63; Compound 2: 15.9; Compound 3: 15.98; Compound 4: 18.94 (Figure 5.4).

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Figure 5.4: HPLC chromatograms of extracts derived from endophyte WF4. Shown are chromatograms when the endophyte was cultured on (A) rice medium and (B) millet medium. The arrows show the retention time of peaks exhibiting antifungal activity.

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Mass analysis showing (M+H+) of the purified antifungal compounds (Figure 5.5A,D,G,J) are shown, alongside the corresponding zoomed-in peaks from extracts of WF4 grown on either millet medium (Figure 5B,E,H,K) or rice medium (Figure 5C,F,I,L). The molecular weights of each of the purified compounds were as follows: Compound 1: 253; Compound 2: 197; Compound 3: 258; Compound 4: 272 (Figure 5.5). Compound 1 could be extracted from WF4 grown on both millet and rice (Figure 5.5B,C), whereas Compound 2 was observed on rice culture only (Figure 5.5E,F). Compounds 3 and 4 could be observed from millet culture only (Figure 5.5H,I,K,L).

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Figure 5.5

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Figure 5.5: Mass spectroscopy of peaks with antifungal activity detected in extracts from endophyte WF4. (A, D, G, J) (M+H+) values of compounds 1, 2, 3 and 4 respectively. The insets show the retention time of each respective compound. (B, E, H, K) Magnification within the whole spectrum of the extract of WF4 cultured on millet medium, showing the absence or presence of peaks corresponding to compounds 1, 2, 3 and 4 respectively. (C, F, I, L) Magnification within the whole spectrum of the extract of WF4 cultured on rice medium, showing the absence or presence of peaks corresponding to compounds 1, 2, 3 and 4 respectively.

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The purified compounds were then subjected to further spectroscopic structure elucidation. Detailed spectroscopic data for the purified compounds are included (1D- NMR, Table A5.5). The IR, mass and 1D-NMR data were as follows:

5.4.5.1 Isolation of Compound 1 (isolated from rice culture and confirmed on millet cultures by LC-MS)

Colorless crystals with molecular formula, C15H11NO3 and molecular weight 253, precipitated from the ethyl acetate-methanol (95:5 v/v) fraction, and then purified by crystallization yielding 2.5 g from 20 g total extract. The crystallization process involved dissolving in minimum volume of chloroform, followed by drop wise addition of hexane until slight turbidity was achieved; the solution was left to re-crystallize at room temperature. The process was repeated three times for further purification, followed by final washing with hexane. The purified compound eluted as a single band on a glass TLC with an Rf value of 0.2 using a solvent mixture of ethyl acetate-hexane (v/v 80:20), and with an Rf value of 0.34 using 100% ethyl acetate as solvent. The IR, 1H NMR, 13C NMR data were as following:

IR: 3861, 2395, 2172, 1637 cm-1.

1H NMR (600 MHz, DMSO): 12.2 (1H, s, OH), 9.5 (1H, s, OH), 9.1 (1H, s, NH), 7.34 (1H, dd, J = 8.0, 1.0 Hz), 7.32 (1H, ddd, J = 8.0, 8.0, 1.0 Hz), 7.29 (1H, dd, J = 8.0, 8.0 Hz), 7.09 (1H,dd, J = 8.0, 1.0 Hz), 7.07 (1H, ddd, J = 8.0, 8.0, 1.0 Hz), 6.82 (1H, m), 6.72 (1H, ddd, J = 8.5, 1.5, 1.0 Hz), 6.71 (1H, dd, J = 2.5, 1.5 Hz).

13C NMR (150 MHz, DMSO): 158.3 (C), 157.2 (C), 142.2 (C), 134.9 (C), 133.1 (C), 129.3 (CH), 126.4(CH), 124.4 (CH), 124.06 (C), 122.1 (CH), 120.9 (C), 120.3 (CH), 116.6 (CH), 115.2 (CH), 114.6 (CH). Comparing these results with reference data (Wei et al., 2011), the compound was confirmed as viridicatol alkaloid (3-Hydroxy-4-(3- hydroxyphenyl)-2-quinolone monohydrate), in the 4-arylquinolin-2(1H)-ones class (Figure 5.6A).

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5.4.5.2 Isolation of Compound 2 (isolated from rice culture)

A yellowish brown amorphous solid with molecular formula, C10H15O3N and molecular weight, 197 g/mol eluted at 52 min, yielding 15 mg from 250 mg. The IR, 1H NMR, 13C NMR data were as follows:

IR: 3864, 3331, 2924, 2361, 1617 cm-1.

1H NMR (600 MHz, d4 methanol): 0.95 (t, J=7.2 Hz, 3H, methyl), 1.01 (d, J= 6.9 Hz, 3H methyl), 1.34 (m, CH2), 1.89 (m, CH), 2.4 (s, 3H, methyl keto), 3.7 (d, CO-CH-N)

13C NMR (150 MHz, d4 methanol): 198.8 (C), 195.01 (C), 178.9 (C), 105.6 (CH), 67.20 (CH), 38.3 (CH), 27.06 (CH2), 24.26 (CH3), 16.65 (CH3), 12.42 (CH3)

Comparing these results with reference data (Mikula et al., 2013) the compound was confirmed as tenuazonic acid (3-acetyl-5-sec-butyl tetramic acid) (Figure 5.6B).

5.4.5.3 Isolation of Compound 3 (isolated from millet culture)

A white amorphous powder with molecular formula, C14H10O5 and molecular weight, 258 g/mol eluted at 22 min, yielding 8 mg from 250 mg. The IR, 1H NMR, 13C NMR data were as follows:

IR: 3500, 2360, 1664, 1164 cm-1.

1H NMR (600 MHz, d4 methanol): 2.8 (3H, s, CH3), 7.3 (d, J=1.8), 6.7 (d, J=2.5), 6.6 (d, J=2.5 Hz) and 6.4 (d, J=1.8).

13C NMR (150 MHz, d6 DMSO): 25.8 (CH3), 99 (C), 100.6(CH), 101.9 (CH), 104.3 (C), 109(C), 118.56 (CH), 139.8(CH), 140 (C), 154.4 (C), 159.8(C), 166.2(C), 166.8 (C), 167 (C).

Comparing these results with reference data (Tan et al., 2008; Ashour et al., 2011), the compound was confirmed as alternariol (Figure 5.6C).

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5.4.5.4 Isolation of Compound 4 (isolated from millet culture)

White crystals with molecular formula, C15H12O5 and molecular weight, 272 g/mol, were eluted at 42 min, yielding 10 mg from 250 mg. The IR, 1H NMR, 13C NMR data were as follows:

IR: 3320, 2925, 2854, 2360, 1666 cm-1.

1H NMR (600 MHz, d4 methanol): 2.8 (3H, s, CH3), 3.9 (3H, s, OCH3), 7.3 (d, J=1.8), 6.7 (d, J=2.5), 6.6 (d, J=2.5 Hz) and 6.59 (d, J=1.8).

13C NMR (150 MHz, d6 DMSO): 25.5 (CH3), 56.3 (OCH3), 98.9 (C), 99.6(CH), 102 (CH), 103.8 (C), 109(C), 118.1 (CH), 138.2(CH), 138.8 (C), 153 (C), 166.6(C), 164.6(C), 165.1 (C), 166.6 (C).

Comparing these results with reference data (Ashour et al., 2011), the compound was confirmed as alternariol-5-O-methyl ether or djalonensone (3,7-Dihydroxy-9-methoxy-1- methyl-6H-dibenzo[b,d]pyran-6-one) (Figure 5.6D).

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Figure 5.6: Structures of the purified anti-Fusarium compounds from endophyte WF4. Shown are the structures for: (A) viridicatol alkaloid, (B) tenuazonic acid, (C) alternariol, and (D) alternariol monomethyl ether.

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5.4.6 Effects of pure endophyte-derived compounds on F. graminearum in vitro

The putative anti-fungal compounds, viridicatol, tenuazonic acid, alternariol and alternariol-mono methyl ether were verified as having anti-F. graminearum activity by using the agar disc diffusion method (diameter of fungal inhibition zone): application of the compounds 1 to 4 (20 µl of 5 mg/ml) caused inhibition zones of 1.8, 2, 1.5, 1.5 mm (respectively) compared to the solvent buffer (0-0.5 mm) (Figure 5.7A, B, C and D), respectively. The purified compounds (20 µl of 5 mg/ml) were co-incubated for 24 h with F. graminearum then stained with neutral red or Evans blue (stains dead cells blue), to examine the in vitro microscopic interactions between the compounds and the pathogen (Figure 5.7E). All the compounds caused apparent breakage of the F. graminearum hyphae (Figure 5.7F, G, H and I) compared to the buffer controls (Figure 5.7J, K, L and M), respectively. Similar to the Proline fungicide control (Figure 5.8A), the fungal hyphae adjacent to each compound stained blue with Evans blue (Figure 5.8C, E, G and I) compared to buffer controls (Figure 5.8B, D, F, H and J), suggesting that hyphae in contact with each endophytic compound died.

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Figure 5.7

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Figure 5.7: The effects of the purified compounds on F. graminearum in vitro using the agar disc diffusion assay and neutral red staining. (A-D) Representative pictures of the agar disc diffusion assay of the purified antifungal compounds, (A) viridicatol, (B) tenuazonic acid, (C) alternariol and (D) alternariol monomethyl ether (n=3). The inhibition of F. graminearum is shown by the clear halo around each disc soaked with the purified compound. (E) Cartoon of the experimental methodology to examine microscopic in vitro interactions between F. graminearum (pink) and each compound (orange) or the buffer control (respective compound solvent). The microscope slides were pre-coated with PDA and incubated for 24 h. F. graminearum hyphae were then stained with the vitality stain, neutral red. Shown are representative microscope slide pictures (n=3) of the interactions of F. graminearum with: (F) viridicatol compared to (J) the buffer control; (G) tenuazonic acid compared to (K) the buffer control; (H) alternariol compared to (L) the buffer control; (I) alternariol monomethyl ether compared to (M) the buffer control. The blue arrows point to areas of apparent breakage of F. graminearum hyphae.

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Figure 5.8

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Figure 5.8: The effects of the purified compounds on F. graminearum in vitro using Evans blue staining. Shown are representative microscope slide pictures (n=3) of the interactions of F. graminearum with: (A) the fungicide PROLINE compared to (B) the buffer control; (C) viridicatol compared to (D) the buffer control; (E) tenuazonic acid compared to (F) the buffer control; (G) alternariol compared to (H) the buffer control; (I) alternariol monomethyl ether compared to (J) the buffer control. In panel (A), the aggregated particles between the hyphae are precipitates of PROLINE.

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5.5 Discussion

I hypothesized that finger millet may host endophytes with anti-fungal activity, including against the pathogenic species of Fusarium, because this crop is known to be resistant to many pathogens (Munimbazi and Bullerman, 1996), including Fusarium ssp. pathogens of Africa where the crop was domesticated (Adipala, 1992; Amata et al., 2010; Saleh et al., 2012) and South Asia (Penugonda et al., 2010; Ramana et al., 2012). To the best of our knowledge, this is the first report of endophyte(s) from finger millet. The putative endophytes were isolated from plants from first generation seeds from India, and then grown on Turface clay rock using hydroponics rather than on soil, suggesting they did not derive from Canadian soil. This growth system might explain why mycorrhizal fungi were also not isolated. Here, extracts from three of the five fungal endophytes (WF4, WF6 and WF7) isolated from finger millet roots inhibited growth of F. graminearum in vitro, along with six other fungal pathogens (Alternaria alternata, Aspergillus flavus, Fusarium lateritium, Fusarium sporotrichioides, Fusarium avenaceum and Trichoderma longibrachiatum), including three other pathogenic Fusarium species (Table 5.1). The extract from endophyte WF4 (predicted to be a Phoma sp.) had the widest anti-fungal target spectrum (7/26 fungal pathogens tested, Table 5. 2) and was the most potent of the extracts against F. graminearum (Figure 5.1I). Furthermore, the extract from endophyte WF4 inhibited the growth of four tested Fusarium pathogens (Table 5.1). For these reasons, we decided to focus the current study on this endophyte. Inoculation of finger millet seedlings with WF4 resulted in no disease symptoms (Figure 5.2); furthermore, Calcofluor staining and confocal microscopy imaging confirmed the ability of WF4 to efficiently colonize root tissues (Figure 5.3). Combined with the fact that this fungus was initially isolated from surface sterilized plants, these results confirmed the endophytic behaviour of WF4 in finger millet. Bio- guided isolation of the active antifungal compounds from the extract of WF4 were conducted using two fermentation media, rice and millet. Inactive fractions, including those that might have compounds active against other fungi, were not subjected to further study. The structural elucidation of the active anti-Fusarium compounds from WF4 revealed two compounds when the fungus was cultured on rice (viridicatol and tenuazonic acid), and three compounds when cultured on millet (viridicatol, alteraniol

159 and alteraniol methyl ether) (Figures 5.4, 5.5, and 5.6) and (Figures A5.1 and A5.2). As different media are enriched in different metabolic precursors, these results were not surprising, as previously shown (Aly et al., 2008). Mechanistically, all the purified compounds caused dramatic breakage of F. graminearum hyphae (Figure 5.7 and 5. 8).

5.5.1 Anti-fungal compounds previously isolated from Phoma species The focus of this study was endophytic fungal strain WF4, predicted to be a Phoma sp. Phoma species have previously been shown to be endophytes of other plants. For example, a Phoma species was isolated as an endophyte from the Chinese medicinal plant Arisaema erubescens (Wang et al., 2012), from the Chinese medicinal plant Taraxacum mongolicum Hand.-Mazz (Zhang et al., 2013b), a Tibetan medicinal plant Phlomis younghusbandii Mukerjee (Zhang et al., 2013a) and from Acer ginnala Maxim, a tree from northeastern China (Qi et al., 2012).

Consistent with our study, anti-fungal compounds have previously been isolated from Phoma species. For example, the compound phomalone, purified from Phoma etheridgei, which was isolated from black galls of Canadian aspen trees, showed antifungal activity against Phellinus tremulae (an aspen decay fungus) (Ayer and Jimenez, 1994). In another study, an α-tetralone derivative, (3S)-3,6,7-trihydroxy-α- tetralone, was purified from a Phoma endophyte, isolated from the Chinese medicinal plant Arisaema erubescens, and shown to have antifungal activities against Fusarium oxysporum and Rhizoctonia solani (Wang et al., 2012). The polyketide, 5- hydroxyramulosin, isolated from an endophytic fungus morphologically similar to Phoma, inhabiting the tree Cinnamomum mollissimum from Malaysia, was shown to have antifungal activity against Aspergillus niger (Santiago et al., 2012). The compounds, epoxydines A and B, epoxydon, (4R,5R,6S)-6-acetoxy-4,5-dihydroxy-2- (hydroxymethyl)cyclohex-2-en-1-one and 2-chloro-6-(hydroxymethyl)benzene-1,4-diol, were purified from a Phoma sp. isolated from Salsola oppositifolia (Amaranth family) following fermentation, and shown to have antifungal activities against the fungus

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Microbotryum violaceum (Qin et al., 2010). Finally, the polyketide xanthepinone, a metabolite with antifungal activity against the fungal pathogens, Pyricularia oryzae, Botrytis cinerea and Phytophthora infestans, was purified from a fungus classified as a Phoma sp. which was collected from French Alpine soil (Liermann et al., 2009). Our study further suggests that it may be useful to further explore diverse Phoma species for new anti-fungal compounds.

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5.5.2 Viridicatol

In this study, I identified the alkaloid viridicatol as one of active anti-fungal compounds from WF4 endophyte extracts fermented on both rice and millet media (Figures 5.5A-C and 5.4A). The ring system of viridicatol is synthesized from phenylalanine and anthranilic acid (precursor to auxin and tryptophan) (Luckner and Mothes, 1962), from benzodiazepine derivatives (Luckner et al., 1969). Viridicatol was shown to be cytotoxic towards various tumor cell lines (Wei et al., 2011). The related compound viridicatin (di-hydroxylated version of viridicatol) was first described in 1953, isolated from Penicillium viridicatum Westling (Cunningham and Freeman, 1953) and subsequently shown to be produced by different Penicillium sp. (Frisvad et al., 2004). Viridicatol has also been reported outside of the Penicillium clade, as it has been purified from Aspergillus versicolor from beach sand in Australia (Fremlin et al., 2009) and from a marine isolate of A. versicolor from Sakhalin Bay, Russia (Yurchenko et al., 2010). Emerging from the literature is that viridicatol is primarily produced by fungi that survive harsh environmental conditions such as pathogen exposure (Cunningham and Freeman, 1953; Sutton, 1982; Payne and Widstrom, 1992) and in extreme environments such as the Mir space station (Kozlovskii et al., 2002) and mangroves (Wei et al., 2011). These observations are consistent with this study, in which viridicatol was isolated from an endophyte of finger millet, a crop that is adapted to extreme stress, including heat, drought and low soil fertility (Uma et al., 1995). To the best of our knowledge, this is the first report of viridicatol from a Phoma species, and the first report of viridicatol having anti-fungal activity. Understanding the full ecological role of viridicatol may come from knocking out or over-expressing the biosynthetic genes that encode this compound.

5.5.3 Tenuazonic acid

In this study, I identified tenuazonic acid (5S,8S)-3-acetyl-5-sec-butylpyrrolidine- 2,4-dione), a tetramic acid analog, as one of the active anti-fungal compounds from the extract of WF4 grown on rice medium (Figures 5.5D, F and 4B). However, the compound was not detected when the fungus was fermented on millet medium (Figure

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5.5E), suggestive of an important underlying plant-endophyte interaction (biochemical precursor or inducer/repressor). Tenuazonic acid is a potent mycotoxin belonging to the 3-acylpyrrolidine-2,4-diones family of tetramic acid analogues (Weidenbörner, 2001). It is biosynthesized from L-isoleucine in Alternaria tenuis (Gatenbeck and Sierankiewicz, 1973). In previous studies, tenuazonic acid showed antibacterial, antiviral and antitumor activities (Gitterman, 1965; Miller et al., 1963). It also inhibited the growth of human adenocarcenoma (Kaczka et al., 1963). Mechanistically, tenuazonic acid suppresses the release of newly formed peptides from the ribosome, resulting in inhibition of protein synthesis (Shigeura and Gordon, 1963). However, the practical application of the compound is restricted due to its extreme toxicity. The compound was reported to be produced by various fungi including Alternaria alternata, A. tenuissima (Davis et al., 1977), Pyricularia oryzae (Iwasaki et al., 1972), Aspergillus sp. (Miller et al., 1963) and Phoma sorghina (Steyn and Rabie, 1976). To the best of our knowledge, this is the first report that tenuazonic acid has anti-Fusarium activity.

5.5.4 Alternariol and alternariol monomethyl ether

In this study, I identified alternariol and alternariol monomethyl ether as anti-fungal polyketide compounds produced by endophyte WF4 when cultured on millet medium (Figure 5.5G, J, H, K and 4C-D). However, these compounds were not detected when the fungus was fermented on rice medium (Figure 5.5I, L), again suggestive of an underlying plant-endophyte interaction. These benzopyranone derivatives are biosynthesized from acetyl-CoA and malonyl-CoA via the polyketide pathway (Tanahashi et al., 2003). Alternariol derivatives are typical examples of the dibenzo-α- pyrones family, members of which are key intermediates in the biosynthesis of pharmacologically active compounds such as cannabinoids (Teske and Deiters, 2008) and progesterone (Edwards et al., 1998). They were reported to be toxic to chickens (Ostry, 2008) and brine shrimp (Zajkowski et al., 1991). Alternariol exhibits remarkable cytotoxicity against L5178Y mouse lymphoma cells (Aly et al., 2008). Further studies showed that alternariol and its monomethyl ether could inhibit topoisomerase I and II by interchelating DNA in human carcinoma cell lines (Fehr et al., 2009). Alternariol

163 monomethyl ether exhibited anti-nematodal activity against Caenorhabditis elegans and inhibited spore germination of the rice blast fungus, Magnaporthe oryzae (Meng et al., 2012). Alternariol monomethyl ether (djalonensone) was previously isolated from the roots of the west African medicinal plant Anthocleista djalonensis and claimed to be a taxonomic marker of this plant species (Onocha et al., 1995). However, some debates have been raised as to whether this metabolite might actually be produced by an endophytic fungus inhabiting it (such as Acremonium sp.) not the plant itself (Hussain et al., 2007). The dibenzo-α-pyrones family is mainly produced by Alternaria species in addition to some other fungi such as Hyalodendriella sp., Cephalosporium acremonium, Microsphaeropsis olivacea, Penicillium verruculosum, Botrytis allii TC 1674 and Phoma sp (Mao et al., 2014). To the best of our knowledge, this is the first report of alternariol and alternariol monomethyl ether from a Phoma sp., and the first report of them having anti-Fusarium activity.

5.6 Study Limitations and Future Experiments

There were some limitations of this study. First, this study confirmed the endophytic behaviour of only one of the fungal strains isolated (WF4); more detailed studies of the other strains will be reported in future papers. Second, only 18S rDNA sequences were used for taxonomic identification of the putative fungal endophytes; as multiple fungal species can share the same 18S sequence (Reiss et al., 1997), the identification of these endophytes will require further validation from additional DNA sequencing. Third, the results reported in this study were based on in vitro assays only, and further studies will be required to test their relevance to the natural plant habitat. In particular, a separate study is required to determine whether the dosages used here in vitro (5 mg/ml) are relevant in planta as they are relatively high compared to the average dosage used for commercial fungicides (Reis et al., 2015). According to previous research, in vitro dosage data has only limited value for predicting field dosages, which varies by fungicide (Reis et al., 2015). This lack of in vitro versus field dosage correlation might be due to variation between fungicides in their uptake, stability under field conditions or degree of degradation by the plant. Field application requires in planta

164 testing followed by further studies to increase the potency/stability of a fungicide, perhaps by chemical/biological derivatization (Kirschning and Hahn, 2012). Similar to the challenge faced by natural products chemists, it is currently not possible to purify these endophytic chemicals in sufficient amounts for in planta testing, especially on large crops. However, these natural products could be used for future synthesis and semi- synthesis as lead compounds (Kobayashi and Harayama, 2009). As for testing of the putative endophytes themselves in planta, finger millet is not susceptible to F. graminearum, so future experiments would need to employ a heterologous host, but the endophytes may not colonize a non-host plant.

We suggest some additional future experiments of interest. First, given that I were able to isolate several putative endophytes from only a single finger millet genotype, it will be valuable to bioprospect diverse finger millet genotypes from Africa and South Asia including ancient and ancestral varieties (Goron and Raizada, 2015). One of the available resources for conducting this type of research is the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) which has a large collection of finger millet landraces from around the world (http://www.icrisat.org/) (Goron and Raizada, 2015). In a previous study, I demonstrated that different genotypes of maize possess a wide diversity of endophytes with potentially useful traits (Johnston-Monje and Raizada, 2011). It may be useful to test this potential diversity of finger millet endophytes for their ability to combat not only Fusarium species, including African isolates of Fusarium verticilliodes, but also Pyricularia oryzae, the fungal agent responsible for blast disease, to which finger millet is highly susceptible (Yaegashi and Asaga, 1981). However, as finger millet is susceptible to P. oryzae, it seems less likely that the putative endophytes isolated here would have anti-P. oryzae activity. Second, prior to using these endophytes or their compounds as commercial biocontrol agents, more detailed experiments are needed to examine the toxicities and safety of the endophyte derived extracts, including at different concentrations, their effects on mammalian models and their persistence in ecosystems. Preliminary experiments using the extract from endophyte WF4 fermented on rice medium showed that it causes variable degrees of

165 toxicity against leaf discs, fruit flies and seeds (Figure A5.3 and Appendix Method A5.1). Third, as F. graminearum has the potential to cause root rot in the seedlings of diverse cereals (Chongo et al., 2001), it is interesting to speculate whether the seedling root endophytes isolated from this study suppress Fusarium root disease in finger millet; this speculative hypothesis will require future testing but might suggest the biological relevance of these endophytes, given that Fusarium diseases in other cereals are most common in shoots and inflorescences.

5.7 Conclusions

To the best of our knowledge, this is the first report of endophyte(s) from finger millet. Here, putative fungal endophytes were isolated from root tissues of Indian finger millet. In vitro, the endophytic extracts showed activity against diverse fungi, including multiple pathogenic species. Using a bio-guided assay, I purified four anti-fungal compounds from two different cultures of the most potent anti-fungal strain (strain WF4, predicted to be a Phoma sp), which was confirmed to behave as an endophyte. Of these compounds, viridicatol, alternariol and alternariol monomethyl ether have not been reported to be produced by Phoma previously. The mode of action of all of these compounds involves apparent breakage of Fusarium hyphae. This study suggests that exploration of endophytes in ancient, subsistence crops may result in the identification of organisms or chemicals with potential to control the diseases of modern crops. These crops have been grown continuously without pesticides including fungicides. I hope the results presented here will encourage other researchers to explore the diversity of finger millet and other ancient crops which have been largely ignored by many Western scientists. Practical application of the endophytic natural products identified from this study will require in planta experiments at the field level.

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Chapter 6: Characterization of antifungal natural products isolated from endophytic fungi of finger millet (Eleusine coracana)

Authors’ contributions

Walaa Kamel Mousa designed and performed all experiments and data analysis and wrote the manuscript

Adrian Schwan contributed chemicals and helped in structural elucidation of the compounds

Manish N. Raizada helped in the experimental design and edited the manuscript

Publication status: pending submission.

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6.1 Abstract

Finger millet is an ancient Afro-Indian crop that is reported to be resistant to many pathogens including the fungal pathogen, Fusarium graminearum. I previously reported the first isolation of putative fungal endophytes from finger millet. I showed that the crude extracts of four putative fungal endophyte strains had anti-Fusarium activity, but only one was characterized in detail. The objectives of this study were to confirm the endophytic lifestyle of the three remaining anti-Fusarium microbes, to identify the underlying major antifungal compounds, and to initially characterize the mode(s) of action of each pure compound. Using confocal microscopy and a plant pathogenesis assay, the three fungal strains were confirmed to be endophytes, based on their ability to colonize finger millet roots without causing disease. Using bio-assay guided fractionation combined with spectroscopic structural elucidation, three anti-Fusarium secondary metabolites were purified and characterized from the crude extracts of these endophytes. These molecules were not previously reported to have antifungal activity nor were they isolated from fungi. The purified antifungal compounds were 5-hydroxy benzofuran, dehydrocostus lactone (guaianolide sesquiterpene lactone), and harpagoside (an iridoide glycoside). Light microscopy combined with vitality staining, used to visualize the in vitro interactions between each pure compound and F. graminearum, suggested a mixed fungicidal/fungistatic mode of action. I conclude that finger millet possesses fungal endophytes that can synthesize anti-fungal compounds that have not previously been reported as bio-fungicides against F. graminearum.

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6.2 Introduction

Finger millet (Eleusine coracana) is an ancient cereal crop widely grown by subsistence farmers in Africa and India. Finger millet was domesticated in Ethiopia and Uganda at 5000 BC then reached India by 3000 BC (Hilu and Wet, 1976). There are some folk remedy reports about the use of finger millet as a diaphoretic and diuretic, and as a remedy for leprosy and liver diseases (Watt and Breyer-Brandwijk, 1932) and for measles, pleurisy, pneumonia, and small pox (Duke and Wain, 1981). Unlike other related cereals, finger millet is well known for its ability to tolerate stress conditions and to resist many pathogens including Fusarium graminearum (Chapter 5) (Munimbazi and Bullerman, 1996; van der Lee et al., 2014; Mousa et al., 2015a; Sundaramari, 2015). F.graminearum is a serious fungal pathogen that causes Fusarium Head Blight (FHB) in wheat and Gibberella Ear Rot (GER) in corn (Miller et al., 2007; Wegulo et al., 2015). Both diseases result in catastrophic losses in grain yield and are associated with mycotoxin accumulation in grains (Wong et al., 1995). Fusarium sp. are ancient fungi that have been dated to 8.8 mya in Africa (O’Donnell et al., 2013), the same continent where finger millet was domesticated.

I hypothesized that the potential long-term co-evolution between finger millet and Fusarium sp. may not only have placed selection pressure on the genome of finger millet but also on its associated microbiome. Specifically, I hypothesized that finger millet may host endophytes that confer resistance to F.graminearum. Endophytes have been reported to help their host plants to combat pathogens (Mousa et al., 2015a; Mousa et al., 2015b; Shehata et al., 2016). Consistent with our hypothesis, in a previous study, I reported the isolation of four putative fungal endophytes (strains WF4-7) from finger millet and showed that their extracts had anti-fungal activity against F.graminearum (Mousa et al., 2015a). Only one of these endophytes, Phoma sp. strain WF4, was analyzed in detail including isolation of the underlying anti-Fusarium compounds.

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The objectives of this study were to confirm the endophytic behaviour of the three remaining, putative, anti-Fusarium fungal endophytes, to identify the underlying antifungal compounds and to study the mode of action of each pure compound.

6.3 Material and methods

6.3.1 Source of biological materials

Putative fungal endophyte strains WF6 and WF7 (Penicillium sp.) were previously isolated from finger millet roots (Genbank: KF957641 and KF957642) (Mousa et al., 2015a). Putative fungal endophytes strain WF5 (Fusarium sp.) was isolated in this study from finger millet shoots (Genbank: KF957640) following a previously described protocol (Mousa et al., 2015a). The F. graminearum strain used in this study (15 Acetyl DON Producer) was obtained from the Agriculture and Agrifood Canada Fungal Type Culture Collection (AAFC Food Research Centre, Guelph, ON, Canada). The effect of the crude culture extract of each fungus on the growth of F. graminearum was previously shown (Figure 6.1H) (Chapter 5) (Mousa et al., 2015a).

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Figure 6.1

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Figure 6.1. Characterization of the putative fungal endophytes previously isolated from finger millet and the antifungal activity of their extracts. (A) Picture of a finger millet plant. (B-D) Pictures of fungal endophytes WF4, WF5 and WF7 isolated from finger millet, respectively. (E) Putative fungal endophyte nomenclatures, alongside the corresponding plant tissue from which they were isolated and best BLAST match taxonomic identification based on 18S rDNA sequencing. (F-H) Example of each endophyte fermented on rice medium as indicated. (I) Quantification of the effects of each endophyte extract on the growth of F. graminearum compared to the fungicide nystatin (diameter of inhibition zone in mm, n=3).

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6.3. 2 Plant pathogenicity assay To confirm that the putative fungal endophytes are not pathogens, a plant pathogenesis assay was conducted. Seeds of finger millet were surface sterilized by washing in 0.1% Triton X-100 detergent (10 min), followed by 3% sodium hypochlorite (20 min, twice). Seeds were planted on sterile Phytagel based medium consisted of: 1 package Murashige and Skoog modified basal salt mixture (Catalog #M571, PhytoTechnology Laboratories, USA), 4 g Phytagel, 1 ml pyridoxine HCl (0.5 mg/ml),

500 µl nicotinic acid (1 mg/ml), 0.332 g CaCl2, 1 ml glycine (2 mg/ml), 10 ml thiamine

HCl (100 mg/L), and 1 ml MgSO4 (18 g/100 ml), per liter. The medium was distributed into sterile glass tubes. Seven seeds were transferred to each tube and allowed to germinate in the dark for seven days, then transferred to light shelves (25˚C, 16 h light). When seedlings were one week old, each endophyte (or control) was applied onto the gel surface (11 mm diameter agar discs), in triplicate. Seedlings that were not inoculated with any fungi served as the negative control. Seedlings that were infected with the fungal pathogen Alternaria alternata were the positive control. Plants were then assessed visually for disease symptoms at 10 days after inoculation with each endophyte or control pathogen.

6.3.3 Root colonization assay

To test if the putative endophytes are able to colonize the roots of finger millet, confocal microscopy imaging was conducted. Finger millet seeds were surface sterilized and planted in glass tubes containing sterile Phytagel based medium (as described above). Each fungal endophyte was applied, in triplicate (100 μl of a 48 h old culture grown in potato dextrose medium) to finger millet seedlings (17 days after germination) and co- incubated with the seedlings at room temperature. The control was finger millet seedlings incubated with potato dextrose medium only. Calcofluor white stain was used to stain fungi (catalogue #18909, Sigma Aldrich, USA), following the manufacturer’s protocol. Thereafter, finger millet roots were scanned with a Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems, Mannheim, Germany) at the Molecular and Cellular Imaging Facility, University of Guelph.

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6.3.4 Bio-guided purification of active anti-Fusarium compounds For large scale fermentation, each endophyte was fermented in 2 L flasks (five flasks per each endophyte) containing 50 g white rice and 100 ml H2O. The flasks were incubated at 25 °C for 30 days without shaking (Figure 6.1E-G). Then, each culture was extracted with ethyl acetate, and washed with water to remove salts and sugars. The extract was dried under vacuum at 45°C and subjected to fractionation between methanol and n-hexane phases to remove long chain fatty acids. To purify the active compounds, the dry residues were separated by flash liquid chromatography. The column was filled with flash grade silica gel (SiliaFlash® P60 230-400, SiliCycle, Canada,Catalog # R12030B) and saturated with the desired mobile phase just prior to sample loading. For each endophyte, 3 g of the dry residue extract was dissolved in chloroform, mixed with silica and evaporated under vacuum. The extract was applied as a dried silica band on top of the column. The mobile phase (gradient combination of hexane/ethyl acetate followed by ethyl acetate/methanol) was then passed through the column under air pressure. Thin layer chromatography (TLC) was used to visualize the bands using different solvent- system combinations and vanillin-H2SO4 reagent. Eluted fractions were tested for anti- Fusarium activity using the dual culture agar diffusion method (as described below). Candidate fractions that inhibited the growth of F. graminearum were subjected to further purification using flash column chromatography and/or preparative TLC. Purified compounds were re-screened for inhibition of F. graminearum growth. The active compounds were subjected to further spectroscopic structural elucidation.

6.3.5 Antifungal assay of purified compounds using agar diffusion assay

To enable the bio-guided purification, the dual culture agar diffusion assay was employed. F. graminearum was grown for 48 h (25˚C, 100 rpm) in liquid potato dextrose broth (Catalog # P6685, Sigma Aldrich, USA), then mycelia were added to melted and cooled PDA media (1 ml of fungal culture into 100 ml of media), mixed and poured into Petri dishes (100 mm x 15 mm), then allowed to re-solidify. Wells (11 mm diameter) were created in this pathogen-embedded agar by puncturing with sterile glass tubes, then a fraction/compound was applied into each well (100 µl per well) in triplicate. The agar

174 plates were incubated at 25˚C for 48 h. The diameter of each one of inhibition was measured (mm). Appropriate solvent mixtures were used as negative controls. Nystatin fungicide (Catalog #N6261, Sigma Aldrich, USA) was used as a positive control at a concentration of 10 μg/ml.

6.3.6 Structure elucidation of active anti-F. graminearum compounds Each purified compound was structurally elucidated using one and two dimensional nuclear magnetic resonance (NMR) techniques in combination with mass spectrometry (MS) methods and IR (infra-red) spectroscopy. NMR analyses were conducted at the University of Guelph NMR Facility using a 600 DPX spectrometer (Bruker, Germany) operating at 600 MHz for 1H and 150 for 13C. Structural assignments were based on spectra resulting from the following NMR experiments: 1H, APT, 1H-1H COSY, 1H-13C direct correlation (HSQC), 1H-13C long range correlation (HMBC). IR spectroscopy was conducted using a Bruker Alpha IR Spectrometer instrument (Bruker, Germany) located in the Department of Chemistry, University of Guelph. MS was conducted in the Mass Spectroscopy Facility of the Advanced Analysis Centre of the University of Guelph using the following acquisition parameters: Ion Source (ESI), Ion Polarity (Positive), capillary exit (Resolution, 140.0 V), Trap Drive (58.9), Accumulation Time (1348 μs), Averages (3 Spectra).

6.3.7 Visualization of the interactions between F. graminearum and the anti-F. graminearum compounds To visualize the interactions between F. graminearum and each pure anti-F. graminearum compound, light microscopy was used combined with vitality staining. Microscope slides were coated with a thin layer of PDA, then 100 µl of F. graminearum culture (48 h old grown in potato dextrose broth at 25˚C, with shaking at 100 rpm) were applied adjacent to 20 µl of each purified compound (5 mg/ml). There were three replicates for each slide. Slides were incubated at 25˚C for 24 h, stained with 100 µl of neutral red (Sigma Aldrich, Catalog #57993) for 3-5 min, then washed 3-4 times with deionized water. Images were taken using a MZ8 Leica microscope (Wetzlar, Germany).

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6.4 Results

6.4.1 Confirming the endophytic behaviour of the putative fungal endophytes

Endophytes are defined as microbes that are able to colonize the internal tissues of their host plants without causing disease (Wilson, 1995). To confirm the endophytic behaviour of the isolated finger millet fungi, two experiments were undertaken:

6.4.1.1 Plant Pathogenicity assay: Seedlings of finger millet were co-incubated with each putative fungal endophyte or with a known pathogen of finger millet, Alternaria alternata (Kumar, 2010). The seedlings inoculated with the pathogen developed disease symptoms including black roots and leaf spots and reduction in plant length (Figure 6.2A), compared to control seedlings that received the buffer only (Figure 6.2B). However, none of the putative fungal endophytes showed any disease symptoms on finger millet seedlings compared to the pathogen control (Figure 6.2C-E).

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Figure 6.2: Plant pathogenicity assay of each putative endophyte following inoculation onto finger millet. (A) Representative picture showing the effect of the known pathogen Alternaria alternata on finger millet seedlings (positive control). (B) Representative picture showing the effect of buffer on finger millet seedlings (negative control). (C-E) Representative pictures showing the effect of the each putative fungal endophyte on finger millet seedlings as indicated.

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6.4.1.2 Root colonization assay: To test the ability of each endophyte to colonize the internal tissues of finger millet roots, confocal microscopy imaging was conducted. Seedlings inoculated with the buffer only (control) showed no observable fungal growth inside the tissues (Figure 6.3A-B). At the early post-inoculation time point used, most of the fungal hyphae were observed on the rhizoplane, however each of the fungi was visualized to initiate colonization of the epidermal and sub-epidermal layers of finger millet roots, providing further support that they are endophytes of finger millet (Figures 6.3C-H).

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Figure 6.3

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Figure 6.3: Test for the ability of the putative fungal endophytes to colonize finger millet roots using confocal scanning laser microscopy. (A-B) Representative pictures of root tissues inoculated with the buffer control. (C- H) Representative pictures of root tissues inoculated with each putative fungal endophyte as indicated. Fungi fluoresce purple-blue due to staining with calcofluor. Plant tissues appear red due to auto-fluorescence. White arrows point to fungi inside the tissues. Abbreviations: PR, primary root; RH, root hair.

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6.4.2 Bio-assay guided purification and structural elucidation of anti-Fusarium compounds

Bio-assay guided purification was conducted to isolate the active anti-fungal compounds from each endophyte extract. Three active anti-Fusarium compounds were purified (Figure 6.4). The diameter of zones of growth inhibition of F. graminearum were 2, 2.5 and 1.5 cm for compound 1, 2 and 3, respectively. The three compounds were subjected to further spectroscopic structure elucidation. Results corresponding to each compound are shown separately below. 1D-NMR data for these three compounds was grouped (Table A6.1). Data for 2D-NMR and MS are not shown.

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Figure 6.4. Structures of the purified anti-Fusarium compounds from fungal endophyte strains WF5, WF6 and WF7. The structures were identified as: (A) 5-hydroxy benzofuranone (from strain WF5), (B) dehydrocostus lactone (from strain WF6), and (C) harpagoside (from strain WF7).

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6.4.3 Structural elucidation of the isolated antifungal compounds

6.4.3.1 Compound 1 (isolated from fungus WF5):

Compound 1 (molecular formula C8 H6 O3) was eluted with hexane-ethyl acetate (90:10) as white amorphous powder, with an Rf value of 0.46, and further moved as a single band in hexane-ethyl acetate (85:15), with an Rf of 0.58. The yield of compound 1 was 50 mg from 3 g of total extract.

IR: 3320.2, 1762, 1604.3, 1503.7, 1474.15, 1240.8, 1077, 948.4, 809.1 cm-1. 1H NMR (600 MHz, DMSO): d 3.83 (2H, s, H-3), 6.65 (1H, dd, J=8.6, 1.6 Hz, H-6), 6.75 (1H, d, J=1.6 Hz, H-4), 6.95 (2H, d, J=8.6 Hz, H-7), 9.32 (1H, s, Ar–OH).

13C NMR (150 MHz, DMSO): 153.9 (C), 146.7 (C), 125.1 (C), 114.2 (CH), 111.8 (CH), 110.5 (CH), 33 (CH2).

Comparing theses spectral data with a previous reference (Venkateswarlu et al., 2006), the compound was confirmed as 5-hydroxy 2(3H)-benzofuranone (Figure 6.4A).

6.4.3.2 Compound 2 (isolated from fungus WF6)

Compound 2 (molecular formula C15H18O2) was eluted from the hexane-ethyl acetate (50:50) fraction as a colorless solid. The compound was then purified by preparative TLC using a solvent mixture of hexane-ethyl acetate (30:70), with an Rf value of 0.73. The yield of compound 2 was 10 mg from 3 g total extract. The IR and 1D- NMR data were as follows:

IR: 2934, 1765, 1257, 1146, 998 cm-1.

1 H-NMR (600MHz, CDCl3): δ 1.56 (1H, m, H-8α), 1.91 (2H, m, H-2), 2.24 (2H, m, H- 8β, 9β), 2.42 (3H, m, H-3, 9α), 2.86 (3H, m, H-1, 5, 7), 3.95 (1H, t, J=9.2Hz, H-6), 4.7 (1H, s, H-15β), 4.87 (1H, s, H-15α), 5.04 (1H, d, J=1.6 H , H-14β), 5.24 (1H, d, J=1.6 Hz, H-14α) 5.46 (1H, d, J=3.6 Hz, H-13β), 6.2 (1H, d, J=3.6 H , H-13α).

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13 C-NMR (150MHz, CDCl3): δ 30.2 (C-3), 30.9 (C-8), 32.4 (C-2), 36.2 (C-9), 45.09 (C- 1), 47.5 (C-7), 52.0 (C-5), 85.2 (C-6), 109.59 (C-14), 112.6 (C-15), 120.19 (C-13), 139.7 (C-11), 149.2 (C-10), 151.2 (C-4), 170.27(C-12).

Comparing these spectral data with reference data (Yuuya et al., 1998), the compound was confirmed as dehydrocostus lactone, an guaianolide sesquiterpene lactone (Figure 6.4B).

6.4.3.3 Compound 3 (isolated from fungus WF7):

Compound 3 (molecular formula C24H30O11) was eluted from the 100% methanol fraction as an amorphous powder. The compound was further purified using preparative TLC with a mobile phase mixture of methanol-water (95:5), with an Rf value of 0.12. The yield of compound 3 was 5 mg from 3 g total extract. The IR and 1D-NMR data were as follows:

IR: 3392, 2884, 1334, 1283, 989, 770 cm-1.

1 \ \ \ \ H-NMR (600MHz, CDCl3): 7.58 (1H-C2 ), 7.47 (1H-C3 ), 7.46 (1H-C4 ),7.3 (1H-C5 ), 7.55 (1H-C6\), 6.14 (s, C1H), 5.6 (d, C3H), 5.5 (d, C4H), 3.3 (d, C6H), 2.9 (m, C9H), 1.33 (s, C10H3). Sugar moiety: 3.98, 3.7, 3.84, 3.51, 3.45, and 3.36 for (C1-C6 protons).

13 C-NMR (150MHz, CDCl3): 94.7 (C-1), 143.4 (C-3), 104.7 (C-4), 71.3 (C- 5), 77.1 (C- 6), 45.0 (C-7), 87.3 (C-8), 53.4 (C-9), 22.4 (C-10), 119 (C- α), 144.9 (C- β), 167.3 (C=O), 134.28 (C-1\), 129 (C-2\), 128.3 (C-3\), 130.3 (C-4\), 128.8 (C-5\), 128.2(C-6\), 76.52 (C- 1 \\), 75.99 (C-2\\), 72.79 (C-3\\), 69.9 (C-4\\), 72.73 (C-5\\), 61.8 (C-6\\).

Comparing these results to the literature (Tong et al., 2006), the compound was identified as harpagoside, an iridoide glycoside (Figure 6.4C).

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6.4.4 Microscopic examination of the interaction between each pure antifungal compound and F. graminearum in vitro

To visualize the interaction between each pure compound and F. graminearum in vitro, light microscopy was used (Figure 6.5). All compounds resulted in reduced hyphal growth associated with frequent hyphal breakage (Figure 6.5B, D and F), compared to the control (Figure 6.5C, E and G), suggestive of a mixed fungistatic/fungicidal mode of action. Interestingly, the hyphae of F.graminearum appeared to bend away from the contact zone with compound 3 (Figure 6.5F).

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Figure 6.5

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Figure 6.5. The effects of the purified endophyte-derived anti-fungal compounds on F. graminearum in vitro using neutral red staining. (A) Cartoon of the experimental methodology used to examine the in vitro interactions between F. graminearum (pink) and each compound (orange) or the buffer control (respective compound solvent). Microscope slides were pre-coated with PDA and incubated for 24 h. F. graminearum hyphae were then stained with neutral red. Shown are representative microscope slide pictures (n=3) of the interactions of F. graminearum with: (B) 5-hydroxy benzofuranone compared to (C) the buffer control; (D) dehydrocostus lactone compared to (E) the buffer control; (F) harpagoside compared to (G) the buffer control. The blue arrows point to areas of apparent breakage of F. graminearum hyphae. Each scale bar equals 25 µm.

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6.5 Discussion

I hypothesized that finger millet may host endophytes with anti-fungal activity, including against the pathogen Fusarium graminearum, because this crop is known to be resistant to many pathogens including F. graminearum (Munimbazi and Bullerman, 1996;Mousa et al., 2015a; Sundaramari, 2015), and its endophytes may have co-evolved with Fusarium pathogens in Africa (Adipala, 1992; Saleh et al., 2012) and South Asia (Penugonda et al., 2010; Ramana et al., 2012). In a previous study (Chapter 5) (Mousa et al., 2015a), I identified four distinct, putative, fungal endophyte species from finger millet (WF4-7) and showed that their corresponding extracts have antifungal activities including against F. graminearum. Only the extract from WF4, a Phoma sp. was characterized in detail, and revealed four anti-Fusarium compounds (viridicatol, tenuazonic acid, alteraniol and alteraniol methyl ether). Here, I confirmed the endophytic behaviour of the three remaining endophytes and identified the compounds underlying their antifungal activity alongside a suggested mode of action. The current anti-Fusarium compounds are different than those I previously identified and broaden the range of bio- fungicides identified from the finger millet microbiome against this important crop pathogen.

6.5.1 Previous reports of non-pathogenic Fusarium and Penicillium sp. as endophytes

One fungal strain reported in this study was predicted to be a Fusarium sp. (WF5). Previous studies involving Fusarium and Asperillus sp. have shown that non-pathogenic fungal strains that belong to the same species as a pathogen can, in some cases, control the ability of the pathogen to cause disease and produce mycotoxins (Cooney et al., 2001; Cotty, 2006). Several competitive exclusion mechanisms have been suggested that explain the biological control ability (Dawson et al., 2004). These mechanisms include blockage of infection sites, competition for limited nutrients in the soil, inhibition of spore germination and induction of host resistance (Larkin and Fravel, 1999; Fravel et al., 2003). In the current study, I showed that the ability of non-pathogenic Fusarium sp. to

188 control pathogenic Fusarium sp. may also be mediated by production of antifungal secondary metabolites.

The two other fungal strains (WF6 and WF7) characterized in this study were closely related to Penicillium sp., including one that most closely resembled Penicillium chrysogenum (WF6) taxonomically. P. chrysogenum was previously isolated as an endophyte of marine red algal species of the genus Laurencia, and was reported to have antifungal activity against the fungus Alternaria brassicae. The antifungal metabolite was identified to be a mono-terpene derivative (Gao et al., 2010). This observation is of interest because the anti-Fusarium compound that I purified from the apparent P. chrysogenum isolate from finger millet is a sesquiterpene lactone, suggesting that the terpenoid pathway may play an important role in the anti-pathogenesis of P. chrysogenum endophytes. Other Penicillium sp. were isolated from diverse hosts including the South Asian medicinal plant Ocimum tenuiflorum (Lai et al., 2013), the Moroccan plant Ceratonia siliqua (El-Neketi et al., 2013), Cannabis sativa L. (Kusari et al., 2013) and Panax ginseng (Zheng et al., 2013). An extract from the Penicillium isolate of Panax ginseng showed anti-fungal activity against Pyricularia oryzae (Zheng et al., 2013). We emphasize this last observation because P. oryzae is the most important fungal pathogen of finger millet, the causal agent of blast disease (Lenné et al., 2007).

6.5.2 Anti-Fusarium compounds purified from finger millet endophytes Here, I have elucidated the structures of three active anti-Fusarium compounds: 5- hydroxy benzofuranone, dehydrocostus lactone, and an iridoide glycoside (harpagoside) (Figure 6.4):

6.5.2.1 4-hydroxy benzofuranone (isolated from Fusarium sp. strain WF5) Simple benzofuranone derivatives have been previously isolated from fungi including Coniothyrium minitans (Machida et al., 2001). Other conjugated benzofuranones have been reported from endophytic fungi such as Pestalotiopsis photiniae, an endophyte that inhabits the Chinese plant, Roystonea regia (Ding et al.,

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2009). Some benzofuranone derivatives were reported to have algicidal, hypotensive and anti-inflammatory activities (Yang et al., 2008; de Souza Nunes et al., 2014). To the best of our knowledge, this is the first report of 4-hydroxy benzofuranone having anti- Fusarium activity.

6.5.2.2 Dehydrocostus lactone (isolated from Penicillium sp. strain WF6) Dehydrocostus lactone derivatives were previously isolated from plant species including Inula racemosa, Centaurea pannonica and Saussurea costus (Rao and Kavitha, 2013)(Julianti et al., 2011; ilošević Ifantis et al., 2013). The compound was reported to have antimicrobial activity including against Mycobacterium sp., Propionibacterium acnes, Staphylococcus aureus, and Malassezia furfur (Cantrell et al., 1998; Rao and Kavitha, 2013). Dehydrocostus lactone was reported to have multiple biological activities including suppression of melanin production, induction of apoptosis in soft tissue sarcoma cell lines, inhibition of migration of prostate cancer cells and anti-trypanosoma activity (Julianti et al., 2011; Kim et al., 2012; Kretschmer et al., 2012; Rao and Kavitha, 2013). To the best of our knowledge, this is the first report of dehydrocostus lactone from a fungal source and the first report of it having anti-Fusarium activity.

6.5.2.3 Harpagoside (isolated from Penicillium sp. strain WF7)

Harpagoside was first isolated from Harpagophytum procumbens (Devil’s claw), a plant widely used in folk medicine for its anti-microbial, anti-inflammatory and analgesic properties (Mncwangi et al., 2012). Subsequently, harpagoside was isolated from diverse plants including Radix scrophulariae, a plant used as a Chinese herbal medicine for treatment of rheumatism (Li et al., 2007). Harpagoside was reported to have anti- inflammatory, antioxidant and analgesic properties (Akdemir et al., 2004; Georgiev et al.; Georgiev et al., 2013). To the best of our knowledge, this is the first report of dehydrocostus lactone from a fungal source and the first report of it having anti-Fusarium activity.

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6.5.3 Future perspectives

A growing number of reports in the literature suggest that endophytic microbes may produce secondary metabolites that are also produced in parallel by their host plants (Hussain et al., 2007; Waqas et al., 2012; Soliman et al., 2015;). Here, I reported the ability of endophytic fungi to produce small bioactive molecules previously reported as plant metabolites. It will be interesting to conduct future experiments to elucidate whether these apparent plant-derived compounds are in fact derived from their microbial inhabitants either exclusively or additively. As a general lesson, this study highlights the value of exploring the orphan crops of subsistence farmers as sources of endophytes and antifungal compounds with potential to combat serious pathogens afflicting mainstream, global agriculture.

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Chapter 7: Root hair-endophyte stacking (RHESt) in an ancient Afro- Indian crop creates an unusual physico-chemical barrier to trap pathogen(s)

Authors’ contributions

Walaa Kamel Mousa designed and performed all experiments, data analysis and wrote the manuscript.

Charles Shearer assisted with the greenhouse experiments.

Victor Limay-Rios performed the DON quantification experiments.

Cassandra Ettinger and Jonathan A. Eisen (University of California, Davis) sequenced the M6 genome and provided gene annotations.

Manish N. Raizada helped in the experimental design and edited the manuscript.

Publication status: pending submission

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7.1 Abstract

The ancient African crop, finger millet, has broad resistance to pathogens including the toxigenic fungus Fusarium graminearum. Here I report the discovery of a novel plant defence mechanism, resulting from an unusual symbiosis between finger millet and a root-inhabiting bacterial endophyte, M6 (Enterobacter sp.). Seed-coated M6 swarms towards Fusarium attempting to penetrate root epidermis, promotes growth of root hairs which then bend parallel to the root axis, then forms biofilm-mediated microcolonies, resulting in a remarkable, multi-layer root hair-endophyte stack (RHESt). RHESt results in a physical barrier that prevents entry and/or traps F. graminearum which is then killed. Thus M6 creates its own specialized killing microhabitat. M6 killing requires c-di-GMP- dependent signalling, diverse fungicides and xenobiotic resistance. Further molecular evidence suggests long-term host-endophyte-pathogen co-evolution. The end-result of this remarkable symbiosis is reduced DON mycotoxin, potentially benefiting millions of subsistence farmers and livestock. RHESt demonstrates the value of exploring ancient, orphan crop microbiomes.

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7.2 Introduction

Finger millet (Eleusine coracana) is a cereal crop widely grown by subsistence farmers in Africa and South Asia (Goron and Raizada, 2015; Thilakarathna and Raizada, 2015). Finger millet was domesticated in Western Uganda and Ethiopia around 5000 BC then reached India by 3000 BC (Hilu and Wet, 1976). With few exceptions, subsistence farmers report that finger millet is widely resistant to pathogens including Fusarium species (Chapter 5) (Mousa et al, 2015a; Sundaramari, 2015). One species of Fusarium, F. graminearum, causes devastating diseases in crops related to finger millet including maize, wheat and barley, associated with accumulation of the mycotoxin deoxynivalenol (DON) which affects humans and livestock (Schaafsma, 2002; Mousa et al, 2015b). However, despite its prevalence as a disease-causing agent across cereals, F. graminearum is not considered to be an important pathogen of finger millet, suggesting this crop has tolerance to this Fusarium species (Munimbazi and Bullerman, 1996; Mousa et al., 2015a).

The resistance of finger millet grain to fungal disease has been attributed to high concentrations of polyphenols (Chandrashekar and Satyanarayana, 2006; Siwela et al., 2010). However, emerging literature suggests that microbes that inhabit plants without themselves causing disease, defined as endophytes, may contribute to host resistance against fungal pathogens (Wilson 1995; Johnston-Monje and Raizada 2011). Endophytes have been shown to suppress fungal diseases through induction of host resistance genes (Waller et al., 2005), competition (Haas and Defago, 2005), and/or production of anti- pathogenic natural compounds (Chapter 3 and 4) (Mousa and Raizada, 2013, 2015).

Fusarium are ancient fungal species, dating to at least 8.8 million years ago, and their diversification appears to have co-occurred with that of the C4 grasses (which includes finger millet), certainly pre-dating finger millet domestication in Africa (O’Donnell et al., 2013). Multiple studies have reported the presence of Fusarium in finger millet in Africa and India (Adipala, 1992; Amata et al., 2010; Pall and Lakhani,

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1991; Penugonda et al., 2010; Ramana et al., 2012; Saleh et al., 2012). A diversity of F. verticillioides (synonym F. moniliforme) has been observed in finger millet in Africa and it has been suggested that the species evolved there (Saleh et al., 2012). These observations suggest the possibility of co-evolution within finger millet between Fusarium and millet endophytes. I previously isolated, for the first time, fungal endophytes from finger millet and showed that their secondary metabolites have anti- Fusarium activity (Mousa et al., 2015a). I could not find reports of bacterial endophytes isolated from finger millet.

The objectives of this study were to isolate bacterial endophytes from finger millet, assay for anti-Fusarium activity and characterize the underlying cellular, molecular and biochemical mechanisms. I report an unusual symbiosis between the host and a root- inhabiting bacterial endophyte.

7.3 Methods

7.3.1 Isolation, identification and antifungal activity of endophytes

Finger millet seeds originating from Northern India were grown on clay Turface in the summer of 2012 according to a previously described method (Mousa et al., 2015a). Tissue pool sets (3 sets of: 5 seeds, 5 shoots and 5 root systems from pre-flowering plants) were surface sterilized following a standard protocol (Mousa et al., 2015a). Surface sterilized tissues were ground in LB liquid medium in a sterilized mortar and pestle, then 50 µl suspensions were plated onto 3 types of agar plates (LB, Biolog Universal Growth, and PDA media). Plates were incubated at 25˚C, 30˚C and 37˚C for 1- 3 days. A total of seven bacterial colonies (M1-M7) were purified by repeated culturing on fresh media. For molecular identification of the isolated bacterial endophytes, PCR was used to amplify and sequence 16S rDNA as previously described (Mousa et al., 2015b), followed by best BLAST matching to GenBank. 16S rDNA sequences were deposited into GenBank. Scanning electron microscopy imaging was used to phenotype

195 the candidate bacterium as previously described (Mousa et al., 2015b) using a Hitachi S- 570 microscope (Hitachi High Technologies, Tokyo, Japan) at the Imaging Facility, Department of Food Science, University of Guelph.

To test the antifungal activity of the isolated endophytes against F. graminearum, agar diffusion dual culture assays were undertaken in triplicate (Mousa et al., 2015c). Nystatin (Catalog #N6261, Sigma Aldrich, USA) and Amphotericin B (Catalog #A2942, Sigma Aldrich, USA) were used as positive controls at 10 μg/ml and 5 μg/ml, respectively, while the negative control was LB medium. Using a similar methodology, additional anti-fungal screening was conducted using the Fungal Type Culture Collection from Agriculture and Agrifood Canada, Guelph, ON (Table S2).

7.3.2 Microscopy imaging 7.3.2.1 In planta colonization of the candidate anti-fungal endophyte M6

In order to verify the endophytic behaviour of the candidate anti-fungal bacterium M6 in maize, wheat and millet, the bacterium was subjected to tagging with green fluorescent protein (GFP) (vector pDSK-GFPuv)(Wang et al., 2007) and in planta visualization using confocal scanning microscopy as previously described (Mousa et al., 2015c) at the Molecular and Cellular Imaging Facility, University of Guelph, Canada.

7.3.2.2 In vitro interaction using light microscopy

Both the fungus and bacterium M6 were allowed to grow in close proximity to each other overnight on microscope slides coated with a thin layer of PDA as previously described (Mousa et al., 2015c). Thereafter, the fungus was stained with the vitality stain, Evans blue which stains dead hyphae blue. The positive control was a commercial biological control agent (Bacillus subtilis QST713, Bayer CropScience, Batch # 00129001) (100 mg/10 ml). Pictures were captured using a light microscope (BX51, Olympus, Tokyo, Japan).

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7.3.2.3 In vitro and in planta interactions using confocal microscopy

All the experiments were conducted using a Leica TCS SP5 confocal laser scanning microscope at the Molecular and Cellular Imaging Facility at the University of Guelph, Canada.

To visualize the interactions between endophyte M6 and F. graminearum inside finger millet, finger millet seeds were surface sterilized and coated with GFP-tagged endophyte, and then planted on sterile Phytagel based medium in glass tubes, each with 4-5 seeds. Phytagel medium was prepared as previously described (Mousa et al., 2015a). At 10 days after planting, finger millet seedlings were inoculated with F. graminearum (50 μL of a 48 h old culture grown in potato dextrose broth) and incubated at room temperature for 24 h. The control consisted of seedlings incubated with potato dextrose broth only. There were three replicate tubes for each treatment. Thereafter, roots were stained with calcofluor florescent stain (catalogue #18909, Sigma-Aldrich), which stains chitin blue, following the manufacturer’s protocol, and scanned.

To visualize the attachment of bacterium M6 to fungal hyphae, GFP-tagged M6 and F. graminearum were inoculated overnight at 30˚C on microscope slides covered with a thin layer of PDA. Thereafter, the fungal hyphae was stained with calcofluor and examined. The same protocol was employed to test if this recognition was disrupted in the Tn5 mutants.

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To visualize biofilm formation by bacterium M6, GFP-tagged M6 were incubated on microscope slides for 24 h at 30˚C, followed by staining with FilmTracer™ SYPRO® Ruby Biofilm Matrix Stain (catalogue #F10318, ThermoFisher Scientific, USA) using the manufacturer’s protocol. The same protocol was employed to test if biofilm formation was disrupted in the Tn5 mutants.

7.3.3 Suppression of F. graminearum in planta and DON accumulation in storage

Bacterium M6 was tested in planta for its ability to suppress F. graminearum in two susceptible crops, maize (hybrid P35F40, Pioneer HiBred) and wheat (Quantum spring wheat, C&M Seeds, Canada), in two independent greenhouse trials as previously described for maize (Mousa et al., 2015c), with modifications for wheat (see Appendix Method A7.1). ELISA analysis was conducted to test the accumulation of DON in seeds after 14 months of storage in conditions that mimic those of African subsistence farmers (temperature ~18-25˚C, with a moisture content of ~40-60%) as previously described (Mousa et al., 2015b). Control treatments consisted of plants subjected to pathogen inoculation only, and plants subjected to pathogen inoculation followed by prothioconazole fungicide spraying (PROLINE® 480 SC, Bayer Crop Science). Results were analyzed and compared using Mann-Whitney t-test (P≤0.05).

7.3.5 Transposon mutagenesis, gene rescues and complementation

To identify the genes required for the antifungal activity, Tn5 transposon mutagenesis was conducted using EZ-Tn5 Tnp TransposomeTM kit (catalogue #TS 08KR, Epicentre, adison, USA) according to the manufacturer’s protocol. The mutants were screened for loss of anti-Fusarium activity using the agar dual culture method compared to wild type. Insertion mutants that completely lost the antifungal activity in vitro, were further tested for loss of in planta activity using maize as a model in two independent greenhouse trials (same protocol as described above). The sequences flanking each candidate Tn5 insertion mutant of interest were identified using

198 plasmid rescues according to the manufacturer’s protocol (see Appendix Method A7.3). The rescued gene sequences were BLAST searched against the whole genome sequence of bacterium M6 (Ettinger et al., 2015). To test if the candidate genes are inducible by F. graminearum or constitutively expressed, real-time PCR analysis was conducted using gene specific primers (see Appendix Method A7.4). Operons were tentatively predicted using FGENESB (Solovyev and Salamov, 2011) from Softberry Inc. (USA). Promoter regions were predicted using PePPER software (University of Groningen, The Netherlands)(de Jong et al., 2012). In order to confirm the identity of the genes discovered by Tn5 mutagenesis, each mutant was complemented with the corresponding predicted wild type coding sequence which was synthesized (VectorBuilder, Cyagen Biosciences, USA) using a pPR322 vector backbone (Novagen, USA) under the control of the T7 promoter. Two μl of each synthesi ed vector was electroporated using 40 μl electro competent cells of the corresponding mutant. The transformed bacterium cells were screened for gain of the antifungal activity against F. graminearum using the dual culture assay as described above.

7.3.6 Mutant phenotyping

7.3.6.1 Transmission electron microscopy (TEM): To phenotype the candidate mutants, TEM imaging was conducted. Wild type strain M6 and each of the candidate mutants were cultured overnight in LB medium (37°C, 250 rpm). Thereafter, 5 µl of each culture were pipetted onto a 200-mesh copper grid coated with carbon. The excess fluid was removed onto a filter, and the grid was stained with 2% uranyl acetate for 10 sec. Images were taken by a F20 G2 FEI Tecnai microscope operating at 200 kV equipped with a Gatan 4K CCD camera and Digital Micrograph software at the Electron Microscopy Unit, University of Guelph, Canada.

7.3.6.2 Motility assay: Wild type or mutant strains were cultured overnight in LB medium (37°C, 225 rpm). The OD595 for each culture was adjusted to 1.0, then 15 µl of each culture were spotted on the center of a semisolid LB plates (0.3% agar) and

199 incubated overnight (37°C, no shaking). Motility was measured as the diameter of the resulting colony. There were ten replicates for each culture. The entire experiment was repeated independently.

7.3.6.3 Swarming test: To examine the ability of the strains to swarm and form colonies around the pathogen, in vitro interaction/light microscopy imaging was conducted. Wild type M6 or each mutant was incubated with F. graminearum on microscope slides covered with PDA (as described above). F. graminearum hyphae was stained with lactophenol blue solution (catalogue #61335, Sigma-Aldrich) then examined under a light microscope (B1372, Axiophot, Zeiss, Germany) using Northern Eclipse software.

7.3.6.4 Biofilm spectroscopic assay: To test the ability of the strains to form biofilms, the strains were initially cultured overnight in LB medium (37°C and 250 rpm), and adjusted to OD600 of 0.5. Cultures were diluted in LB (1:100), thereafter, 200 µl from each culture were transferred to a 96 well microtitre plate (3370, Corning Life Sciences, USA) in 6 replicates. The negative control was LB medium only. The microtitre plate was incubated for 2 days at 37°C. The plate was emptied by aspiration and washed three times with sterile saline solution. Adherent cells were fixed with 200 µl of 99% methanol for 15 min then air dried. Thereafter, 200 µl of 2% crystal violet (94448, Sigma) were added to each well for 5 min then washed with water, and left to air dry. Finally, 160 µl of 33% (v/v) glacial acetic acid were added to each well to solubilise the crystal violet stain. The light absorption was read by a spectrophotometer (SpectraMax plus 348 microplate reader, Molecular Devices, USA) at 570 nm (Stepanović et al., 2000). The entire experiment was repeated independently.

7.3.6.5 Fusaric acid resistance: To test the ability of M6 wild type or EwfR::Tn5 to resist fusaric acid, the strains were allowed to grow on LB agar medium supplemented with

200 different concentrations of fusaric acid (0.01, 0.05 and 0.1%,Catalog #F6513, Sigma Aldrich) as previously reported (Bacon et al., 2006).

c-di-GMP chemical complementation: To test if the Tn5 insertion in the predicted guanylate cyclase gene could be complemented by exogenous c-di-GMP, M6 wild type or EwgC::Tn5 strains were grown on LB agar medium supplemented with c-di-GMP (0.01 or 0.02 mg/ml) (Catalog # TLRL-CDG, Cedarlane, Canada). After 24 h, the agar diffusion method was used to test for antifungal activity.

7.3.6.6 Colicin V assay: To verify that the Tn5 insertion in the predicted colicin V production gene caused loss of colicin V secretion, M6 wild type or EwvC-4B9::Tn5 strains were inoculated as liquid cultures (OD600 = 0.5) into holes created in LB agar medium pre-inoculated after cooling with an E.coli strain sensitive to colicin V (MC4100, ATCC 35695) (Gérard et al., 2005).

7.3.7 Biochemical and enzymatic assays

7.3.7.1 Detection of anti-fungal phenazine derivatives: For phenazine detection, bio- guided fractionation combined with LC-MS analysis was undertaken. Wild type M6 or mutant bacterial strains were grown for 48 h on Katznelson and Lochhead liquid medium (Paulus and Gray, 1964), harvested by freeze drying, then the lyophilized powder from each strain was extracted by methanol. The methanolic extracts were tested for anti- Fusarium activity using the agar diffusion method as described above. The extracts were dried under vacuum and dissolved in a mixture of water and acetonitrile then fractionized over a preparative HPLC C18 column (Nova-Pak HR C18 Prep Column, 6 µm, 60 Å, 25 x 100 mm prepack cartridge, part # WAT038510, Serial No 0042143081sp, Waters Ltd, USA). The solvent system consisted of purified water (Nanopure, USA) and acetonitrile (starting at 99:1 and ending at 0:100) pumped at a rate of 5 ml/min. The eluted peaks were tested for anti-Fusarium activity. Active fractions were subjected to LC-MS

201 analysis. Each of the active fractions was run on a Luna C18 column (Phenomenex Inc, USA) with a gradient of 0.1% formic acid in water and 0.1% formic in acetonitrile. Peaks were analyzed by mass spectroscopy (Agilent 6340 Ion Trap), ESI, positive ion mode. LC-MS analysis was conducted at the Mass Spectroscopy Facility, McMaster University, ON, Canada.

7.3.7.2 GC-MS to detect production of 2,3 butanediol: To detect 2,3 butanediol, wild type strain M6 was grown for 48 h on LB medium. The culture filtrate was analyzed by GC- MS (Mass Spectroscopy Facility, McMaster University, ON, Canada) and the resulting peaks were analyzed by searches against the NIST 2008 database.

7.3.7.3 Chitinase assay: Chitinase activity of wild type strain M6 and the putative chitinase Tn5 mutant was assessed using a standard spectrophotometric assay employing the Chitinase Assay Kit (catalogue #CS0980, Sigma Aldrich, USA) according to the manufacturer’s protocol. There were three replicates for each culture, and the entire experiment was repeated independently.

7.4 Results

7.4.1 Isolation, identification and antifungal activity of endophytes

A total of seven bacterial endophytes were isolated from surface-sterilized tissues of finger millet, strains M1 to M7 (Figure 7.1A,B and Table A7.2). BLAST searching of the 16S rDNA sequences against Genbank suggested that strains M1 and M6 resemble Enterobacter sp. while M2 and M4 resemble Pantoea sp.., and M3, M5, and M7 resemble Burkholderia sp.. GenBank accession numbers for strains M1, M2, M3, M4, M5, M6 and M7 are KU307449, KU307450, KU307451, KU307452, KU307453, KU307454, and KU307455, respectively. Interestingly, five of the seven strains showed anti-fungal activity against F. graminearum in vitro (Figure 7.1B). Strain M6 from millet

202 roots showing the most potent activity and hence was selected for further study including whole genome sequencing, which resulted in a final taxonomic classification (Enterobacter sp., strain UCD-UG_FMILLET) (Ettinger et al., 2015). M6 was observed to inhibit the growth of 5 out of 20 additional crop-associated fungi, including pathogens, suggesting it has a wider target spectrum (Table A7.3). As viewed by electron microscopy, M6 showed an elongated rod shape with a wrinkled surface (Figure 7.11E). Following seed coating, GFP-tagged M6 localized to finger millet roots intercellularly and intracellularly (Figure 7.1F-G). In addition, colonization of finger millet with M6 did not result in pathogenic symptoms (Figure A7.1). Combined, these results confirm that M6 is an endophyte of finger millet.

To determine whether strain M6 has anti-F.graminearum activity in planta, related susceptible cereals (maize and wheat) were used as model systems (Figure 7.1H-R), since finger millet itself is not reported to be susceptible to this pathogen. Seed-coated GFP-tagged M6 was shown to colonize the internal tissues of maize (Figure 7.1H) and wheat (Figure 7.1N) suggesting it can also behave as an endophyte in these crop relatives. M6 treatments (combined seed coating and foliar spray) caused statistically significant (P≤0.05) reductions in F. graminearum disease symptoms in maize (Gibberella Ear Rot, Figure 7.1I-L) and wheat (Fusarium Head Blight) (Figure 7.1O-Q) ranging from 70 to 90% and 20-30%, respectively in two greenhouse trials, compared to plants treated with Fusarium only (yield data in Figure A7.2A-B; Table A7.4). Foliar spraying alone with M6 resulted in more disease reduction compared to seed coating alone, though this effect was not statistically significant, at P≤0.05 (Figure A7.2C). Following extended storage to mimic those of African subsistence farmers (ambient temperature and moisture), treatment with M6 resulted in dramatic reductions in DON accumulation, with DON levels declining from ~3.4 ppm to 0.1 ppm in maize, and from 5.5 ppm to 0.2 ppm in wheat, equivalent to 97% and 60% reductions, respectively, compared to plants treated with Fusarium only, at P≤0.05 (Figure 7.1M and R; Table A7.5).

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Figure 7.1 204

Figure 7.1: Isolation, identification and antifungal activity of finger millet endophytes. (A) Picture showing finger millet grain head. (B) Mixed culture of endophytes isolated from finger millet. (C) Quantification of the inhibitory effect of finger millet endophytes or fungicide controls on the growth of F. graminearum in vitro. For these experiments, n=3. (D) M6 endophyte suppresses the growth of F. graminearum hyphae (white) using the dual culture method. (E) Imaging of M6 viewed by scanning electron microscopy. (F-G) GFP-tagged M6 inside roots of finger millet viewed by scanning confocal microscopy. (H) GFP-tagged M6 inside roots of maize (stained with propidium iodide). (I) Effect of M6 treatment on suppression of F. graminearum disease in maize in two greenhouse trials. (J-L) (left to right) Representative ears from M6, fungicide and Fusarium only, treatments. (M) Effect of M6 or controls on reducing DON mycotoxin contamination in maize grain during storage following the two greenhouse trials. (N) GFP- tagged M6 inside roots of wheat viewed by confocal microscopy. (O) Effect of M6 treatment on suppression of F. graminearum disease in wheat in two greenhouse trials. (P) Picture of a healthy wheat grain. (Q) Picture of an infected wheat grain. (R) Effect of M6 or controls on reducing DON mycotoxin contamination in wheat grain during storage following the two greenhouse trials. Scale bars in all pictures equal 5 µm. For greenhouse disease trials, n=20 for M6, and n=10 for the controls. For DON quantification, n=3 pools of seeds. The whiskers (I, O) indicate the range of data points. The error bars (C, M, Q) indicate the standard error of the mean. For all graphs, letters that are different from one another indicate that their means are statistically different (P≤0.05, Mann Whitney t-test).

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7.4.2 Microscopic imaging of M6-Fusarium interactions in finger millet roots and in vitro

Since F. graminearum has been reported to infect cereal roots (Chongo et al., 2001), and since endophyte M6 was originally isolated from the same tissue, finger millet roots were selected to visualize potential interactions between M6 and F. graminearum (Figure 7.2). GFP-tagged M6 was coated onto millet seed. Following germination, GFP- M6 showed sporadic, low population density distribution throughout the seminal roots (Figure 7.2B). Following inoculation with F. graminearum, which was visualized using calcofluor staining, GFP-M6 accumulated at sites of attempted entry by Fusarium, creating a remarkable, high density layer of microcolonies of M6 along the entire root epidermal surface, the rhizoplane (Figure 7.2C-D). External to the M6 rhizoplane barrier was a thick mat of root hairs (RH) (Figure 7.2C-D). RH number and length were much greater at sites of M6 accumulation compared to the opposite side (Figure 7.2C-D), and M6 was shown in vitro to produce auxin (Appendix Method A7.2, Figure A7.3), a known RH-growth promoting plant hormone (Pitts et al., 1998). Interestingly, most RH were bent, parallel to the root axis (Figure 7.2D). The RH mat appeared to obscure M6 cells, but when observed in a low RH density area (Figure 7.2E), M6 cells were clearly visible and appeared to attach onto RH and engulf Fusarium hyphae (Figure 7.2F). Imaging at deeper confocal planes below the surface of the RH mat (Figure 7.2G,H) revealed that the mat did not consist only of RH, but rather that M6 cells were intercalated between bent RH strands forming an unusual, multi-layer root hair- endophyte stack (RHESt). Within the RHESt, F. graminearum hyphae appeared to be trapped (Figure 7.2H). By imaging only the M6-Fusarium interaction within the RHESt, M6 microcolonies were observed to be associated with breakage of the fungal hyphae (Figure 7.2I,J).

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Figure 7.2

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Figure 7.2: Confocal imaging of M6-Fusarium interactions in finger millet roots. (A) Picture of millet seedling showing primary root (PR) zone used for confocal microscopy. (B) Control primary root that was seed coated with GFP-M6 (green) but not infected with F. graminearum. As a control, the tissue was stained with fungal stain calcofluor to exclude the presence of other fungi. Root following seed coating with GFP-tagged endophyte M6 (M6, green) following inoculation with F. graminearum (Fg, purple blue, calcofluor stained) showing interactions with root hairs (RH, magenta, lignin autofluoresence). (C) Low and (D) high magnifications to show the dense root hair and endophyte barrier layers. (E) Low and (F) high magnifications at the edge of the barrier layers. (G) Low and (H) high magnifications in a deeper confocal plane of the root hair layer shown in (D) showing root hair endophyte stacking (RHESt) with trapped fungal hyphae. (I) Low and (J) high magnifications of the interaction between M6 (green) and F. graminearum in the absence of root hair-lignin autofluorescence showing breakage of fungal hyphae.

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To confirm that the endophyte actively kills Fusarium, Evans blue vitality stain, which stains dead hyphae blue, was used following co-incubation on a microscope slide. The fungal hyphae in contact with strain M6 stained blue and appeared broken (Figure 7.3A) in contrast to the control (F. graminearum exposed to buffer only) (Figure 7.3B). The M6 result was similar to the well known fungicidal activity of the commercial biocontrol agent, B. subtilis (Figure 7.3C). Combined, these results suggest that M6 cooperates with RH cells to create a specialized killing microhabitat (RHESt) that protects millet roots from invasion by F. graminearum.

Since M6, in the absence of Fusarium, was sporadically localized in planta, but then accumulated at sites of Fusarium hyphae, it was hypothesized that M6 actively seeks Fusarium. To test this hypothesis, GFP-tagged M6 and F. graminearum were spotted adjacent to one another on a microscope slide coated with agar; as time progressed, M6 was observed to swarm towards Fusarium hyphae (Figure 7.3D-H), confirming its ability to seek Fusarium. Upon finding the pathogen, M6 cells were observed to physically attach onto F. graminearum hyphae (Figure 7.3G-H). At the endpoint of these interactions, dense microcolonies of M6 were observed to break the hyphae (Figure 7.3J). Transmission electron microscopy showed that M6 possesses multiple peritrichous flagella (Figure 7.3K). Since this interaction was observed in vitro, independent of the host plant, the data show that M6 alone is sufficient to exert its fungicidal activity. To test if the attachments of M6 observed in vitro and in planta are mediated by biofilm formation, the proteinaceous biofilm matrix stain, Ruby Film Tracer (red), was used in vitro. Red staining, indicating biofilm formation, was observed associated with M6 in the absence of Fusarium (Figure 7.3L). In the presence of F. graminearum, biofilm was also observed on the hyphal surfaces (Figure 7.3M). Combined, these results suggest that M6 cells swarm towards Fusarium hyphae attempting to penetrate the root epidermis, induces root hair growth and bending (directly or indirectly), resulting in formation of RHESt within which M6 cells form biofilm-mediated microcolonies which attach, engulf and kill Fusarium.

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Figure 7.3

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Figure 7.3: Behavior and interactions of endophyte M6 and F. graminearum in vitro on microscope slides. (A-C) Light microscopy of interactions between F. graminearum (Fg) and M6 following staining with Evans blue, which stains dead hyphae blue. Shown are: (A) Fg following overnight co-incubation with M6, (B) Fg, grown away from M6 (control), and (C) Fg following overnight co-incubation with a commercial biological control agent. (D-H) Time course to illustrate the swarming and attachment behaviour of GFP-tagged M6 (green) to Fg (blue, calcofluor stained) viewed at 0.5, 1.5, 3, 6, 8 h following co-incubation, respectively. Fg and M6 shown in (D) and (E) were inoculated on the same slide distal from one another at the start of the time course but digitally placed together for these illustrations. (J) M6 shown breaking Fg hypha. (K) Transmission electron microscope picture of M6 showing its characteristic flagella. (L, M) Biofilm formed by M6 as viewed by staining with Ruby film tracer (red) in the (L) absence of Fg or (M) presence of Fg.

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7.4.4 Identification of strain M6 genes required for anti-Fusarium activity

Since the fungicidal activity of M6 was observed to occur independently of its host plant, M6 was subjected to Tn5 mutagenesis, and then candidate Tn5 insertions were screened in vitro for loss of fungicidal activity against F. graminearum. Out of 4800 Tn5 insertions that were screened in triplicate, sixteen mutants were isolated that resulted in loss or reduction in the diameter of inhibition zones of F. graminearum growth (Figure A7.4A). The mutants that resulted in complete loss of the antifungal activity in vitro were validated for loss of anti-Fusarium activity in planta in two independent greenhouse trials in maize (Figure A7.4B-C), demonstrating the relevance of the in vitro results. Rescue of the Tn5-flanking sequences followed by BLAST searching against the whole genome wild type M6 (Ettinger et al., 2015), resulted in the successful identification of 13 candidate genes in 12 predicted operons (Table A7.6 and A7.7). Based on gene annotations and the published literature, four regulatory and/or anti-microbial mutants of interest were selected, complemented (Figure A7.4D) and subjected to detailed characterization. The selected genes encode two LysR family transcriptional regulators, a diguanylate cyclase, and a colicin V biosynthetic enzyme:

7.4.4.1 LysR transcription regulator in a phenazine operon (ewpR-5D7::Tn5)

The ewpR mutant strain showed complete loss of the antifungal activity in vitro (Figure A7.4A) and reduced activity in planta (Figure 7.4A) The Tn5 insertion was localized to an operon (ewp, Figure 7.4B) that included tandem paralogs of phzF (ewpF1 and ewpF1) which were predicted to encode trans-2,3-dihydro-3-hydroxyanthranilate isomerase (EC # 5.3.3.17), a homodimer enzyme that catalyzes the core skeleton of phenazine, a potent anti-fungal compound (Parsons et al., 2004). The insertion occurred within a member of the LysR transcriptional regulator family (ewpR), which has been previously reported to induce phenazine biosynthesis (Lu et al., 2009). Three lines of evidence suggest the LysR gene is an upstream regulator of the ewp operon. First, the genomic organization showed that LysR was transcribed in the opposite direction as the operon. Second, the LysR canonical binding site sequence (TN11A) was observed

212 upstream of the phzF (ewaF) coding sequences (Goethals et al., 1992) (Figure 7.4B). Finally, and most important, real time PCR revealed that the expression of ewpF1 and ewpF2 were dramatically down-regulated in the LysR mutant compared to wild type (Figure 7.4C). Combined, these results suggest that EwpR is a regulator of phenazine biosynthesis in strain M6. The crude methanolic extract from the ewpR mutant strain lost anti-Fusarium activity in vitro in contrast to extracts from wild type M6 or randomly selected Tn5 insertions that otherwise had normal growth rates (Figure 7.4E). Anti-F. graminearum bioassay guided assay fractionation using extracts from M6 showed two active fractions (FrA, FrB) (Figure 7.3F), each containing a compound with a diagnostic fragmentation pattern of [M+H]+=181.0, corresponding to a phenazine nucleus

(C12H8N2, MW = 180.08) (Wang et al., 2011), and molecular weights (M+Z= 343.3 and

356.3) indicative of phenazine derivatives [griseolutein A (C17H14N2O6, MW= 342.3) and

D-alanyl-griseolutein (C18H17N3O5, MW=355.3), respectively] (Giddens et al., 2002; Kitahara et al., 1982; Nakamura et al., 1959) (Figure 7.4F-G). Surprisingly, the ewpR mutant showed a significant reduction in motility (Figure 7.4H) and swarming (Figure 7.4I) compared to the wild type (Figure 7.4J), concomitant with a ~60 % reduction in flagella (Figure 7.4K) compared to wild-type (Figure 7.3K), loss of attachment to Fusarium hyphae (Figure 7.4L) compared to wild-type (inset in Figure 7.4L), as well as reductions in biofilm formation (Figure 7.4M-N). Combined, these results suggest that ewpR is required for multiple steps in the anti-fungal pathway of M6 including phenazine biosynthesis.

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Figure 7.4

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Figure 7.4: Continued

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Figure 7.4: Characterization of phenazine mutant ewpR-5D7::Tn5. (A) Effect of M6 mutant strain ewpR- on suppression of F. graminearum (Fg) in maize compared to wild type M6 and Fg-only control, with corresponding, representative maize ear pictures. (B) Genomic organization of the predicted phenazine biosynthetic operon showing the position of the Tn5 insertion and putative LysR binding site within the promoter (P). (C) Quantitative real time PCR (qRT-PCR) gene expression of the two core phenazine genes (ewpF1 and ewpF2) in wild type M6 (+) and mutant (-) (ewpR-) backgrounds. (D) Illustration of the phenazine biosynthetic pathway. (E) Agar diffusion assay showing the inhibitory effect of different methanol extracts on the growth of Fg from wild type M6, two wild type fractions (FrA, FrB), the ewpR- mutant (mutant), a random Tn5 insertion or buffer. (F-G) Mass spectroscopy analysis of putative phenazine derivatives from wild type M6 fractions A and B. (H) Quantification of ewpR- mutant strain motility compared to wild type M6, with representative pictures (inset) of motility assays on semisolid agar plates. (I, J) Light microscopy image showing loss of swarming and colony formation of (I) ewpR- mutant strain around Fg hyphae stained with lactophenol blue, compared to (J) wild type M6. (K) Electron microscopy image of ewpR- mutant strain. (L) Confocal microscopy image showing attachment pattern of GFP-tagged ewpR- mutant strain (green) to Fg hyphae stained with calcofluor stain, compared to wild type M6 (inset). (M) Confocal microscopy image showing reduced proteinaceous biofilm matrix stained with Ruby film tracer (red) associated with GFP-tagged ewpR- mutant strain compared to wild type M6 (inset). (N) Spectrophotometric quantification of biofilm formation associated with wild type M6 compared to the ewpR- mutant strain, with representative biofilm assay well pictures (left and right, respectively; 3 replicates shown). For graphs shown in (A, C, H, N) letters that are different from one another indicate that their means are statistically different (P≤0.05, Mann Whitney t-test), and the whiskers represent the standard error of the mean.

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7.4.4.2 LysR transcriptional regulator in a fusaric acid resistance pump operon (ewfR-7D5::Tn5)

This mutant allele resulted in a significant loss of the antifungal activity in vitro (Figure A7.4A). The Tn5 insertion occurred in an operon (ewf, Figure 7.5A) that included genes that encode membrane proteins required for biosynthesis of the fusaric acid efflux pump including a predicted fusE-MFP/HIYD membrane fusion protein and fusE (ewfD and ewfE, respectively) and other membrane proteins (ewfB, ewfH and ewfI) (Toyoda et al., 1991; Utsumi et al., 1991). Fusaric acid (5-butylpyridine-2-carboxylic acid) is a mycotoxin antibiotic that is produced by Fusarium which interferes with bacterial growth and metabolism and also alters plant physiology (Bacon et al., 2006; Marre et al., 1993). Bacterial-encoded fusaric acid efflux pumps promote resistance to fusaric acid (Borges-Walmsley et al., 2003; Hu et al., 2012). Consistent with expectations, the ewf mutant strain failed to grow on agar supplemented with fusaric acid compared to the wild type (Figure 7.5B-C). The Tn5 insertion specifically occurred within a member of the LysR transcriptional regulator family (ewfR). Similar to ewpR above and a previously published fusaric acid resistance operon (Hu et al., 2012), the regulator was transcribed in the opposite direction as the ewf operon, with this genomic organization suggesting that ewfR may be an upstream regulator of the operon. Indeed, the LysR canonical binding site sequence (TN11A) was observed upstream of the ewfB-J coding sequences (Goethals et al., 1992) (Figure 7.5A). Finally, real time PCR revealed that the expression of ewfD and ewfE were dramatically downregulated in the LysR mutant compared to wild type (Figure 7.5D). Combined, these results suggest that EwfR is a positive regulator of the ewf fusaric acid resistance operon in strain M6. In addition, the ewf mutant showed a significant reduction in motility (Figure 7.5E) and swarming (Figure 7.5F) compared to the wild type (Figure 7.5G), concurrent with a ~30 % reduction in flagella (Figure 7.5H) compared to the wild-type (Figure 7.3K). The mutant also showed loss of attachment to Fusarium hyphae (Figure 7.5I) compared to wild-type (inset) and reductions in biofilm formation (Figure 7.5J-K). Combined, these results suggest that strain M6 expression of resistance to fusaric acid is a pre-requisite step that enables subsequent anti-fungal steps.

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Figure 7.5

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Figure 7.5: Continued

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Figure 7.5: Characterization of fusaric acid resistance mutant ewfR-7D5::Tn5. (A) Genomic organization of the predicted fusaric acid resistance operon showing the position of the Tn5 insertion and putative LysR binding site within the promoter (P). (B-C) The inhibitory effect of fusaric acid embedded within agar on the growth of the ewfR- mutant compared to wild type M6. (D) Quantitative real time PCR gene expression of two protein-coding genes required for the formation of the fusaric acid efflux pump (ewfD and ewfE) in wild type M6 (+) and mutant (-) (ewfR-) backgrounds. (E) Quantification of ewfR- mutant strain motility compared to wild type M6, with representative pictures (inset) of motility assays on semisolid agar plates. (F, G) Light microscopy image showing decreased swarming and colony formation of (F) the ewfR- mutant strain around Fg hyphae stained with lactophenol blue, compared to (G) wild type M6. (H) Electron microscopy image of the ewfR- mutant strain. (I) Confocal microscopy image showing the attachment pattern of the GFP-tagged ewfR- mutant strain (green) to Fg hyphae stained with calcofluor stain, compared to wild type M6 (inset). (J) Confocal microscopy image showing a proteinaceous biofilm matrix stained with Ruby film tracer (red) associated with GFP-tagged ewfR- mutant strain compared to wild type M6 (inset). (K) Spectrophotometric quantification of biofilm formation associated with wild type M6 compared to the ewfR- mutant strain, with representative biofilm assay well pictures (left and right, respectively; 3 replicates shown).qRT-PCR analysis of: (L) wild type ewfR expression in a wild type M6 background, (M) wild type ewpR in an ewfR- mutant background, and (N) wild type ewpR in a wild type M6 background. For graphs shown in (D, E, K, L- N) letters that are different from one another indicate that their means are statistically different (P≤0.05; Mann Whitney t- test, in the case of L-N, within a time point), and the whiskers represent the standard error of the mean.

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7.4.4.3 Interaction between the phenazine biosynthetic operon (ewp) and the fusaric acid resistance operon (ewf)

The expression of ewfR (LysR regulator of fusaric acid resistance) increased two- fold in the presence of Fusarium mycelium in vitro after 1 h of co-incubation, and tripled after 3 h (Figure 7.5L). Expression of ewfR was also up-regulated by fusaric acid alone (Figure 7.5L), demonstrating that the resistance operon is inducible. Fusaric acid has been shown to suppress phenazine biosynthesis through suppression of quorum sensing regulatory genes (van Rij et al., 2005). Interestingly, expression of the putative LysR regulator of phenazine biosynthesis (ewpR, see above) was downregulated by fusaric acid at log phase (2.5-3 h), but only when fusaric acid resistance was apparently lost (ewfR mutant) (Figure 7.5M) compared to wild type (Figure 7.5N), suggesting that fusaric acid normally represses phenazine biosynthesis in M6. These results provide evidence for an epistatic relationship between the two LysR genes required for the anti-Fusarium activity.

7.4.4.4 Diguanylate cyclase (ewgS-10A8::Tn5)

This mutant allele was associated with a significant loss of the antifungal activity in vitro (Figure A7.4A). The Tn5 insertion occurred in a coding sequence encoding diguanylate cyclase (EC 2.7.7.65) (ewgS), an enzyme that catalyzes conversion of 2- guanosine triphosphate to c-di-GMP, a secondary messenger that mediates quorum sensing and virulence traits (Römling et al., 2013) (Figure 7.6A). Addition of exogenous c-di-GMP to the growth medium restored the antifungal activity of the mutant (Figure 7.6B). Real time PCR showed that ewgS is not inducible by Fusarium (Figure 7.6C). Consistent with its predicted upstream role in regulating virulence traits, the ewg mutant showed dramatic losses in motility (Figure 7.6D), swarming (Figure 7.6E, F) and flagella formation (40% reduction, Figure 7.6G compared to wild-type, Figure 7.3K). Attachment to Fusarium hyphae (Figure 7.6H) and biofilm formation (Figure 7.6I-J) appeared to have been lost completely.

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Figure 7.6

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Figure 7.6: Continued

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Figure 7.6: Characterization of di-guanylate cyclase mutant ewgS-10A8::Tn5. (A) Illustration of the enzymatic conversion of 2 guanosine phosphate to c-di- GMP catalyzed by di-guanylate cyclase. (B) Complementation of the putative ewgS- mutant with respect to inhibition of F. graminearum (Fg) by addition of c-di-GMP (0.01 and 0.02 mg/ml), compared to wild type strain M6. (C) qRT-PCR analysis of wild type ewgS+ expression in a wild type M6 background. (D) Quantification of ewgS- mutant strain motility compared to wild type M6, with representative pictures (inset) of motility assays on semisolid agar plates. (E, F) Light microscopy image showing decrease in swarming and colony formation of (E) ewgS- mutant strain around Fg hyphae stained with lactophenol blue, compared to (F) wild type M6. (G) Electron microscopy image of ewgS- mutant strain. (H) Confocal microscopy image showing attachment pattern of GFP-tagged ewgS- mutant strain (green) to Fg hyphae stained with calcofluor stain, compared to wild type M6 (inset). (I) Confocal microscopy image showing loss of proteinaceous biofilm matrix stained with Ruby film tracer (red) associated with GFP-tagged ewgS- mutant strain compared to wild type M6 (inset). (J) Spectrophotometric quantification of biofilm formation associated with wild type M6 compared to the ewgS- mutant strain, with representative biofilm assay well pictures (left and right, respectively; 3 replicates shown). For graphs shown in (C, D, J), letters that are different from one another indicate that their means are statistically different (P≤0.05, Mann Whitney t-test), and the whiskers represent the standard error of the mean.

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7.4.4.5 Colicin V production protein (EwvC-4B9::Tn5)

This mutant strain showed significant loss of the antifungal activity in vitro (Figure A7.4A). The Tn5 insertion occurred in a minimally characterized gene required for colicin V production (ewvC) orthologous to cvpA in E.coli (Fath et al., 1989). Colicin V is a secreted peptide antibiotic (Fath et al., 1994). Consistent with the gene annotation, the ewvC mutant strain failed to inhibit the growth of an E. coli strain that is sensitive to colicin V, compared to wild type M6 (Figure 7.7A). Real time PCR showed that the expression of wild type ewvC increased 6-fold after co-incubation with Fusarium at log phase (Figure 7.7B). The mutant showed only limited changes in other virulence traits, including reductions in motility (Figure 7.7C) and swarming (Figure 7.7D,E), but with only a ~10% minor reduction in flagella formation (Figure 7.7F, compared to wild-type, Figure 7.3K), no obvious change in attachment to Fusarium hyphae (Figure 7.7G), and only a slight reduction in biofilm formation (Figure 7.5H,I).

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Figure 7.7

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Figure 7.7: Characterization of colicin V mutant ewvC-4B9::Tn5. (A) Dual culture agar diffusion assay showing loss of antagonism against the colicin V sensitive E coli strain (MC4100) by the ewvC- mutant compared to wild type M6. (B) qRT-PCR analysis of ewvC+ expression in a wild type M6 background. (C) Quantification of ewvC- mutant strain motility compared to wild type M6, with representative pictures (inset) of motility assays on semisolid agar plates. (D, E) Light microscopy image showing decreases in swarming and colony formation of (D) the ewvC- mutant strain around Fg hyphae stained with lactophenol blue, compared to (E) wild type M6. (F) Electron microscopy image of the ewvC- mutant strain. (G) Confocal microscopy image showing the attachment pattern of the GFP-tagged ewvC- mutant strain (green) to Fg hyphae stained with calcofluor stain, compared to wild type M6 (inset). (H) Confocal microscopy image showing the proteinaceous biofilm matrix stained with Ruby film tracer (red) associated with the GFP-tagged ewvC- mutant strain compared to wild type M6 (inset). (I) Spectrophotometric quantification of biofilm formation associated with wild type M6 compared to the ewvC- mutant strain, with representative biofilm assay well pictures (left and right, respectively; 3 replicates shown). For graphs shown in (B, C, I) letters that are different from one another indicate that their means are statistically different (P≤0.05, Mann Whitney t- test), and the whiskers represent the standard error of the mean.

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7.5 Discussion

7.5.1 RHESt as a novel plant defence mechanism

I hypothesized that the ancient African cereal finger millet hosts endophytic bacteria that contribute to its resistance to Fusarium, a pathogenic fungal genus that has been reported to share the same African origin as its plant target. Here, I report that a microbial inhabitant of finger millet (M6) actively swarms towards invading Fusarium hyphae, analogous to mobile immunity cells in animals, to protect plant cells that are immobile, consistent with a hypothesis that we recently proposed (Soliman et al., 2015). Endophyte M6 then builds a remarkable physico-chemical barrier resulting from root hair-endophyte stacking (RHESt) at the rhizosphere-root interface that prevents entry and/or traps Fusarium for subsequent killing. Mutant and biochemical data demonstrate that the killing activity of M6 requires genes encoding diverse regulatory factors, compounds and xenobiotic resistance. The RHESt consists of two lines of defence, a dense layer of intercalated root hairs and endophyte microcolonies followed by a long, continuous endophyte barrier layer on the root epidermal surface (Figure A7. 5). RHESt represents a plant defence mechanism that has not been previously captured to the best of our knowledge and is an unusual example of host-microbe symbiosis.

The epidermal root surface where microbes reside is termed the rhizoplane (Compant et al., 2010; Germaine et al., 2009; Glaeser et al., 2015; Marasco et al., 2012; Reinhold-Hurek et al., 2015). Soil microbes have previously been reported to form biofilm-mediated aggregates on the rhizoplane (Dandurand et al., 1997; Foster, 1986; Fukui et al., 1994; Newman and Bowen, 1974; Rovira, 1956; Rovira and Campbell, 1974) sometimes as part of their migration from the soil to the root endosphere (Compant et al., 2010; Mercado-Blanco and Prieto, 2012) and may prevent pathogen entry (Compant et al., 2010; Danhorn and Fuqua, 2007). However, here I report that an endophyte, not soil microbe, forms a pathogen barrier on the rhizoplane. The previously reported soil microbes on the rhizoplane are thought to take advantage of nutrient-rich root exudates (Danhorn and Fuqua, 2007), whereas RHESt is a de novo, inducible structure that only forms in the presence of Fusarium in coordination with root hairs.

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M6 creates its own specialized killing microhabitat by inducing growth of local root hairs which are then bent to form the RHESt scaffold, likely mediated by biofilm formation and attachment. In vitro, I observed that M6 synthesizes auxin (IAA), a hormone known to stimulate root hair growth (Lee and Cho, 2014) and that can be synthesized by microbes (Patten and Glick, 1996). Root hair bending associated with RHESt might be an active process, similar to rhizobia-mediated root hair curling (Oldroyd, 2013), and/or an indirect consequence of micro-colonies attachment to adjacent root hairs.

7.5.2 Regulatory signals within the anti-fungal pathway

Some of the M6 mutants that lost anti-fungal activity had pleiotropic phenotypes, including loss of swarming, attachment and/or biofilm formation, which was a surprising result. These mutants included the transcription factors associated with operons for phenazine biosynthesis, fusaric acid resistance, as well as formation of cyclic-di-GMP. One interpretation of these results is that the underlying genes help to regulate the early steps of the anti-fungal RHESt pathway. Indeed, phenazines have been reported to act as signaling molecules that regulate the expression of hundreds of genes including those responsible for motility and defense (Du et al., 2015). Our results showed an epistatic relationship between the two transcription factors regulating phenazine biosynthesis and fusaric acid resistance, with the latter required for the former (Figure 7.4M,N), suggesting that the pleiotropic phenotypes observed in the fusaric acid resistance regulatory mutant may have been mediated by a reduction in phenazine signaling. Finally, c-di-GMP as a sensor of the environment and population density (Flemming and Wingender, 2010; Srivastava and Waters, 2012), and a secondary messenger involved in transcriptional regulation of genes encode virulence traits such as motility, attachment, and biofilm formation (Römling et al., 2013) (Landini et al., 2010; Srivastava and Waters, 2012; Steinberg and Kolodkin-Gal, 2015), all activities that would be logically required for the RHESt-mediated anti-fungal pathway. Genome mining of strain M6 also revealed the presence of a biosynthetic cluster for 2, 3 butanediol which is a hormone known to elicit plant defences (Cortes‐Barco et al., 2010). Production of 2, 3 butanediol

229 was confirmed by LC-MS analysis (Figure A7.6A). Butanediol is thus a candidate signalling molecule for M6-millet cross-talk.

7.5.3 Fungicidal compounds required for M6 killing activity

In addition to RHESt formation, I gained evidence that bacterial endophyte M6 evolved multiple biochemical strategies to actively break and kill Fusarium hyphae (Figure 7.2J and Figure 7.3A,J) involving diverse classes of compounds, including phenazines, colicin V, chitinase and potentially other metabolites.

7.5.3.1 Phenazines: Phenazines are heterogeneous nitrogenous compounds produced exclusively by bacteria (Mavrodi et al., 2013). Phenazines exhibit potent antifungal activity, in particular against soil pathogens including Fusarium species (Gurusiddaiah et al., 1986; Anjaiah et al., 1998; Ligon et al., 2000; Bloemberg and Lugtenberg, 2003). Here M6 was observed to produce at least two distinct phenazines, griseolutein A and D- alanyl-griseolutein (Figure 4F-G), previously shown to have anti-microbial activities (Hori et al., 1978). Phenazines lead to the accumulation of reactive oxygen species in target cells, due to their redox potential (Hassan and Fridovich, 1980; Hassett et al., 1995). Phenazines were also reported to induce host resistance (Audenaert et al., 2002). For bacteria to survive inside a biofilm, where oxygen diffusion is limited, molecules with high redox potential such as phenazines are required (Fu et al., 1994; Hernandez and Newman, 2001; Price-Whelan et al., 2006; Stewart, 2003; Wang and Newman, 2008). Furthermore, the redox reaction of phenazine releases extracellular DNA which enhances surface adhesion and cellular aggregation of bacteria to form a biofilm (Das et al., 2013; Das et al., 2015; Das and Manefield, 2012; Das et al., 2010; Petersen et al., 2005; Whitchurch et al., 2002). Consistent with these roles, phenazines are known to be produced inside biofilms (Price-Whelan et al., 2006), and are required for biofilm formation (Pierson III and Pierson, 2010), which might explain the diminished biofilm formation observed with the phenazine-associated LysR mutant. Since M6 was observed to produce biofilm around its fungal target (Figure 7.3M) which is killed within RHESt (Figure 7.2J), phenazines may be part of the killing machinery.

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7.5.3.2 Colicin V: An exciting observation from this study is that colicin V also appears to be required for the fungicidal activity of strain M6 (Figure 7.7). Colicin V is a small peptide antibiotic belonging to the bacteriocin family which disrupts the cell membrane of pathogens resulting in loss of membrane potential (Gérard et al., 2005). Bacteriocins are generally known for their antibacterial activities (Gillor and Ghazaryan, 2007). Indeed, I could find only one previous report of colicin V having anti-fungal activity (Hammami et al., 2011). Our results suggest that this compound may target a wider spectrum of pathogen targets than previously thought.

7.5.3.3 Chitinase: A Tn5 insertion in a gene (ewc-3H2::Tn5) encoding chitinase (EC 3.2.1.14) also resulted in a significant loss of the antifungal activity (Figure A7.4A). I confirmed that the mutant showed a significant reduction in production of chitinase in vitro, compared to wild type M6 (Figure A7.6B). Chitinase hydrolyzes chitin, a principle component of the fungal cell wall (Dahiya et al., 2005a; Dahiya et al., 2005b). Chitin-producing microbes were previously reported to have antifungal activity against diverse fungal pathogens (Dahiya et al., 2005a; Dahiya et al, 2005). Hydrolysis of chitin produces N-acetyl glucosamine that has also been shown to induce plant host resistance (Gohel et al., 2006).

7.5.3.4 Other putative M6 anti-fungal metabolites: Two additional putative Tn5 mutants suggest that other metabolites may be involved in the anti-Fusarium activity of strain M6 within the RHESt, specifically phenylacetic acid (PAA) and P-amino-phenyl-alanine antibiotics (PAPA). The requirement for PAA was suggested by a putative Tn5 insertion in a gene encoding phenylacetic acid monoxygenase which resulted in loss of anti- Fusarium activity (m2D7, Figure A7.4A). This enzyme catalyzes the biosynthesis of hydroxy phenylacetic acid, derivatives of which have been shown to act as anti-fungal compounds (Burkhead et al., 1998; Slininger et al., 2004). In an earlier report, an Enterobacter sp. that was used to control Fusarium dry rot during seed storage (Schisler et al., 2000) was shown to require phenylacetic acid, indole-3-acetic acid (IAA) and

231 tyrosol (Slininger et al., 2004). In addition to PAA being implicated here, wild type strain M6 was shown to produce IAA in vitro (Figure A7.3). The requirement for PAPA was suggested by a putative Tn5 insertion (ewt-15A12::Tn5, Figure A7.4A) which appears to have disrupted a gene encoding a permease transport protein that is a part of an operon responsible for biosynthesis of PAPA and 3-hydroxy anthranilates. PAPA is the direct precursor of well known antibiotics including chloramphenicol and obafluorin (Herbert and Knaggs, 1992; He et al., 2001; Lewis et al., 2003).

7.5.4 M6-Fusarium-millet co-evolution and the fusaric acid-phenazine arms race

I previously demonstrated that finger millet also hosts fungal endophytes that secrete complementary anti-F. graminearum natural products including polyketides and alkaloids (Mousa et al., 2015a). These observations, combined with loss of function bacterial mutants from this study that demonstrate that no single anti-fungal mechanism is sufficient for M6 to combat Fusarium, and that diverse classes of anti-fungal compounds are required (phenazine metabolites, colicin V peptide antibiotic, chitinase enzyme), suggests that the endophytic community of finger millet and Fusarium have been engaged in a step-by-step arms race that resulted in the endophytes having a diverse weapons arsenal, presumably acting within RHESt. Consistent with this interpretation, mutant analysis showed that the anti-fungal activity of M6 requires a functional operon that encodes resistance to the Fusarium mycotoxin, fusaric acid. Furthermore, our results show a novel epistatic regulatory interaction between the fusaric acid resistance and phenazine pathways, wherein an M6-encoded LysR activator of fusaric acid resistance prevents fusaric acid from suppressing expression of the M6-encoded LysR regulator of phenazine biosynthesis (Figure 7.5M). Fusaric acid has previously been shown to interfere with quorem sensing-mediated biosynthesis of phenazine (van Rij et al., 2005). I propose that these data, in particular the phenazine-fusaric acid arms race, provide a molecular and biochemical paleontological record that M6 and Fusarium co-evolved.

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I show how this tripartite co-evolution likely benefits subsistence farmers not only by suppressing Fusarium entry and hence disease in plants, but also in seeds after harvest. Specifically, under poor seed storage conditions that mimic those of subsistence farmers, M6 caused dramatic reductions in contamination with DON (Figure 7.1M,R), a potent human and livestock mycotoxin. Hence, in the thousands of years since this ancient crop was domesticated, farmers may have been inadvertently selecting for the physico-chemical RHESt barrier activity of endop

hyte M6, simply by selecting healthy plants and their seeds. I have shown here that the benefits of 6 are transferable to two of the world’s most important modern crops, maize and wheat (Figure 7.1I and O), which are severely afflicted by F. graminearum and DON. In addition to F. graminearum, M6 inhibited the growth of five other fungi including two additional Fusarium species (Table A7.3), suggesting that RHESt- mediated plant defence may contribute to the broad spectrum pathogen resistance of finger millet reported by subsistence farmers. Despite its importance, finger millet is a scientifically neglected crop (Goron and Raizada, 2015; Thilakarathna and Raizada, 2015). Therefore, our study suggests the value of exploring the microbiome-host interactions of other scientifically neglected, ancient crops.

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Chapter 8: Bacterial endophytes from wild maize suppress Fusarium graminearum in modern maize and inhibit mycotoxin accumulation

Authors’ contributions

Walaa K Mousa designed and carried out all experiments, performed all data analyses, and wrote the manuscript.

Charles S Chearer helped in the greenhouse experiments.

Victor Limy-Rois performed DON assay.

Dr. Manish N. Raizada designed the study and edited the manuscript.

Publication status: published.

Mousa WK, Shearer C, Limay-Rios V, Zhou T and Raizada MN (2015). Bacterial endophytes from wild maize suppress Fusarium graminearum in modern maize and inhibit mycotoxin accumulation. Frontiers in Plant Science 6, 805.

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8.1 Abstract

Wild maize (teosinte) has been reported to be less susceptible to pests than their modern maize (corn) relatives. I hypothesized that the wild relatives of modern maize may host endophytes that combat pathogens. Fusarium graminearum is the fungus that causes Gibberella Ear Rot (GER) in modern maize and produces the mycotoxin, deoxynivalenol (DON). In this study, 215 bacterial endophytes, previously isolated from diverse maize genotypes including wild teosintes, traditional landraces and modern varieties, were tested for their ability to antagonize F. graminearum in vitro. Candidate endophytes were then tested for their ability to suppress GER in modern maize in independent greenhouse trials. The results revealed that three candidate endophytes derived from wild teosintes were most potent in suppressing F. graminearum in vitro and GER in a modern maize hybrid. These wild teosinte endophytes could suppress a broad spectrum of fungal pathogens of modern crops in vitro. The teosinte endophytes also suppressed DON mycotoxin during storage to below acceptable safety threshold levels. A fourth, less robust anti-fungal strain was isolated from a modern maize hybrid. Three of the anti-fungal endophytes were predicted to be Paenibacillus polymyxa, along with one strain of Citrobacter. Microscopy studies suggested a fungicidal mode of action by all four strains. Molecular and biochemical studies showed that the P. polymyxa strains produced the previously characterized anti-Fusarium compound, fusaricidin. Our results suggest that the wild relatives of modern crops may serve as a valuable reservoir for endophytes in the ongoing fight against serious threats to modern agriculture. I discuss the possible impact of crop evolution and domestication on endophytes in the context of plant defense.

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8.2 Introduction

Modern maize, belonging to the genus Zea, was domesticated in southern Mexico 9,000 years ago from wild, annual tropical grasses called teosintes, with the primary ancestor being Parviglumis (Zea mays ssp. parviglumis) which survives today in the wild (Matsuoka et al., 2002). There are additional species of teosintes that continue to grow in the wild in Mexico and Central America including the perennial Zea diploperennis (Iltis and Doebley, 1980). Following its domestication into an edible crop (Z. mays ssp. mays), maize was bred and spread by indigenous farmers throughout the Americas to give rise to diverse traditional landraces (Matsuoka et al., 2002). In the 20th Century, scientists created improved, commercial inbreds and hybrids such as the temperate hybrid, Pioneer 3751 (Smith et al., 2004). Wild maize has been reported to be more resistant to pests than their modern counterparts, perhaps due to loss of defense alleles and/or loss of the protective casing (fruitcase) enclosing the grains in modern varieties, as a result of breeding and domestication (Wang et al., 2005; Lange et al., 2014).

Though the increased disease susceptibility of modern maize has been attributed to changes in the plant genome, there may be additional explanations. Endophytes are microbes that inhabit the internal tissues of plants, including seeds, without causing disease symptoms (Johnston-Monje and Raizada, 2011; Mousa and Raizada, 2013). Some endophytes have been shown to help their host plants to combat pathogens (Mousa and Raizada, 2013, 2015). During maize evolution, domestication, breeding and migration, some endophytes were lost (Johnston-Monje and Raizada, 2011; (Johnston- Monje et al., 2014), and it is also possible that endophytic genomes may have been modified - phenomena that might contribute to the increased disease susceptibility of modern maize.

Modern maize is susceptible to various pathogens including Fusarium graminearum, the fungus that causes Gibberella Ear Rot (GER). GER is a serious global disease particularly in Europe, the United States and Canada (van der Lee et al., 2014).

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In grain, F. graminearum produces deoxynivalenol (DON), a mycotoxin that inhibits DNA and protein synthesis, resulting in various toxicity effects in both humans and animals (Munkvold, 2003b; Voss, 2010; Hassan et al., 2015). In a three year survey, 59% of maize samples tested from around the world were found to be contaminated with DON (Rodrigues and Naehrer, 2012).

Although the current disease management strategies to combat F. graminearum rely on breeding for resistance genotypes, optimizing cultural practices or use of fungicides, these strategies have achieved low to moderate success (Munkvold, 2003a; Reid et al., 2009). A promising alternative strategy to manage Fusarium outbreaks and reduce mycotoxin contamination may be through the use of biological antagonists (Eilenberg, 2006; Chulze et al., 2014). We have recently reported that wild, traditional and modern maize possess endophytes that combat pathogens including F. graminearum in vitro (Johnston-Monje and Raizada, 2011; Johnston-Monje et al., 2014). Other studies have identified other biological control agents that combat F. graminearum including Bacillus and Pseudomonas sp. (Moussa et al., 2013; Shi et al., 2014). However, most of this research is preliminary, and effective commercial biological control is not currently available.

Here, I tested the hypothesis that the wild relatives of maize may possess endophytes that help their hosts to naturally combat F. graminearum. A library of bacterial endophytes, previously isolated from diverse maize genotypes including wild, traditional and modern varieties (Johnston-Monje and Raizada, 2011; Johnston-Monje et al., 2014), were screened for their ability to inhibit the growth of F. graminearum in vitro and suppress GER in planta.

8.3 Materials and Methods

8.3.1 Source of bacterial endophytes

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A library of 215 bacterial endophytes was previously isolated in our lab to study the diversity of maize microbial endophytes (Johnston-Monje and Raizada, 2011). The endophytes were isolated from 14 diverse Zea genotypes, including wild teosintes (Zea mays ssp. parviglumis, Zea mays ssp. mexicana, Zea diploperennis, Zea nicaraguensis), ancient and traditional Mexican landraces of modern maize (Zea mays ssp. mays: Gaspe yellow Flint, Cristalino de Chihuahua, Chapalote, Mixteco, Bolita, Jala, Nal-Tel, Tuxpeno) and modern maize varieties (Zea mays ssp. mays: Pioneer 3751 and B73). The endophytes analyzed from wild, traditional and modern genotypes represented 46%, 33% and 21% of the library, respectively, with approximately half the library isolated from non-wild, post-domesticated maize (54%) (Table A8.3).

8.3.2 Antifungal screening

Overnight cultures of each endophyte were used to screen endophytic bacteria for in vitro inhibition of growth of F. graminearum (obtained from Agriculture and Agrifood Canada, Guelph, ON) using the dual culture method. Each bacterial endophyte was cultured in liquid broth (LB, Luria-Bertani, composed of 10 g NaCl, 5 g yeast extract, 10 g tryptone, per liter), grown for 1-3 days at 37 ˚C with shaking at 225 rpm then centrifugation for 5 min, followed by resuspension in PBS buffer to an OD600 of 0.4-0.6 (Spectromax, serial # MN03135, USA). F. graminearum was grown for 48 h (25˚C, 100 rpm) in liquid potato dextrose broth media (Catalog # P6685, Sigma Aldrich, USA), then mycelia was added to melted, cooled PDA media (1 ml of fungus into 100 ml of media), mixed and poured into Petri dishes (100 mm x 15 mm), then allowed to re-solidify. Wells (11 mm diameter) were created in this pathogen-embedded agar by puncturing with sterile glass tubes, into which the endophyte cultures were applied (200 µl per well). The agar plates were incubated at 30˚C for 48 h in darkness. The radius of each one of inhibition was measured (mm). The commercial broad-spectrum fungicides, Amphotericin B (Catalog #A2942, Sigma Aldrich, USA) and Nystatin (Catalog #N6261, Sigma Aldrich, USA), were used as positive controls at concentrations of 5 μg/ml and 10 μg/ml, respectively. LB was used as a negative control. Each endophyte was screened in three independent replicates.

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8.3.3 Anti-fungal target spectrum of the candidate endophytes Endophytes that tested positive for activity against F. graminearum were re- screened for activity against a diversity of other fungal species including crop pathogens (from the Agriculture and Agrifood Canada Fungal Type Collection, Guelph, ON, Canada) using the dual culture method (described above) to characterize the activity spectrum of each endophyte. The crop fungi tested included: Alternaria alternata, Alternaria arborescens, Aspergillus flavus, Aspergillus niger, Bionectria ochroleuca, Davidiella (Cladosporium) tassiana, Diplodia pinea, Diplodia seriata, Epicoccum nigrum, Fusarium lateritium, Fusarium sporotrichioides, Fusarium avenaceum (Gibberella avenacea, two isolates), Nigrospora oryzae, Nigrospora sphaerica, Paraconiothyrium brasiliense, Penicillium expansum, Penicillium afellutanum, Penicillium olsonii, Rosellinia corticium, Torrubiella confragosa, Trichoderma hamatum and Trichoderma longibrachiatum.

8.3.4 Molecular identification of candidate endophytic bacteria using 16S rDNA and 23S rDNA For taxonomic identification of candidate endophytic bacteria, a standard PCR protocol was used (Johnston-Monje and Raizada, 2011). Bacterial genomic DNA was extracted (GenElute Bacterial Genomic DNA kit, NA2110-1KT, Sigma) and quantified using a Nanodrop machine (Thermo Scientific, USA). The extracted DNA was used to amplify 16S rDNA and 23S rDNA using PCR.

For 16S rDNA amplification: A PCR master mix (20 μl) was made as follows: 2.5 μl Standard Taq Buffer (10X) (New England Biolabs), 0.5 μl of 25 m dNTP mix, 1 μl of 10 m 1492r primer with sequence GGTTACCTTGTTACGACTT, 1 μl of 10 m 799f primer with sequence AACMGGATTAGATACCCKG (Where M is A or C and K is G or T), 0.25 μl of 50 m gCl2, 0.25 μl of Standard Taq (10 U/μl, New England Biolabs), 50 ng DNA were added to the total reaction volume (2.5 ng/µl was the final DNA concentration in the PCR reaction) and double distilled water up to 20 μl total.

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For 23S rDNA amplification: A PCR master mix (20 μl) was made as described above using 1 μl of 10 m 23S 6F primer with sequence 5′- GCGATTTCYGAAYGGGGRAACCC and 1μl of 10 m 23S R primer with sequence 5′- TTCGCCTTTCCCTCACGGTACT (where Y is C or T and R is A or G) (Anthony et al., 2000).

For both 16S rDNA and 23S rDNA, the PCR amplification conditions were: 96°C for 3 min, followed by 35 amplification cycles (94°C for 30 sec, 48°C for 30 sec, 72°C for 90 sec), and a final extension at 72°C for 7 min, using a PTC200 DNA Thermal Cycler (MJ Scientific, USA). Finally, the PCR products were separated on 1.5 % agarose gels at ≤5 V/cm, then the bands were visualized under UV light; 700 bp and 400 bp bands were excised for 16S rDNA and 23S rDNA, respectively and eluted from the gels (Illustra GFX 96 PCR Purification kit, GE Healthcare, USA). The purified DNA was sequenced at the Genomic Facility Laboratory at the University of Guelph. For 16S, primers 1492r and 799f were used for sequencing, while for 23S, primer 23S 6F was used. Bacterial strains were identified based on 16S rDNA and 23S rDNA sequence comparisons using BLAST searches to GenBank. To assist with taxonomic identification, the full length 16S rDNA sequences for the three Paenibacillus sp. were used to generate a phylogenetic tree using Phylogeny.fr (Dereeper et al., 2008; Dereeper et al., 2010).

8.3.5 Scanning electron microscope (SEM) imaging of endophytes

Scanning electron microscopy imaging was conducted to visualize the external appearance of candidate bacteria following a standard protocol (Hayat, 1974). Bacterial cultures were plated on LB plates, incubated for 24 h then suspended and washed in phosphate buffer (pH 7). A drop of the suspension was placed on a carbon disc and left to dry for 1 h. The dried bacteria was washed with phosphate buffer then fixed by adding 2% glutaraldehyde for 1 h. The fixed bacteria was then treated with 1% osmium tetroxide for 30 min, then gradually dehydrated using an ethanol series (50%, 70%, 80%, 90% and 100%) followed by critical point drying. The dried bacterial films were coated with gold and examined by SEM (Hitachi S-570 SEM, Hitachi High Technologies, Tokyo, Japan) at the Imaging Facility, Department of Food Science, University of Guelph.

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8.3.6 In vitro interaction between each endophyte and F. graminearum

The in vitro interaction between F. graminearum and each bacterial endophyte was studied microscopically. Each microscope slide was coated with a thin layer of PDA, then 50 µl of bacterial endophyte culture (grown overnight in LB incubated at 37ºC, 250 rpm) was applied adjacent to 50 µl of F. graminearum mycelia (grown for 24-48 h in potato dextrose broth at 25˚C, 100 rpm). Each slide was incubated at 25ºC for 24 h then stained with the vitality stain, neutral red (Sigma Aldrich, Catalog #57993) or Evans blue (Sigma Aldrich, Catalog # E2129) by placing 100 µl of stain on the slide, followed by a 3-5 min incubation at room temperature, then washing 3-4 times with deionized water. A commercial biological control agent with a fungicidal mode of action (Bacillus subtilis QST713, Bayer CropScience, Batch #00129001) was used as a positive control (100 mg/10 ml). Pictures were taken using light microscopes (MZ8, Leica, Wetzlar, Germany for neutral red staining; and a BX51 microscope, Olympus, Tokyo, Japan for Evans blue staining). There were 3-4 replicates for each slide.

8.3.7 PCR based approach to detect the fusaricidin biosynthetic gene in the Paenibacillus endophyte strains

In order to detect the presence of a candidate fusaricidin synthetase gene (Eric et al.) in the Paenibacillus endophyte strains, two oligonucleotides (FusAF and FusAR) were designed based on the fusA sequence (GenBank accession #EU184010) using Primer3 software. For fusA amplification, a PCR master mix (20 μl) was made as follows: 2.5 μl Standard Taq Buffer (10X) (New England Biolabs), 0.5 μl of 25 m dNTP mix, 0.25 μl of 50 m gCl2, 0.25 μl of Standard Taq (10 U/μl, New England Biolabs), 1 μl of primer FusAF with sequence 5′- AGGCAAGCTTTGACTTGGAA -3′ and 1 μl of primer FusAR with sequence 5′- CGCTTGCTCAGACCATACAA -3′, 50 ng DNA were added to the total reaction volume (2.5 ng/µl was the final DNA concentration in the PCR reaction) and double distilled water up to 20 μl total. The PCR amplification conditions were: 96°C for 3 min, followed by 35 amplification cycles (94°C for 30 sec, 48°C for 30 sec, 72°C for 90 sec), and a final extension at 72°C for 7 min, using a

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PTC200 DNA Thermal Cycler (MJ Scientific, USA). The PCR products were separated on 1.5 % agarose gels at ≤5 V/cm, then the bands were visuali ed under UV light; bands were excised and eluted from the gels (Illustra GFX 96 PCR Purification kit, GE Healthcare, USA). The purified DNA was sequenced at the Genomic Facility Laboratory at the University of Guelph. The corresponding gene was identified based on best BLAST matches to Genbank.

8.3.8 Biochemical detection of fusaricidins in endophyte culture filtrates using LC- MS

To detect the presence of fusaricidins biochemically in Paenibacillus sp., endophytes were grown for 48 h on Katznelson and Lochhead liquid medium (Paulus and Gray, 1964), harvested by freeze drying, then the lyophilized powder from each strain was extracted by methanol. The methanolic extracts were run on a Luna C18 column with a gradient of 0.1% formic acid and 0.1% formic in acetonitrile. Peaks were analyzed by mass spectroscopy (Agilent 6340 Ion Trap), ESI, positive ion mode. LC-Mass analysis was conducted at the Mass Spectroscopy Facility, McMaster University, Ontario, Canada. The m/z ratios were compared to the published literature (Beatty and Jensen, 2002; Kajimura and Kaneda, 1996, 1997).

8.3.9 GFP-tagging for ecological tracking in planta

In order to test the ability of candidate anti-Fusarium endophytes to colonize a modern maize hybrid, the endophytes were subjected to tagging with green fluorescent protein (GFP) followed by in planta visualization using confocal scanning microscopy.

To prepare competent cells: One liter of LB broth was inoculated with 10 ml bacterial o culture grown overnight (37 C at 250 rpm) until early log phase (OD600 = 0.4-0.6). The cells were harvested by chilling for 15 min on ice and centrifuged at 4000 x g for 15 min

242 at 4˚C. The pellets were then re-suspended in cold water and centrifuged two times. Finally, the pellets were re-suspended in cold 10% glycerol, centrifuged and re- suspended in 3 mL of 10% glycerol from which 40 µl aliquots were made and frozen at - 80oC.

GFP plasmid transformation: A wide-host promoter plasmid, pDSK-GFPuv (Wang et al., 2007) was used to transform E.coli DH5α (Catalog #EC6P095H, Epicenter, Madison, USA), which was then stored at -80oC. The plasmid was extracted from E.coli using a standard protocol (Catalog # 732-6100, BioRad Inc, USA) then introduced into bacterial cells by electroporation (Calvin and Hanawalt, 1988). Suspensions of 40 μl cold competent cells were mixed with 1 μl of plasmid DNA (240 ng/μl) then electroporated at 1.6 KV for 1 sec using a Bio-Rad Gene Pulser 200/2.0 (Bio-Rad Hercules, USA). After electroporation, cells were incubated for 1 h in 1 ml of LB at 37°C with shaking at 250 rpm. Transformed cells were plated on LB agar containing Kanamycin (35 μg/μl) and incubated for 24 h at 37°C, then the plate was examined for fluorescent colonies (Illumatool, #LR 92240, Lightools Research, USA).

To visualize GFP-tagged endophyte cells inside maize tissues: Modern maize seeds (Ontario maize hybrid P35F40, see below) were surface sterilized, coated with GFP- tagged endophyte (see below for details), and planted on wet paper towels. One-week-old seedlings were stained with propidium iodide (1 mg/ml) (Sigma Aldrich, Catalog #P4170) then washed with deionized water. The seedlings were screened by a TCS SP2 confocal laser scanning microscope (Leica Microsystems, Mannheim, Germany) at the Imaging Facility, University of Guelph. The conditions for confocal microscopy were as follows: excitation at 488 nm with an Argon laser and at 543 nm with a green helium laser (emission ranges = 504-532 nm and 524-699 nm, respectively), pinhole [Au] = 1.0 airy, objective lens = 63x oil immersion, and frame average = 3 times.

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8.3.10 Suppression of GER in greenhouse trials

The candidate endophytes that suppressed the growth of F. graminearum in vitro were tested for their ability to suppress GER in greenhouse trials (Crop Science Greenhouse Facility, University of Guelph):

Seed treatment: Seeds of a susceptible commercial maize hybrid P35F40 were surface sterilized as follows: seeds were washed in 0.1% Triton X-100 detergent for 10 min with shaking; the detergent was decanted, 3% sodium hypochlorite was added for 10 min, followed by rinsing with autoclaved, distilled water, washing with 95% ethanol for 10 min; and finally the samples were washed 5-6 times with autoclaved, distilled water. Effective surface sterilization was ensured by inoculating the last wash on LB and PDA plates at 37°C and 25°C, respectively; all washes showed no growth. The sterilized seeds were then coated with endophytic inoculants (on the day of planting). To prepare endophytic bacterial inoculants, bacteria were grown for 24 h at 37˚C in liquid LB medium, centrifuged, washed and suspended in PBS buffer to an OD600 of 0.5. Thereafter, 500 μl of each bacterial suspension were mixed with 10 ml polyvinyl pyrrolidine (PVP, Catalog # 9003398, Sigma Aldrich, USA) as a seed-coating agent; then incubated with the seeds for 2 h on a horizontal shaker (Serial #980216M, National Labnet Company, USA). Seed coated with the endophytes or buffer control were then germinated on wet paper towels and kept in the dark for 7 days; uniformly sized seedlings were transferred into pots containing Turface clay (Turface Athletics Inc, USA) in the greenhouse under the following growth conditions: (28˚C/20˚C, 16h:8 h, ≥800 μmol m-2 s-1 at pot level, with high pressure sodium and metal halide lamps supplemented with GroLux bulbs) using drip irrigation with modified Hoagland’s solution until maturity (Gaudin et al., 2011).

Pathogen introduction: Pathogen spores were prepared as follows: the liquid medium used for spore suspension was prepared with the following composition per liter in distilled water: 2 g KH2PO4, 2 g KNO3, 1 g MgSO4, 1 g KCl, 1 g dextrose, 20 mg/100 ml

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(of each) of minor elements (FeCl2, MnSO4, ZnSO4). Approximately 350 ml of the liquid medium was added to 2 L flasks and autoclaved at 121°C for 10 min. After cooling to room temperature, either 3-4 PDA plugs of F. graminearum isolate or 10 ml of liquid conidia suspension was added aseptically to each flask, which was incubated on a shaker table at room temperature under 12:12 h UV light: dark cycle for approximately two weeks. Using a haemocytometer, the solution was standardized to 20,000 spores/ml before being stored in the fridge or used in the greenhouse directly. One ml of F. graminearum spore suspension was applied first to silks beginning after their emergence.

Endophyte silk spray treatment: To ensure high titre of the endophytes, they were introduced a second time, by spraying 1 ml of each endophyte (OD600 of 0.5, grown in LB) simultaneously with the pathogen inoculant, and then again at three days after pathogen inoculation.

Control treatments: For the positive control group, seeds were coated only with PVP followed by prothioconazole fungicide spraying (PROLINE® 480 SC, Bayer Crop Science) at the post-silking stage prior to infection with the fungal pathogen. The negative control was seeds coated only with PVP, then sprayed at silking with 1 ml of F. graminearum spore suspension only.

Experimental design: There were 20 plants arranged in a randomized block design per each treatment group. The trial was repeated independently in the summers of 2012 and 2013. In the second trial, to increase disease severity, the humidity around the ears was artificially increased by placing plastic bags around the ears. The plants were grown until full maturity.

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Disease assessments: At full maturity stage, ears were phenotyped visually for the percentage of apparent infection, scored as the length of diseased area from the ear tip (infection site) relative to the total length of each respective ear. The other phenotype measured was average grain yield per plant (g) at harvest. Results were analyzed and compared by Mann-Whitney t-test (P≤0.05).

8.3.11 Suppression of DON Production

Maize kernels were ground for 40 sec to a texture that would pass through a 20- mesh sieve using an M2 Stein mill (Fred Stein Lab, Inc. Atchinson, KS, USA). Ground samples (5 g) were diluted in distilled water at a ratio of 1:5 (w/v) and shaken vigorously for 3 min using a bench top reciprocal Eberbach shaker equipped with a flask carrier (Eberbach Corp, Ann Arbor, MI). A 2 ml aliquot of the suspension fluid was transferred into a microcentrifuge tube and spun at 8000 rpm for 60 sec. Sample aliquots were subsequently diluted in distilled water when appropriate. ELISA analysis was carried out with the EZ-ToxTM DON Test (Diagnostix Ltd., Mississauga, ON, Canada) following the manufacturer’s protocol with a detection limit of 0.1 µg/g. There were three replicates for each treatment. Results were analyzed and compared by the Mann-Whitney t-test (P≤0.05).

8.3.12 DON detoxification

To test for the ability of the candidate endophytes to directly detoxify DON into epi- DON, in vitro liquid chromatography coupled to a diode array detector (LC-DAD) was used. Endophytes were grown in 5 ml LB for 48 h at 250 rpm with 20-ppm deoxynivalenol (DON, Catalog #D0156, Sigma-Aldrich, USA). The cultures were diluted to 2 ppm DON with Mili Q water. Then, DON was extracted from the bacterial cultures using monoclonal antibody-based affinity chromatography (VICAM, DONtestTM HPLC, G1005) then subjected to HPLC analysis to detect DON and epi-DON compared to control buffers. The samples were run on a C18 column (250 x 4.6 mm, product # 00G-

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4396-E0, Phenomenex Inc, USA) with an isocratic water- acetonitrile (90:10) system. Peaks were detected by photodiode array spectrophotometer equipped within an Agilent 1200 Infinity Series HPLC (Agilent, USA). There were three replicates for each endophyte tested.

8.3.13 Statistical analyses

All statistical analysis was performed using Prism Software version 5 (GraphPad Software, USA).

8.4 Results

8.4.1 Antifungal screening

The dual culture method was used to screen 215 bacterial endophytes, previously isolated from diverse maize genotypes (Figure 8.1A-B), for their ability to suppress the growth of F. graminearum. Zones of inhibition of F. graminearum were measured after 24-48 h of co-incubation (Figure 8.1C-D). The results revealed that four bacterial endophytes could consistently inhibit the growth of F. graminearum (strains 1D6, 3H9, 4G12 and 4G4). Strain 1D6 resulted in the greatest growth inhibition, while 3H9 caused the least growth inhibition to F. graminearum (Figure 8.1D). Three of these endophytes were isolated from wild maize genotypes (teosintes): strain 1D6 from Z. diploperennis and strains 4G12 and 4G4 from Parviglumis, the direct ancestor of modern maize (Figure 8.1A, Figure 8.2A). The remaining candidate endophyte (strain 3H9) was isolated from a modern commercial variety (Z. mays ssp mays, Pioneer 3751 hybrid).

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Figure 8.1

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Figure 8.1: Origin of endophytes used in this study and results of the in vitro anti- Fusarium screen. (A) A map showing the origin of maize genotypes previously used to isolate the endophytic library. (B) Examples of endophytes from the library isolated from different Zea genotypes as indicated. (C) Example of an endophyte culture showing suppression of F. graminearum hyphae (white) using the dual culture method. (D) Quantification of the inhibitory effect of the endophytes or fungicide controls on the growth of F. graminearum in vitro. For these experiments, n=3. The error bars indicate the standard error of the mean. The black asterisk indicates that the treatment means are significantly different from the fungicide Nystatin at p≤0.05. The green asterisk indicates that the treatment means are significantly different from the fungicide Amphotericin at P≤0.05, Mann Whitney t-test.

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8.4.2 Anti-fungal target spectrum of the candidate endophytes Using the dual culture method, endophytes that tested positive for activity against F. graminearum, were re-screened for activity against a collection of fungi including crop pathogens. Each candidate endophyte was screened in three independent replicates. Endophytes 1D6, 4G12 and 4G4 from the wild teosintes showed the highest spectrum of activity as they inhibited the growth of 20, 19 and 20 fungi, respectively, out of 20 tested fungi (other than F. graminearum). Endophyte 3H9 from modern maize showed a narrow activity spectrum as it inhibited the growth of only one fungus in addition to F. graminearum (Table 8.1).

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Table 8.1: Effect of the candidate bacterial endophytes on the growth of diverse crop fungal pathogens in vitro.

Target fungal Mean diameter of inhibition zone with each endophyte species Nystatin Amphotericin 1D6 3H9 4G4 (10 µg/ml) (5 µg/ml) 4G12

Alternaria alternata 0.0±0.0 0.0±0.0 3.0±0.2*# 0.0±0.0 4.5±0.2*# 5.0 ±0.0*# Alternaria arborescens 0.0±0.0 0.0±0.0 5.5±0.2*# 0.0±0.0 5.5±0.2*# 5.0±0.2*# Aspergillus flavus 2.0±0.2 0.0±0.0 5.5±0.3*# 0.0±0.0* 3.5±0.2*# 4.0±0.0*# Aspergillus niger 0.0±0.0 2.0±0.0 6.5±0.3*# 0.0±0.0# 5.0±0.0*# 7.0±1.0*# Bionectria ochroleuca 2.0±0.2 0.5±0.2 5.5±0.2*# 3.5±0.3*# 6.0±0.2*# 6.5±0.2*# Davidiella tassiana 1.5±0.2 0.5±0.3 5.0±0.3*# 0.0±0.0*# 4.5±0.7*# 5.0±0.0*# Diplodia pinea 2.5±0.2 3.0±0.2 6.5±0.3*# 0.0±0.0*# 5.5±0.2*# 6.0±0.0*# Diplodia seriata 3.0±0.2 2.0±0.2 3.0±0.2# 0.0±0.0*# 0.0±0.0*# 1.5±0.2*# Epicoccum nigrum 0.0±0.0 0.0±0.0 1.5±0.2*# 0.0±0.0 4.0±0.0*# 3.0±0.2*# Fusarium avenaceum 2.5±0.3 3.0±0.6 7.0±0.2*# 0.0±0.0*# 4.5±0.2*# 3.0±0.2* (isolate 1) Fusarium 1.5±1.6 0.0±0.0 6.5±0.3*# 3.0±0.2*# 6.0±0.4*# 5.0±0.5*# graminearum Fusarium lateritium 0.0±0.0 1.0±0.2 1.5±0.3*# 0.0±0.0# 4.0±0.5*# 5.5±0.3*# Fusarium 1.0±0.2 1.0±0.2 4.0±0.0*# 0.0±0.0*# 5.5±0.7*# 4.0±0.0*# sporotrichioides Fusarium avenaceum 0.0±0.0 0.0±0.0 3.5±0.2*# 0.0±0.0 2.0±0.0*# 6.5±0.2*# (isolate 2) Nigrospora oryzae 0.0±0.0 0.0±0.0 6.0 ±0.4*# 0.0±0.0 4.0±0.5*# 3±0.2*# Nigrospora sphaerica 0.0±0.0 0.0±0.0 6.0±0.6*# 0.0±0.0 6.0±0.2*# 3.5±0.0*# Paraconiothyrium 0.0±0.0 0.0±0.0 5.0±0.3*# 0.0±0.0 4.0±0.0*# 4.0±0.0*# brasiliense Penicillium 3.0±0.2 3.0±0.2 6.0±0.6*# 0.0±0.0*# 2.0±0.5*# 5.0±0.2*# afellutanum Penicillium expansum 2.0±0.2 5.0±0.2 3.0±0.2*# 0.0±0.0*# 4.0±0.0*# 5.5±0.5*# Penicillium olsonii 1.5±0.3 3.5±0.3 1.0±0.2*# 0.0±0.0*# 1.5±0.2# 3.0±0.6*# Rosellinia corticium 2.0±0.2 4.5±0.3 7.0±0.6*# 0.0±0.0*# 3.0±0.2*# 7.0±0.2*# *indicates significant different from nystatin, # indicates significant different from amphotericin (P≤0.05, Mann Whitney t-test)

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8.4.3 Molecular identification of candidate endophytic bacteria 16S rDNA and 23S rDNA sequencing were used for taxonomic identification of endophytic bacteria. BLAST searching against the Genbank database suggested that three of the candidate endophytes, 1D6, 3H9 and 4G4, most closely resemble Paenibacillus sp. while 4G12 resembles a Citrobacter sp. (Figure 8.2, Table A8.1). GenBank accession numbers for strains 1D6, 3H9, 4G12 and 4G4 using 16S rDNA are KM104866, KM104867, KM104868 and KM104869, respectively. GenBank accession numbers for strains 1D6, 3H9, 4G12 and 4G4 using 23S rDNA are KM387727, KM387728, KM387729 and KM387730, respectively. Phylogenetic tree data suggested that that the three Paenibacillus strains are P. polymxa (Figure 8.2B), and further suggested that strain 4G4 is different than 1D6 and 3H9.

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Figure 8.2

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Figure 8.2: continued

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Figure 8.2: Taxonomic characterization of candidate anti-Fusarium endophytes. (A) Details of the taxonomic identification of the anti-Fusarium endophytes using 16S rDNA and 23S rDNA, and the tissue and host from which the endophytes were originally isolated. (B) 16S rDNA based phylogenetic tree of the three predicted Paenibacillus sp. Blue arrows point to endophytes in this study. Scale bar equals 0.1.

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8.4.4 Scanning electron microscope (SEM) imaging of endophytes Scanning electron microscopy was used to visualize the external appearance of the candidate endophytes (Figure 8.3). Of the 3 strains predicted to be P. polymxa, strain 1D6 was an elongated rod with an apparent smooth surface; strain 3H9 was an elongated rod with a rough surface; while strain 4G4 was a cylindrical rod with an apparently rough surface. The Citrobacter-predicted strain 4G12 had a rhomboidal hexagonal rod phenotype.

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Figure 8.3: Electron microscope images of the anti-fungal endophyte strains. (A- D) correspond to strains 1D6, 3H9, 4G12 and 4G4, respectively.

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8.4.5 In vitro interaction between each endophyte and F. graminearum

To better understand the anti-fungal mode of action of the candidate endophytes, the in vitro interactions between F. graminearum and each endophyte were visualized following their co-incubation on a microscope slide and subsequent staining with the vitality stains, neutral red and Evans blue. All the four endophytes caused apparent dramatic breakage of F. graminearum hyphae when compared to the control zone on the other side of the microscope slide (that was exposed to only LB media) (Figure 8.4). Upon staining with Evans blue (which stains dead cells in blue), fungal hyphae in contact with the commercial biological control or each of the four endophytes stained blue (Figure 8.5A, C, E, G and I) compared to the buffer controls (Figure 8.5B, D, F, H and J), suggesting that hyphae in contact with each bacterial endophyte died. Combined, these results suggest that all four endophytes have fungicidal activity.

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Figure 8.4

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Figure 8.4: Microscopic in vitro interactions between each anti-fungal endophyte and F. graminearum. (A) Cartoon of the experimental methodology to examine microscopic in vitro interactions between F. graminearum (pink) and each endophyte (orange) or the buffer control (LB medium). The microscope slides were pre-coated with PDA and incubated for 24 h. F. graminearum hyphae were then stained with neutral red. Shown are representative microscope slide pictures (n=3) of the interactions between F. graminearum and: (B) Strain 1D6 compared to (C) the buffer control; (D) Strain 3H9 compared to (E) the buffer control; (F) Strain 4G12 compared to (G) the buffer control; (H) Strain 4G4 compared to (I) the buffer control.

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Figure 8.5

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Figure 8.5: The effects of the candidate endophytes on F. graminearum in vitro using the vitality stain, Evans blue. Shown are representative microscope slide pictures (n=3) of the interactions of F. graminearum with: (A) the commercial biological control agent, Bacillus subtilis (100 mg/10 ml) compared to (B) the buffer control; (C) Strain 1D6 compared to (D) the buffer control; (E) strain 3H9 compared to (F) the buffer control; (G) Strain 4G12 compared to (H) the buffer control; (I) Strain 4G4 compared to (J) the buffer control.

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8.4.6 Candidate fungicide mechanism of action

A candidate gene approach was undertaken to help understand the fungicide mode of action of the endophytes. Paenibacillus are well known to produce fusaricidin compounds that combat various fungal pathogens including F. graminearum; the compound is in fact named after Fusarium (Beatty and Jensen, 2002; Choi et al., 2008; Kajimura and Kaneda, 1996, 1997)To detect the presence of fusaricidin biosynthetic genes in the three predicted Paenibacillus strains, PCR primers were designed based on the fusaricidin synthase gene sequence (GenBank accession # EU184010). Each genome amplified a single band that was sequenced; the results revealed that all three of the Paenibacillus endophyte genomes encode a putative fusA ortholog, with DNA sequence identities ranging from 92-94% (Figure 8.6A, Table A8.2). GenBank accession numbers for fusA sequences amplified from strains 1D6, 3H9, 4G4 were KT343965, KT343966 and KT343967, respectively. FusA is a non-ribosomal peptide synthetase with relaxed substrate specificity that can incorporate different amino acids, resulting in different fusaricidin derivatives (Han et al., 2012). To confirm that the fusA orthologs were expressed by the endophytes, and to identify the specific fusaricidin derivatives produced, LC/MS was employed. Peaks with M+Z similar to fusaricidin C (947.6), fusaricidin B (883) and fusaricidin D (961.7) were detected in the liquid cultures of strains 1D6, 3H9 and 4G4, respectively (Figure 8.6B-D) (Beatty and Jensen, 2002; Kajimura and Kaneda, 1996, 1997). Combined, these results demonstrate that the well- known anti-Fusarium compound fusaricidin is encoded and expressed by each of the three Paenibacillus endophytes.

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Figure 8.6

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Figure 8.6: Molecular and biochemical detection of the candidate anti-fungal compound, fusaricidin, in Paenibacillus strains. (A) Details of fusA gene orthologs isolated from the candidate Paenibacillus endophytes by PCR amplification. (B-D) Combined ion chromatogram/mass spectrum for candidate fusaricidin derivatives detected in the cultures of the Penibacillus endophytes as indicated.

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8.4.7 Suppression of Gibberella ear rot (GER) in planta

Greenhouse experiments were undertaken to determine if the endophytes could suppress Gibberella ear rot (GER) in planta using a modern maize hybrid, P35F40, which is susceptible to this disease. To confirm that the candidate bacterial strains originally isolated from the two evolutionarily distant maize genotypes (Z. diploperennis and Parviglumis) could colonize the internal tissues of this modern hybrid, thus behaving as endophytes, GFP tagging was conducted. Attempts were made to GFP tag all endophytes, but unfortunately, only strain 4G12 from ancestral Parviglumis, was successfully tagged. GFP-tagged 4G12 was visualized by scanning confocal microscopy and shown to colonize maize roots (Figure 8.7A, B), confirming its behavior as an endophyte in the modern maize relative. All four endophytes were then tested for their ability to suppress GER under greenhouse conditions in two independent trials (Figures 8.7 and 8.8). The main entrance routes for F. graminearum in maize are exposed silks where the ascospores can germinate and grow towards the developing ear (Kebebe et al., 2015; Munkvold, 2003b; Sutton, 1982a). Therefore, the disease severity was scored as the length of diseased area, measured from the ear tip where Fusarium spores were introduced, relative to the total length of the ear (Figure 8.7I). Kernel yields were also quantified:

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Figure 8.7

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Figure 8.7: Greenhouse trial 1 to test for the ability of the candidate endophytes to suppress Gibberella Ear Rot (GER) in a modern hybrid. (A, B) GFP- tagged endophyte strain 4G12 visualized inside maize roots, in the (A) absence or (B) presence of propidium iodide that outlines the cell with red color. Representative ears from each treatment. (I) Picture of an ear to illustrate the methodology of scoring disease severity: The fungal pathogen was introduced to the tip of the ear, indicated by the asterisk. Therefore, the disease was scored as the ratio of the length of the diseased ear tip portion relative to total ear length, multiplied by 100 to give a percentage. (J, K) Quantification of the effect of different treatments on GER suppression, as: (J) percent ear infection, and (K) average grain yield per plant. For both measurements, n=20 per treatment (n=10 for both controls). The whiskers indicate the range of data points. The black asterisk indicates that the treatment means were significantly different from the Fusarium only treatment The green asterisk indicates that the treatment means were significantly different from prothioconazole fungicide (Proline) treatment (P≤0.05, Mann Whitney t-test).

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Figure 8.8

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Figure 8.8: Greenhouse trial 2 to test for the ability of the candidate endophytes to suppress Gibberella Ear Rot in a modern hybrid. (A-F) Representative ears from each treatment. (G) Picture of an ear to illustrate the methodology of scoring disease severity: the fungal pathogen was introduced to the tip of the ear, indicated by the asterisk. Therefore, the disease was scored as the ratio of the length of the diseased ear tip portion relative to total ear length, multiplied by 100 to give a percentage. (H, I) Quantification of the effect of different treatments on GER suppression, as (H) percent ear infection, and (I) average grain yield per plant. For both measurements, n=20 per treatment (n=10 for both controls). The whiskers indicate the range of data points. The black asterisk indicates that the treatment means were significantly different from the Fusarium only treatment at p≤0.05. The red asterisk indicates that the treatment means were significantly different from prothioconazole fungicide (Proline) treatment at p≤0.05.

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First greenhouse trial (Summer 2012): Representative pictures of treated ears are shown (Figure 8.7C-H). Treatment with three of the four endophytes caused significant reductions (P≤0.05) in GER disease severity ranging from 12-38%: strain 1D6 resulted in the greatest disease suppression followed by strain 4G12 and then strain 4G4, while the effect of strain 3H9 on GER suppression was statistically insignificant when compared to the Fusarium treatment only, at P≤0.05 (Figure 8.7J, Table 8.2). None of the endophyte treatments caused a significant change in grain yield, at P≤0.05 (Figure 8.7K, Table 8.2).

Second greenhouse trial (Summer 2013): In the second trial, the disease pressure was increased by raising the humidity. Representative pictures of treated ears are shown (Figure 8.8A-F). Treatments with all four of the endophytes caused significant reductions (P≤0.05) in GER disease severity ranging from 59-84%: strains 1D6, 4G12 and 4G4 resulted in the statistically greatest disease suppression, while again the effect of strain 3H9 on GER suppression was the lowest, but this time statistically significant compared to the Fusarium treatment only, at P≤0.05 (Figure 8.8H, Table 8.3). All of the endophyte treatments caused dramatic 3-4-fold increases in grain yield compared to the Fusarium treatment only (Figure 8.8I, Table 8.2).

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Table 8.2: Suppression of Gibberella Ear Rot by the candidate endophytes in two replicate greenhouse trials

Treatment % infection % disease Average % of yield (mean ± SEM)* reduction yield per increase plant (g)* Greenhouse trial 1 Fusarium only 33.7±2.3 a 0 41.7±1.3 a 0 Proline fungicide 23.1±2.0 b 32 50.9±1.2 b 22 1D6 20.9±1.7 b 38 43.5±1.2 a 4 3H9 32.4±1.4 a 39 43.1±1.3 a 3 4G12 24.1±2.1 b 29 38.3±2.5 a -8 4G4 29.7±1.7 d 12 42.5±2.9 a 2 Greenhouse trial 2 Fusarium only 88.5±3.8 a 0 7.7±1.2 a 0.0 Proline fungicide 41.4±4.5 b 53 40.8±1.2 b 430 1D6 14.6±1.9 c 84 33.5±2.1 c 335 3H9 36.1±6.2 b 59 37.5±3.3 b 387 4G12 16±1.1 c 81 37.4±3.2 b 386 4G4 14.6±1.3 c 82 39.8±3.0 b 417

*Letters that are different from one another indicate that their means are statistically different (P≤0.05, Mann Whitney t-test)

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8.4.8 Effect of the endophyte treatments on DON contamination In order to quantify DON levels in maize seeds, ELISA-based testing was conducted. Immediately after harvest, only traces of DON were detected in plants treated with Fusarium only (~0.1 ppm) while all other treatments did not show any detectable levels of DON (data not shown). Seeds were stored at room temperature inside closed envelopes for one year, then the samples were analyzed again for DON content. Consistently in both trials, all four endophyte treatments caused dramatic reductions in DON accumulation during storage, with DON levels declining from ~3.5 ppm to 0.1-1.0 ppm (Table 8.3, Figure 8.9,). The majority of the endophyte treatments resulted in a DON content of only 0.1 ppm, equivalent to a 97% reduction compared to the Fusarium- only control.

8.4.9 DON detoxification In order to test the ability of the candidate endophytes to directly detoxify DON into epi-DON in vitro, a standard HPLC based method was used. However, the results revealed that none of the candidate endophytes could directly detoxify DON into epi- DON (Figure A8.1).

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Table 8.3: Reduction of DON mycotoxin accumulation during storage following treatment with the candidate endophytes

Treatment DON content (ppm) % of DON reduction (mean ± SEM)* relative to Fusarium only treatment* Greenhouse trial 1 Fusarium only 3.4±0.4 a 0 Proline fungicide 0.7±0.4 b 80 1D6 0.1±0.0 c 97 3H9 1.0±0.8 d 71 4G12 0.1±0.0 c 97 4G4 0.1±0.0 c 97 Greenhouse trial 2 Fusarium only 3.5±0.3 a 0 Proline fungicide 0.1±0.0 b 97 1D6 0.1±0.0 b 97 3H9 0.1±0.0 b 97 4G12 0.1±0.0 b 97 4G4 0.2±0.1 b 94 * Letters that are different from one another indicate that their means are statistically different (P≤0.05, Mann Whitney t-test).

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Figure 8.9: Test for the ability of the candidate endophytes to reduce DON mycotoxin accumulation in maize grain during storage. DON measurements after storage of maize grain from: (A) greenhouse trial 1 (summer 2012), and (B) greenhouse trial 2 (summer 2013). For both trials, n=3 pools of seeds. The black asterisk indicates that the treatment means were significantly different from the Fusarium only treatment. The green asterisk indicates that the treatment means were significantly different from the prothiocona ole fungicide (Proline) treatment at p≤0.05, Mann Whitney t-test.

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8.5 Discussion

I hypothesized that the wild relatives of modern crops, which grow without fungicides, may host beneficial endophytes that help their hosts to naturally combat fungal pathogens including F. graminearum. In this study I found support for this hypothesis. In vitro screening of 215 maize bacterial endophytes (Figure 8.1A-B) identified four candidate endophytes that could inhibit the growth of F. graminearum and its DON mycotoxin to within the acceptable levels (Figure 8.9). Despite the screen containing 116 endophytes from non-wild maize genotypes (45 from modern maize and 71 from traditional landraces, totaling 54% of the library), the three most potent endophytes were isolated from their wild counterparts (representing 46% of the endophytes screened) (Figure 8.1, Figure 8.7, Figure 8.8). Specifically, anti-Fusarium endophyte strain 1D6 was originally isolated from Z. diploperennis, and strains 4G12 and 4G4 were isolated from Parviglumis (Figure 8.2A). The remaining candidate endophyte (strain 3H9) was isolated from a modern maize variety (Z. mays ssp mays, Pioneer 3751 hybrid). The suggested mode of action of all four endophytes was fungicidal, not fungistatic (Figures 8.4 and 8.5). Plants treated with these endophytes showed a remarkable reduction in DON contamination during storage (Figures 8.9) which might be attributed to an initial reduction in F. graminearum inoculum, as none of the endophytes were able to directly inactivate DON in vitro (Figure A8.1). The permitted level of DON mycotoxin contamination in maize grain is 2 ppm in food and 5 ppm in animal feed (Jelinek et al., 1988). However, the dietary value permitted for swine feed in Canada and the USA is only 1 ppm (Schaafsma et al., 2009). Except for strain 3H9, the endophytes consistently reduced DON to within the 1 ppm level during storage.

8.5.1 Host environmental history

Interestingly, the anti-Fusarium endophytes isolated from the wild teosintes (1D6, 4G12 and 4G4) showed an exceptionally broad spectrum of anti-fungal activities (Table 8.1). Parviglumis teosinte appears to have been adapted for thousands of years in the seasonal tropical forest region of the Central Balsas Valley of southwestern Mexico

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(Piperno et al., 2009). Zea diploperennis originated from the Sierra de Manantlan region of Jalisco, in Southern Mexico, which has both dry and wet climates but with very high levels of total rainfall (1700 mm) (Iltis and Doebley, 1980; Sánchez‐Velásquez et al., 2002). Perhaps the broad spectrum activity of the endophytes from these Zea genotypes is the result of co-evolutionary selection by their host plants for endophytes that could combat fungal pathogens which are especially problematic in regions of high humidity. In contrast, the candidate endophyte (strain 3H9) isolated from the modern hybrid, which was bred under temperate conditions, showed the weakest anti-Fusarium activities (Figure 8.1D, Figure 8.7J, Figure 8.8H, Figure 8.9A) as well as the narrowest target spectrum of anti-fungal activity (Table 8.1).

Unfortunately, there has been no systematic comparison of fungal resistance in teosintes versus modern maize, though one Fusarium species (Gibberella fujikori) has been reported to infect both (Lange et al., 2014). In a survey concerning the incidence of Fusarium species in maize seeds worldwide, Fusarium species were the most abundant fungi detected (39-62%) with the most frequent species being F. moniliforme (MacDonald and Chapman, 1997). Fusarium species were reported in 10% of wild teosinte seeds collected from Mexico, Nicaragua and Guatemala, with F. moniliforme and F. subglutinans reported to be the most abundant (Desjardins et al., 2000). However, the authors noted that whereas Fusarium species were detected in 100% of modern maize seeds (Z. mays sp. mays), their incidence in teosinte seeds was only 4% when grown under the same conditions, and in general, at least anecdotally, teosintes appeared to have much lower rates of Fusarium infection than modern maize in Mexico (Desjardins et al., 2000). It may be that there has been three-way co-evolutionary selection within the teosintes between the host plant, its endophytes and Fusarium species.

8.5.2 Host life strategy

This study involved Zea genotypes which encompassed three critical life strategy transitions: (1) the evolutionary transition from wild perennial to annual growth habit; (2)

277 the agricultural transition from wild to domesticated primitive plant; and (3) the transition from a domesticated primitive plant to modern cultivars (Dávila-Flores et al., 2013; Rosenthal and Dirzo, 1997). As noted above, the three consistently robust anti-fungal endophytes were isolated from Z. diploperennis, which is a wild perennial teosinte, and from Parviglumis, which is a wild annual teosinte. These two Zea genotypes were previously shown to be more resistant to insects compared to domesticated modern maize (an annual), with Z. diploperennis showing more resistance than Parviglumis (Dávila- Flores et al., 2013; Rosenthal and Dirzo, 1997). These results are consistent with other reports that domestication reduces resistance to insects (Lange et al., 2014) and herbivores (Chen et al., 2015). To explain these results, Rosenthal and Dirzo (1997) suggested the resource allocation hypothesis in which metabolic resources are diverted away from plant defense as a result of selection for faster plant growth rates (associated with annualism) and higher grain yields (associated with domestication and breeding). This hypothesis is supported by results from other crops (Benrey et al., 1998; Gols et al., 2008). Given the results of this study, it is interesting to pose a parallel hypothesis: as plants diverted precursors for defense compounds to enable faster plant growth and higher grain yield, they may have also prevented their endophytes from producing defense compounds, thus reducing the reasons to support such endophytes. Modern breeding under conditions of fungicide inputs may have also caused crops to no longer devote metabolic resources to support endophytes with redundant pesticide function. Alternatively, it may be that selection by humans against plant-derived toxins during domestication and breeding may have also involved selection against anti-pathogen endophytes with indiscriminate toxicity, by altering plant loci that promote the colonization of specific endophytes. These three hypotheses (host-endophyte resource allocation hypothesis, pesticide-endophyte-redundancy hypothesis, endophyte-toxin- selection hypothesis) require further investigation. As this study involved only two modern maize genotypes (B73, Pioneer 3751) focusing on only a single pathogen, future studies should involve a more balanced number of plant genotypes across the evolutionary spectrum.

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8.5.3 Paenibacillus polymyxa strains span the evolutionary transitions of Zea

In this study, despite testing 215 diverse bacterial endophyte strains, three out of four candidate endophytes with anti-Fusarium activity were predicted to be Paenibacillus polymyxa (strains 1D6, 3H9, and 4G4) with a fourth strain identified as a Citrobacter sp. (strain 4G12) (Figure 8.2, Table A8.1). The three P. polymyxa appear to be distinct, when all the data are taken into account [16S rDNA phylogenetic tree data (Figure 8.2B), fusA gene sequence information (Figure 8.6A, Table A8.2), biochemical profiles (Figure 8.6) and anti-Fusarium results (e.g. Figure 8.7)]. Bacterial endophytes previously isolated from different maize varieties belong to diverse genotypes including Paenibacillus and Citrobacter, but also Bacillus, Clostridium, Enterobacter, Pantoea, Methylobacteria, Pseudomonas, Burkholderia, Erwinia and Microbacterium (Johnston- Monje and Raizada, 2011)((Cotta et al., 2014; Johnston-Monje et al., 2014).

As already noted, the putative P. polymyxa endophytes appear to span hundreds of thousands of years of evolutionary transitions of Zea (Matsuoka et al., 2002), since they were present in a wild Mexican perennial (Z. diploperennis), a wild Mexican annual (Parviglumis) and a modern temperate hybrid (Pioneer 3751) (Table 8.2). Therefore, these anti-Fusarium Paenibacilli cross host boundaries of evolution, domestication, migration and breeding, suggesting a tight conserved host-endophyte relationship. Consistent with this observation, P. polymyxa was previously reported as a ubiquitous, conserved endophyte across diverse Zea genotypes including wild perennial and annual teosintes, traditional farmer landraces and modern genotypes (Johnston-Monje and Raizada, 2011). P. polymyxa is well known as a good plant colonizer, which is in part due to its robust ability to form biofilms (Haggag and Timmusk, 2008; Timmusk et al., 2005). Furthermore, consistent with our results, P. polymyxa was previously reported to antagonize numerous plant pathogens (Timmusk et al., 2009; Xu and Kim, 2014) including F. graminearum (He et al., 2009). P. polymyxa was further shown to decrease DON production under greenhouse conditions (He et al., 2009), consistent with the results of this study.

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Previous reports have shown that the anti-fungal mechanism of action of Paenibacillus sp. involves production of potent antifungal compounds including polymyxins, fusaricidins, colistins, volatiles and lytic enzymes (He et al., 2007; Naghmouchi et al., 2012; Raza et al., 2008; Raza et al., 2009; Raza et al., 2015). In particular, Paenibacillus is well known to combat F. graminearum by employing fusaricidin, a compound named after Fusarium as noted earlier (Beatty and Jensen, 2002; Choi et al., 2008; Kajimura and Kaneda, 1996, 1997). Consistent with the literature, our results show that the three P. polymyxa strains characterized in this study produce fusaricidins (Figure 8.6, Table A8.2). However, we do not exclude other compounds as the genus Paenibacillus is well known for its ability to produce an arsenal of antimicrobial compounds including polyketides and non-ribosomal peptides. Fusaricidin production, together with the in vitro microscopic interaction data (Figures 8.4, 8.5 and 8.6), suggests a fungicidal mechanism of action. P. polymyxa has also been shown to induce systemic host resistance (Mei et al., 2014). P. polymyxa was also shown to alter plant metabolism by enhancing the production of flavonoids such as apigenin-7-O- glucoside (Schmidt et al., 2014) or reducing cinnamic acid in root exudates. These compounds suppress the development of pathogen conidia, thereby significantly reducing their ability to colonize plants (Ling et al., 2011). It will be useful to investigate if the predicted Paenibacilli endophytes from this study also employ these modes of action. It will also be useful to test whether these strains are resistant to fusaric acid, an antibiotic produced by Fusarium; resistance to this compound has been shown to be essential for effective biological control against Fusarium species (Chulze et al., 2014; Eilenberg, 2006).

8.5.4 The emerging importance of Citrobacter sp.

As already noted, the anti-Fusarium endophyte strain 4G12 is predicted to be a Citrobacter species (Figure 8.2). Citrobacter species were previously reported as endophytes of an ancient Mexican landrace (Nal Tel) and teosinte (e.g. Z. nicaraguensis) (Johnston-Monje and Raizada, 2011), in addition to other plants including Brazilian sugarcane (Magnani et al., 2013) and the legume tree, Conzattia multixora which is

280 exclusively found in Guatemala and Mexico (Wang et al., 2006). Citrobacter species were claimed to moderately control some fungal plant pathogens such as Monilinia fructicola, the causal agent of brown rot in stone fruits (Janisiewicz et al., 2013). However, to the best of our knowledge, Citrobacter was not previously reported to effectively control Fusarium species, suggesting that this genus may have a wider spectrum of anti-fungal activity than previously thought (Table 8.1).

8.6 Conclusions

This study has identified candidate endophytes that could suppress the serious, toxigenic pathogen F. graminearum in maize. The endophytes reduced DON mycotoxin concentrations during storage to levels significantly below acceptable safety thresholds, a promising result that requires field level validation for further practical applications. The most potent of the candidate strains were derived from wild teosinte genotypes including the ancestor of modern maize. Further bioprospecting of wild relatives of modern crops for beneficial microbes may open a promising avenue for biocontrol against the most devastating diseases afflicting modern agriculture. Teosintes are under threat from deforestation, urbanization, and cattle, and it is hoped that this study will assist in efforts to conserve these wild species. These results, combined with the literature, have led us to several host-endophyte hypotheses with respect to plant defense that require further investigation.

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Chapter 9: An endophytic bacterium isolated from wild maize suppresses Fusarium head blight and DON mycotoxin in modern wheat

Authors’ contributions

Walaa K Mousa designed and carried out all experiments, performed all data analyses, and wrote the manuscript.

Charles S Shearer helped with the greenhouse experiments.

Victor Limay-Rios performed DON quantification assays.

Dr. Manish N. Raizada helped to design the study and edited the manuscript.

Publication status: pending submission

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9.1 Abstract

Previously, I isolated Paenibacillus and Citrobacter endophytes from wild and modern maize (corn) as antagonists in modern maize against Fusarium graminearum. In wheat, this pathogen causes Fusarium Head Blight (FHB) with subsequent accumulation of deoxynivalenol (DON) mycotoxin. Subsistence farmers dry or store grain improperly leading to additional post-harvest DON accumulation. Here I tested whether these maize endophytes could cross host boundaries to control this pathogen in wheat and control DON under conditions that mimic those of subsistence farmers. The endophytes were applied to wheat infected with F. graminearum in greenhouse trials. DON was quantified using ELISA. All the endophytes suppressed FHB and reduced DON in wheat after long- term storage under ambient conditions. The endophytes isolated from the wild ancestor of maize (parviglumis) showed the greatest efficacy, with one strain reducing DON to within acceptable safety levels. I conclude, Parviglumis possesses the most potent endophytes that reduced FHB and/or DON in wheat following storage conditions that mimic those of subsistence farmers. Wild relatives of crops may be untapped reservoirs of endophytes that combat pathogens and reduce their toxins across host boundaries.

Key words: Citrobacter, Paenibacillus, Fusarium graminearum, Fusarium Head Blight (FHB), wheat, teosinte, endophyte, deoxynivalenol (DON)

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9.2 Introduction

Endophytes are primarily bacteria and fungi that comprise the non-pathogenic, endogenous plant microbiome (Wilson 1995; Johnston-Monje and Raizada 2011). Endophytes have been shown to suppress fungal diseases through a variety of biochemical and molecular mechanisms (Mousa and Raizada, 2013, 2015).

Wheat is highly susceptible to Fusarium graminearum, the causal fungal agent of Fusarium Head Blight (FHB), a devastating disease worldwide (van der Lee et al., 2014), with successive epidemics in North America (McMullen et al., 1997; Sutton, 1982; Gilbert and Tekauz, 2000). Recently, in Canada, FHB has become more severe as the F. graminearum population structure has shifted towards more toxic and versatile strains (Ward et al., 2008) that might have been accidently imported from Europe (Gale et al., 2007; van der Lee et al., 2014). FHB results in seeds with inferior quality, low grain yield and contamination with sesquiterpenoid trichothecene mycotoxins, including deoxynivalenol (DON); these mycotoxins represent a serious hazard to human and animal health (Osborne and Stein, 2007; Pestka, 2010). In infected grain, DON can accumulate during grain storage (Magan et al., 2003). Of particular concern is that millions of subsistence wheat growers around the world (e.g. in Ethiopia, Egypt, Middle East, Himalayas) are unable to properly dry and store grain (Ayalew et al., 2006), which leads to persistent growth of the pathogen and further DON accumulation (Piacentini et al., 2015). Low grain yield, along with DON contamination associated with FHB, is the direct cause of catastrophic economic losses to farmers. For example, in 1993, an FHB epidemic in Canada and the United States resulted in $1 billion USD in losses (McMullen et al., 1997). In 1996, an FHB epidemic in Ontario resulted in $100 million CDN in losses (Schaafsma, 2002).

Despite some progress in breeding for resistance, as well as the use of fungicides and other cultural practices, to date there is no single effective strategy to manage FHB in wheat (Yuen and Schoneweis, 2007; Walter et al., 2010; Wegulo et al., 2015). However,

284 integrated management strategies including the use of microbial antagonists have been suggested (Wegulo et al., 2015). F. graminearum develops an intercellular cortical hyphal network complex in the spikelet and rachis prior to intracellular colonization (Brown et al., 2010), which makes foliar sprays less effective (Mesterházy et al., 2003). Given that endophytes can live inside plant tissue, mostly intercellular (Wilson, 1995), this characteristic may make them ideal to control FHB.

We have reported that wild and modern maize possess bacterial endophytes that combat pathogens including F. graminearum in vitro (Johnston-Monje et al., 2014; Johnston- Monje and Raizada, 2011). Recently, I have identified four candidate endophytes isolated from these maize genotypes (Figure 9.1A) that suppress F. graminearum in planta in modern maize (Figure 9.1B) and suppress DON mycotoxin after grain storage (Chapter 8) (Mousa et al., 2015b). Specifically, these endophytes were isolated from the extant Mexican ancestor of modern maize (Zea mays ssp. parviglumis (Matsuoka et al., 2002), a perennial Zea diploperennis that grows in Central America and Mexico (Iltis and Doebley, 1980), and a temperate modern hybrid, Pioneer 3751 (Smith et al., 2004).

It is not known whether the endophytes isolated from maize, including its wild ancestors, can protect against pathogens in other modern crops such as wheat. The purpose of this study is to test the ability of candidate anti-Fusarium endophytes isolated from wild and modern maize to combat FHB in wheat and inhibit DON accumulation following grain drying and storage conditions that mimic those of subsistence wheat farmers.

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9.3 Materials and Methods

9.3.1 Sources of biological materials

The four candidate anti-Fusarium bacterial endophytes used in this study were originally isolated from Z. diploperennis (strain 1D6), a modern maize hybrid (strain 3H9) and Z. mays ssp. parviglumis (strains 4G12 and 4G4) (Figure 9.1A) (Johnston- Monje and Raizada, 2011). These endophytes were classified taxonomically as Paenibacillus ssp. (Strains 1D6, 3H9 and 4G4) and a Citrobacter sp. (Strain 4G12) (Chapter 8) (Mousa, et al., 2015b). The GenBank accession numbers for the corresponding 16S rDNA sequences are as follows: 1D6 (#KM104866), 3H9 (#KM104867), 4G12 (#KM104868) and 4G4 (#KM104869). The F. graminearum strain used in this study (15 Acetyl DON Producer) was obtained from Ridgetown College (University of Guelph) and was previously isolated from maize kernels during an outbreak in Ontario, Canada, of fusariosis in 2006. For wheat, spring wheat cultivar (Quantum Spring Wheat) was used.

9.3.2 GFP-tagging to test endophyte behavior in wheat

To test the ability of bacterial endophytes isolated from maize to colonize wheat, the endophytes were subjected to tagging with green fluorescent protein (GFP) followed by in planta visualization using confocal scanning microscopy.

Competent cell preparation: One liter of LB broth was inoculated with a 10 ml overnight o bacterial culture (grown at 37 C at 250 rpm) until early log phase (OD600 = 0.4-0.6). The cells were chilled for 15 min on ice and centrifuged at 4000 x g for 15 min at 4˚C. The pellets were then re-suspended in cold water and centrifuged twice. Finally, the cells were re-suspended in cold 10% glycerol, centrifuged and re-suspended in 3 mL of 10% glycerol; 40 µl aliquots were frozen at -80oC.

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GFP vector transformation: The wide-host promoter vector, pDSK-GFPuv (Wang et al., 2007) was employed to transform E.coli DH5α (Catalog #EC6P095H, Epicenter, Madison, USA). Plasmid DNA was extracted from E.coli using a Quantum Prep Plasmid Miniprep Kit (Catalog # 732-6100, BioRad Inc, USA) then transformed into bacterial cells using electroporation (Calvin and Hanawalt, 1988). Cold competent cells (40 μl) were added to 1 μl of plasmid DNA (240 ng/μl) then electroporated at 1.6 KV for 1 sec in a Bio-Rad Gene Pulser 200/2.0 (Bio-Rad Hercules, USA). Subsequently, cells were allowed to recover for 1 h at 37°C in 1 ml of LB with shaking at 250 rpm. The cells were plated on kanamycin- LB agar (35 μg/μl), incubated for 24 h at 37°C, then the plate was checked for fluorescent colonies (Illumatool, #LR 92240, USA).

To visualize GFP-tagged endophyte cells inside wheat tissues: Spring wheat seeds were surface sterilized (see below for details on method and chemical used in the sterilization), coated with GFP-tagged endophyte (see coating procedure below), and germinated on wet paper towels. The seedlings were screened using a TCS SP2 confocal laser scanning microscope (Leica Microsystems, Mannheim, Germany) at the Imaging Facility, University of Guelph. The conditions for the confocal scanning were as follows; excitation at 488 nm Argon laser and 543 nm green helium laser, Emission ranges = 504- 532 nm and 524-699 nm, Pinhole [Au] = 1.0 airy, objective lens = 63x oil immersion and frame average = 3 times.

9.3.3 Suppression of FHB in greenhouse trials

The candidate anti-Fusarium endophytes were tested for their ability to suppress FHB in replicated greenhouse trials (Crop Science Greenhouse Facility, University of Guelph):

Seed treatment: Seeds of spring wheat were surface sterilized as follows: seeds were washed in 0.1% Triton X-100 detergent with shaking for 10 min; the detergent was

287 poured off, 3% sodium hypochlorite was then added (twice for 10 min), which was followed by rinsing with distilled, autoclaved water, washing with 95% ethanol for 10 min; and finally the seeds were washed 5-6 times with distilled, autoclaved water. Complete surface sterilization was verified by inoculating the last wash on PDA and LB plates at 25°C and 37°C, respectively; none of these washes showed growth. The seeds were then coated with the endophytes. To prepare endophyte inoculants, bacteria were incubated at 37˚C for 24 h in LB medium, centrifuged, washed and re-suspended in PBS buffer to an OD600 of 0.5. Afterwards, 500 μl of each bacterial culture was rocked for 2 h on a horizontal shaker (Serial #980216M, National Labnet Company, USA) with 10 ml polyvinyl pyrrolidine (PVP, Catalog # 9003398, Sigma Aldrich, USA) which was used as a seed-coating agent. Endophyte- or buffer-coated seeds were germinated on wet paper towels in the dark for 7 days; uniformly sized seedlings were moved to pots containing Turface clay (Turface Athletics Inc, USA) in a greenhouse.

Pathogen treatment: Pathogen spores were prepared as follows: the liquid spore suspension medium was prepared using distilled water as follows (per L): 2 g KNO3, 2 g

KH2PO4, 1 g MgSO4, 1 g KCl, 1 g dextrose, 20 mg/100 ml (of each) of minor elements

(FeCl2, ZnSO4, MnSO4). Subsequently, 350 ml of the medium was added to 2 L flasks, then sterilized at 121°C for 10 min. Followed by cooling at room temperature, 3-4 PDA plugs of F. graminearum or 10 ml liquid conidia suspensions were added to each flask under sterile conditions, and these were subsequently incubated on shaker tables under a 12:12 h UV light:dark cycle for ~two weeks at room temperature. The spore solution was standardized to 20,000 F. graminearum spores/ml with a haemocytometer, then refrigerated or used to treat plants directly. Per plant, 1 ml of the F. graminearum spore suspension (20,000 spores/ml) was then sprayed directly onto wheat heads at the flowering stage with the emergence of anthers (flowers in anthesis, as anthers are the primary sites of infection). No surfactant or other formulation agents were used.

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Endophyte treatment: Each endophyte was introduced twice more to ensure its high titre: the first endophyte spray was performed simultaneously with the pathogen (at the flowering stage with the emergence of anthers), while the second spray was the endophyte alone, 3 days later. Each endophyte was grown overnight in LB medium then

1 ml of OD600 0.4-0.6 was used for spraying. No surfactant or other formulation agents were used.

Control treatments: For the positive control, seeds were coated with PVP followed by fungicide spraying (PROLINE® 480 SC Foliar Fungicide, Bayer Crop Science), at concentration of 200 g ai/Ha, on mature wheat heads prior to infection with the fungal pathogen; no endophytes were introduced. The negative control consisted of seeds coated with PVP followed by repeated spraying with 1 ml LB medium (as buffer control), along with the pathogen inoculation (as described above).

Experimental design and growth conditions: Each treatment group consisted of 20 plants arranged in a randomized block design. Two independent greenhouse trials were conducted in the summer of 2013. The growth conditions were as follows: 28˚C/20˚C, 16h:8 h, ≥800 μmol m-2 s-1 at pot level, with metal halide and high pressure sodium lamps supplemented with GroLux lighting, fertilized using drip irrigation containing modified Hoagland’s solution, until seed maturity (Gaudin et al., 2011). During the Fusarium or endophyte spray treatments, the greenhouse temperature was 28-30˚C. To promote Fusarium growth, the greenhouse was sprayed twice per week with water to achieve a typical relative humidity of ~60-80%.

Post-maturation conditions: Following grain head maturation, watering was ceased and plants were allowed to dry for an average of 18 days in the greenhouse under the same conditions as the growth phase (28˚C/20˚C, 16h:8 h) but without added humidity (all spraying was ceased). Grains were then threshed, and then stored in sealed paper

289 envelopes for 14 months under ambient conditions in a laboratory (temperature ~18- 250C, with a moisture content of ~40-60%).

Disease scoring: At full maturity, following seed drying, disease was scored as the percentage of grains per plant with visually observable disease symptoms relative to the total number of grains per plant. The infected seeds were defined as having a rough, shriveled surface and/or white hyphae (Schmale and Bergstrom, 2003). Total dry grain weight at harvest per plant was also measured. Results were analyzed and compared using Mann-Whitney t-test (P≤0.05). Prism Software v5 (GraphPad Software, USA) was used for the statistical analysis.

9.3.4 DON Assay

Wheat seeds were pulverized for 40 sec using a M2 Stein mill (Fred Stein Lab, Inc. Atchinson, KS, USA) to a fine texture sufficient to pass through a 20 mesh sieve. The samples (5 g) were then diluted in distilled water 1:5 (w/v) ratio and shaken intensely for 3 min using a bench top reciprocal Eberbach shaker equipped with a flask carrier (Eberbach Corp, Ann Arbor, MI).

Each liquid suspension (2 ml aliquots) was poured into a microcentrifuge tube and centrifuged for 60 sec at 8000 rpm. Each sample was diluted in distilled water as needed. The EZ-ToxTM DON Test (Diagnostix Ltd., Mississauga, ON, Canada) was used for ELISA analysis following the manufacturer’s protocol with a detection limit of 0.1 µg/g. There were three replicates per treatment, and the data was compared using the Mann- Whitney t-test (P≤0.05). Prism Software v5 (GraphPad Software, USA) was used for the statistical analysis.

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9.4 Results

9.4.1 Colonization of maize endophytes in wheat

To confirm that the candidate anti-Fusarium bacterial strains originally isolated from maize (Z. diploperennis and parviglumis) could colonize the internal tissues of wheat, thus behaving as endophytes, GFP tagging was conducted. Unfortunately, only strain 4G12 was successfully tagged, despite attempts to tag all endophytes. GFP-tagged 4G12 was visualized using scanning confocal microscopy and shown to colonize wheat tissues (Figure 9.1C-E), suggesting it behaves as an endophyte in wheat.

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Figure 9.1

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Figure 9.1: Origin of endophytes used in this study, activity against F. gramineraum in maize and GFP-tagging of the endophytes to test wheat colonization. (A) A map showing the origins of maize genotypes used to isolate the candidate anti-Fusarium endophytic bacteria. (B) A graph showing the activity of the candidate endophytes in suppressing F. graminearum in maize (Chapter 8) (Mousa et al, 2015b). Letters that are different from one another indicate that their means are statistically different (P≤0.05, Mann Whitney t-test). (C-E) GFP-based tracking of endophyte strain 4G12: (C) picture of a representative wheat seedling coated with GFP-4G12; (D) GFP- 4G12 visualized inside wheat roots; (E) magnification of image shown in (D).

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9.4.2 Greenhouse trials

First greenhouse trial: Treatment with all four endophytes caused statistically significant reductions (P≤0.05) in FHB disease severity, with reductions ranging from 44 to 56% compared to Fusarium-only treated plants (P = 0.047, 0.002, 0.001, 0.014 and 0.003 for treatment with PROLINE, 1D6, 3H9, 4G12 and 4G4, respectively) (Figure 9.2C, Table 9.1). There was no statistical difference between the endophyte treatments with respect to the alleviation in disease symptoms (P≤0.05). Strain 4G4 nearly doubled grain yield per plant (an increase of 107.5%), but this was the only endophyte that caused a significant change in this metric compared to Fusarium-only treated plants (P= 0.010); the effect of 4G4 was significantly different than all other treatments (P=0.003, 0.005, 0.040 and 0.020 compared to PROLINE, 1D6, 3H9 and 4G12, respectively) (Figure 9.2D, Table 9.1).

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Table 9.1: Suppression of Fusarium Head Blight by anti-Fusarium endophytes in two replicate greenhouse trials relative to Fusarium only treatment.

Treatment Host plant % infection % disease Average % yield (mean ± reduction yield* increase SEM)* Greenhouse trial 1 Fusarium only 52.0±6.3 a 0 10.6±2.6 a 0 PROLINE® fungicide 32.0±4.1 b 38 10.9±1.3 a 3 1D6 (Paenibacillus sp.) Z. diploperennis 24±2.1 b 54 12.7±1.3 a 20 3H9 (Paenibacillus sp.) Z. mays ssp. 23±3.5 b 56 13.9±0. 8 a 31 mays (hybrid Pioneer 3751) 4G12 (Citrobacter sp.) Z. mays ssp. 29±3.4 b 44 11.6±1.9 a 10 parviglumis 4G4 (Paenibacillus sp.) Z. mays ssp. 23±2.2 b 56 22±3.6 b 108 parviglumis Greenhouse trial 2 Fusarium only 54.0±2.6 a 0 17.7±1.5 a 0 PROLINE® fungicide 32±2.6 b 41 20.1±2.1 a 14 1D6 (Paenibacillus sp.) Z. diploperennis 34.0±2.0 b 37 20.6±2.8 a 17 3H9 (Paenibacillus sp.) Z. mays ssp. 26.0±0.9 b 52 19.3±1.2 a 9 mays (hybrid Pioneer 3751) 4G12 (Citrobacter sp.) Z. mays ssp. 33.0±2.9 b 39 25.5±3.1∆ 44 parviglumis 4G4 (Paenibacillus sp.) Z. mays ssp. 24.0±2.3 c 56 41.57±0.2 c 135 parviglumis *Letters that are different from one another indicate that their means are statistically different (P≤0.05, Mann-Whitney t- tests.). ∆ symbol indicates statistical significant different to Fusarium treatment only and 4G4 treatment but not to the other treatments.

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Figure 9.2: Greenhouse trial 1 to test for the ability of the candidate endophytes to suppress Fusarium Head Blight in spring wheat. (A) Picture of a healthy wheat grain compared to, (B) a picture of an infected wheat grain. (C, D) Quantification of the effect of different treatments on FHB suppression, as (C) the percent of diseased seeds per plant, and (D) the average grain yield per plant. For both measurements, n=20 per treatment (n=10 for both controls). The whiskers indicate the range of data points. Letters that are different from one another indicate that their means are statistically different (P≤0.05, Mann Whitney t-test).

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Second greenhouse trial: Treatments with all four of the endophytes caused significant reductions in FHB disease severity when compared to the Fusarium only treatment (P= 0.0001, 0.0001, 0.0001, 0.0007 and 0.0001 for treatment with PROLINE, 1D6, 3H9, 4G12 and 4G4, respectively). All endophyte treatments resulted in reduction in disease severity by 37-55% compared to Fusarium-only treated plants (Figure 9.3A, Table 9.1). Treatment with strain 4G4 resulted in a statistically significant reduction in disease severity when compared to other treatments (P = 0.050, 0.016, 0.050 and 0.036 compared to PROLINE, 1D6, 3H9 and 4G12, respectively). Treatment with endophyte strains 4G12 and 4G4 caused statistically significant increases in average grain yield per plant (increases of 44% and 135%, respectively) compared to the Fusarium-only treatment (P= 0.0430 and 0.0001, respectively) (Figure 9.3B, Table 9.1). However, strain 4G4 resulted in a significant increase in grain yield when compared to strain 4G12 (P =0.004). Hence, strain 4G4 doubled grain yield per plant in both greenhouse trials under Fusarium infection conditions. Wheat heads treated with endophyte strain 4G4 showed statistically less disease severity and higher grain yield compared to the other endophytes (P≤0.05) (Table 9.1).

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Figure 9.3: Greenhouse trial 2 to test for the ability of the candidate endophytes to suppress Fusarium Head Blight in spring wheat. Quantification of the effect of different treatments on FHB suppression, as (A) the percent of diseased seeds per plant, and (B) the average grain yield per plant. For both measurements, n=20 per treatment (n=10 for both controls). The whiskers indicate the range of data points. Letters that are different from one another indicate that their means are statistically different (P≤0.05, Mann Whitney t- test) while ∆ symbol indicates statistical significant different to Fusarium treatment only and 4G4 treatment but not to the other treatments.

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9.4.3 Effect of the endophyte treatments on DON contamination

Health Canada has set acceptable levels of DON contamination in wheat to 2.0 ppm for non-staple foods and 1.0 ppm in baby food (Canada, 2012). In order to quantify DON levels in wheat grain, ELISA-based testing was conducted. Immediately following harvest, only trace levels of DON (~0.1 ppm) were detected in plants treated with Fusarium only, while all other treatments did not show any detectable levels of DON (data not shown). The grain was stored under ambient conditions inside sealed envelopes for 14 months, and then the samples were re-analyzed for DON contents:

Greenhouse trial 1 treatments: Plants that had been treated with endophyte strain 4G12 showed a dramatic reduction in DON concentration in their grain following storage, to within acceptable levels (0.9 ppm) compared to the Fusarium-only control (5.5 ppm), equivalent to an 84% reduction (Figure 9.4A, Table 9.2). However, all other endophyte treatments caused statistically significant reductions in DON accumulation, with DON levels declining from 5.5 to 1.3-2.2 ppm, equivalent to 76.3-60% reductions compared to the Fusarium-only control (P = 0.040, 0.010, 0.010 and 0.010 for treatments with 1D6, 3H9, 4G12 and 4G4, respectively) (Figure 9.4A, Table 9.2).

Greenhouse trial 2 treatments: Strain 4G12 consistently caused dramatic reduction in the post-storage DON concentration, shifting it to within acceptable levels (0.4 ppm) compared to the Fusarium-only control (7.6 ppm), equivalent to a 95% reduction, which was statistically significant (P= equals 0.010) (Figure 9.4A, Table 9.2). However, all other endophyte treatments caused statistically significant reductions in DON accumulation, with DON levels declining from 7.6 to 1.8-4.0 ppm equivalent to 76-47% reductions compared to the Fusarium-only control (P= 0.010, 0.010 and 0.010 for 1D6, 3H9 and 4G4, respectively) (Figure 9.4B, Table 9.2).

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Table 9.2: Reduction of DON mycotoxin accumulation during storage following treatment with anti-Fusarium endophytes compared to Fusarium only treatment.

Treatment Host plant DON content % of DON (ppm) reduction (mean ± SEM)* Greenhouse trial 1 Fusarium only 5.5±0.7 a 0 PROLINE® fungicide 1.3±0.9 b 76 1D6 Zea diploperennis 2.2±1.7 b 60 3H9 Z. mays ssp. mays 1.3±0.8 b 76 (hybrid Pioneer 3751) 4G12 Z. mays ssp. parviglumis 0.9±0.5 b 84 4G4 Z. mays ssp. parviglumis 2.2±1.0 b 60 Greenhouse trial 2 Fusarium only 7.6±0.3 a 0 PROLINE® fungicide 0.5±0.15 b 93 1D6 Z. diploperennis 1.8±0.4 c 76 3H9 Z. mays ssp. mays 4.0±0.9 d 47 (hybrid Pioneer 3751) 4G12 Z. mays ssp. parviglumis 0.4±0.2 b 95 4G4 Z. mays ssp. parviglumis 2.3±0.5 c 70 *Letters that are different from one another indicate that their means are statistically different (P≤0.05, Mann-Whitney t-tests).

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Figure 9.4: Test for the ability of the candidate endophytes to reduce DON mycotoxin accumulation in wheat grain during storage. DON measurements after storage of wheat grain from (A) greenhouse trial 1 and (B) greenhouse trial 2. For both trials, n=3 pools of seeds. Letters that are different indicate that the treatment means were significantly different at P≤0.05, Mann Whitney t-test. The error bars represent the standard error of the mean (SEM).

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9.5 Discussion In a previous study, I identified four bacterial endophytes isolated from wild, pre- domesticated and modern maize that could inhibit the growth of F. graminearum and reduce DON accumulation in modern maize (Mousa et al., 2015). Here, I asked if the same endophytes could inhibit F. graminearum in wheat. The results showed that the four endophytes suppressed FHB and reduced DON in wheat, with strains isolated from one wild maize genotype (Z. mays ssp. parviglumis), being the most potent at increasing grain yield (4G4, 4G12) and reducing DON accumulation after storage (strain 4G12) following exposure to the pathogen (Figure 9.1A, 9.2C, 9.3B and Figure 9.4; Tables 9.1 and 9.2). Consistent with these results, earlier, the strains isolated from wild maize (4G12, 4G4 and 1D6) showed the most consistent disease suppression ability in maize compared to the strain isolated from a modern maize variety (strain 3H9) (Figure 9.1B). The effect of strain 3H9 was statistically significant compared to all other endophyte treatments. However, all endophyte strains resulted in a statistically significant decrease in disease severity when compared to the Fusarium only treatment. In maize, all four strains dramatically reduced DON levels to 0.1-1.0 ppm. Moreover, all four strains resulted in 3-4-fold increases in grain yield compared to the control (Chapter 8) (Mousa et al., 2015b). The variation between the endophytes to suppress F. graminearum in a different host may be, in part, attributed to differences in host-endophyte interactions including the variable ability of each endophyte to colonize a heterologous host (Partida- Martínez and Heil, 2011).

9.5.1 The impact of improper grain storage on DON accumulation

The impact of poor drying and storage on mycotoxin accumulation is often over- looked by Western researchers. Subsistence farmers in Africa, South Asia and elsewhere typically dry grain in the field or on linen sheets, then store the grain in pits or above- ground granaries (mud, thatch, clay jars) under ambient temperature and humidity (FAO 1994). In general, improper grain drying and storage leads increased levels of mycotoxins including DON (Bhatt and Miller 1991; Wagacha and Muthomi 2008) due to persistent growth and/or metabolism of the toxigenic fungi (Piacentini et al., 2015). Here I

302 mimicked the storage conditions of poor subsistence farmers to study its effect on mycotoxin accumulation in wheat grains. In our study, DON accumulation during storage appeared to be directly related to the (pre-storage) percentage of infection (Wegulo et al., 2012). Consistent with the literature, both of these measurements were higher in Fusarium-only treated plants than each endophyte treatment.

9.5.2 An endophyte isolated from a wild crop relative crosses host boundaries As noted above, both strains 4G4 and 4G12 were previously isolated from Z. mays ssp. Parviglumis, a wild teosinte, which is the direct, primary ancestor of all modern maize (Matsuoka et al., 2002). Evidence suggests that parviglumis may have thrived for thousands of years in the Central Balsas Valley of southwestern Mexico, which is a seasonal tropical forest (Piperno et al., 2009). The potency of the endophytes from this plant may be the result of evolutionary selection by the host for beneficial microbes that could combat fungal pathogens that are especially a concern in areas of high humidity. Indeed, parviglumis was previously shown to have greater resistance to pathogens and insects compared to modern maize (De la Paz et al., 2010; Lange et al., 2014). Interestingly, whereas Fusarium species were detected in 100% of modern maize seeds (Z. mays ssp. mays), their incidence in teosinte seeds was only 4% under the same growth conditions; anecdotally, teosintes appeared to show lower rates of Fusarium infection than modern maize growing in Mexico (Desjardins et al.,, 2000). Parviglumis was also shown to be more resistant to Colletotrichum graminicola, the causal fungal agent of anthracnose disease in maize and wheat (Lange et al., 2014). In another study, the disease severity and infection rate of Ustilago maydis, the causal fungal agent of corn smut in maize and teosinte, was reported to be very low in parviglumis compared to modern maize (Munkacsi et al., 2008). It is interesting to speculate the existence of three-way co- evolution involving teosinte, its endophytes and its fungal pathogens. More generally, the current results suggest endophytes from one wild crop relative can be used to control diseases in a related modern crop belonging to the same family (the grass family, Poaceae). If true, then wild relatives of modern crops may be an untapped reservoir of endophytic microbes that can be used across entire crop families – a hypothesis that requires broad testing.

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9.5.3 Paenibacillus sp. as antagonist microbes against FHB Three of the bacterial endophytes identified in this study were previously classified taxonomically as Paenibacillus ssp. (strains 1D6, 3H9 and 4G4). In previous research, microbial antagonists have been reported to suppress FHB, though none of these were apparently commercialized; these antagonists include Paenibacillus ssp. ( He et al., 2009; El Daim et al., 2015) as well as other bacterial species of Bacillus, Cryptococcus, Pseudomonas, Brevibacillus, Streptomyces and Lysobacter (Khan et al., 2001; Jochum et al., 2006; Palazzini et al., 2007; Schisler et al., 2014; Yoshida et al., 2012), in addition to fungi such as Aureobasidium pullulans and Clonostachys rosea (Hue et al., 2009; Wachowska et al., 2012). Paenibacillus ssp. were previously reported as endophytes of diverse grasses within the family Poaceae including wheat (Mano and Morisaki, 2008; Shi et al., 2013; Durán et al., 2014; Mareque et al., 2015). In wheat, the anti-FHB Paenibacillus sp. were primarily isolated from soil and were shown to reduce DON in planta and increase grain yield (He et al., 2009). Moreover, paenibacilli have been reported to show broad spectrum anti-fungal activity against numerous plant pathogens (Timmusk et al., 2009; Xu and Kim, 2014).

Mechanistically, the Paenibacillus genome is characterized by the presence of several gene clusters that encode the biosynthesis of antimicrobial polyketides and non- ribosomal peptides (Kim and Timmusk, 2013; El Daim et al., 2015; Mousa and Raizada, 2015). This arsenal of antimicrobial compounds includes polymyxins, colistins and derivatives of fusaricidin which are bacterial secondary metabolites named after Fusarium (He et al., 2007; Raza et al., 2008; Raza et al., 2009; Naghmouchi et al., 2012; Raza et al., 2015) . The efficacy of Paenibacillus may be, in part, due to its efficient plant colonization, which might be attributed to biofilm formation (Timmusk et al., 2005; Haggag and Timmusk, 2008). As the current Paenibacillus strains were isolated from a novel source (parviglumis), it will be interesting to perform comparative genomic and biochemical analyses between the new anti-Fusarium isolates and those reported in the literature.

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9.5.4 Citrobacter as antagonist microbes against FHB The last anti-FHB strain isolated in this study (strain 4G12) was taxonomically predicted to be a Citrobacter species. Citrobacter sp. were previously reported as endophytes of various plants including related grasses such as sugarcane (Magnani et al., 2013), as well as legumes (Wang et al., 2006). I could find no reports of Citrobacter ssp. acting as Fusarium-antagonists in planta. However, it was reported that Citrobacter ssp. could control other fungal plant pathogens such as Monilinia fructicola, the causal agent of brown rot in stone fruits (Janisiewicz et al., 2013). Mechanistically, a Citrobacter ssp. was shown to produce chitinase (Meruvu and Donthireddy, 2014a), and could effectively inhibit the growth of fungal plant pathogens such as Aspergillus flavus and A. niger (Meruvu and Donthireddy, 2014b). Citrobacter ssp. have been reported to express a Type 3 fimbriae gene that is associated with biofilm formation (Cheryl-lynn et al., 2010).

In addition to its anti-FHB activity, strain 4G12 was the most potent of the endophytes in suppressing DON mycotoxin in grain after storage. A Citrobacter strain was previously reported as one constituent, along with several other species, of an enriched culture that could effectively detoxify DON into epi-DON in vitro, but the exact contribution of Citrobacter to this activity was not clear (Islam et al., 2012). Enzymatic pathways for DON detoxification includes, but not limited to, epimerization, de- epoxidation, and glycosylation (Zhou et al., 2008; Karlovsky, 2011). It will be interesting in the future to understand the anti-Fusarium and anti-DON mechanisms of action of strain 4G12.

9.6 Implications and future experiments This study has identified endophytes isolated from maize that could suppress the serious, toxigenic fungal pathogen F. graminearum in wheat. One endophytic strain reduced DON mycotoxin in grain to within acceptable safety levels following grain drying and extended storage conditions that mimicked those of subsistence farmers -- a promising result that requires field level validation for further practical applications. As we move into a more practical phase, some future experiments will be required. Specifically: (1) whether seed surface sterilization is required prior to the endophyte

305 treatments must be determined; (2) the most effective route of application for each endophyte must be quantified separately to determine if the strains act as endophytes or epiphytes; (3) endophyte survival within the grain must be assayed quantitatively (e.g. by qPCR); and (4) Fusarium infection and DON should be measured at different time intervals during grain storage.

In this study, the most potent of the anti-Fusarium strains were derived from parviglumis teosinte, the wild ancestor of modern maize suggesting that endophytes from wild relatives of one grass may benefit other grasses. Based on our results, we encourage other scientists to explore other wild relatives of modern crops for their endophytes, as they may have potential to reduce crop pathogens and their toxins across host boundaries.

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Chapter 10: General Discussion

Ancient crops and their wild relatives, grown without fungicides, are under- explored sources of beneficial microbes. I hypothesized that these plants may host endophytes that co-evolved to help their hosts naturally combat fungal pathogens including F. graminearum. The overall objectives of the thesis were: (1) to identify endophytes that can combat F. graminearum in vitro and in planta from the ancient crop, finger millet (Eleusine coracana), and from diverse maize genotypes, including wild relatives and ancient landraces; and (2,3) to characterize the underlying cellular, biochemical and/or molecular antifungal mechanisms. The results of this study support my hypothesis as discussed below.

10.1 Summary of the major finding in the thesis

10.1.1 Endophytes isolated from finger millet have anti-Fusarium activity (Chapters 5, 6, and 7) In this thesis, I report the first ever isolation of endophytes from finger millet. Seven fungal endophytes were isolated from finger millet roots and shoots. Four fungal-derived extracts (WF4, WF5, WF6 and WF7) inhibited the growth of F. graminearum in vitro, along with other fungal pathogens. These fungi belong to species of Fusarium, Aspergillus and Penicillium. Seven bacterial endophytes were isolated from different finger millet tissues. One bacterial endophyte (strain M6, an Enterobacter sp.) showed potent activity against F. graminearum. M6 suppressed both Gibberella ear rot in maize and Fusarium head blight in wheat.

10.1.2 Endophytes isolated from maize having anti-fungal activity (Chapters 8 and 9) Screening of 215 maize bacterial endophytes, previously isolated from diverse maize genotypes, identified four candidate endophytes that could inhibit the growth of F. graminearum and its DON mycotoxin to within the acceptable levels. Despite the screen

307 containing 116 endophytes from non-wild maize genotypes (45 from modern maize and 71 from traditional landraces, totalling 54% of the library), the three most potent endophytes were isolated from the wild counterparts of modern maize (representing 46% of the endophytes screened). Specifically, anti-Fusarium endophyte strain 1D6 was originally isolated from Z. diploperennis, and strains 4G12 and 4G4 were isolated from Parviglumis teosinte, the direct ancestor of modern maize. The remaining candidate endophyte (strain 3H9) was isolated from a modern maize variety (Z. mays ssp mays, Pioneer 3751 hybrid). All the four candidate endophytes suppressed both Gibberella ear rot in maize and Fusarium head blight in wheat.

10.1.3 Biochemical antifungal mechanisms (Chapter 5, 6, 7 and 8)

Bio-guided isolation of the active antifungal compounds from finger millet fungal extracts resulted in purification of seven anti-Fusarium compounds. The structures of these compounds were fully characterized. These compounds are viridicatol, tenuazonic acid, alteraniol, alteraniol methyl ether, a hydroxy benzofuranon derivative, harpagoside and dehydrocostus lactone. All of these compounds were reported here to have anti- Fusarium activity for the first time. Two phenazine derivatives were detected in the culture of Enterobacter sp. isolated from finger millet. Three fusaricidin derivatives were detected in the culture of three Paenibacillus strains isolated from maize.

10.1.4 Molecular antifungal mechanisms (Chapter 7) Bacterial strain M6, isolated from finger millet roots, showed the most potent anti- Fusarium activity in vitro and in greenhouse trials. As a result, it was selected to study its underlying anti-fungal molecular mechanisms. Thirteen candidate genes encoding the antifungal activity were identified. Real time PCR showed that most of the genes are inducible by Fusarium. The candidate anti-Fusarium genes from M6 include operons that encode phenazine (a potent anti-fungal metabolite), butanediol (an elicitor of host plant defences), and a fusaric acid resistance protein (FARP). Fusaric acid is a metabolite produced by Fusarium pathogens to inhibit the biosynthesis of bacterial phenazine (van Rij et al., 2005); FARP biosynthesized by M6 bacteria is an apparent efflux transporter for fungal fusaric acid (Utsumi et al., 1991). Since both Fusarium and finger millet

308 appear to be ancient to Africa, the phenazine-butanediol-fusaric acid-FARP interaction network may represent a fascinating example of three-way co-evolution between an endophyte, a pathogen and a host plant.

10.1.5 Cellular antifungal mechanism(s) (Chapter 7) Using confocal imaging, endophyte M6 cells were observed to construct a remarkable physical barrier resulting from root hair-endophyte stacking (RHESt) at the rhizosphere-root interface that prevents entry and/or traps Fusarium for subsequent killing. The RHESt structure was observed to contain two distinct lines of defence: a layer of intercalated root hairs and endophyte microcolonies, residing above a dense, continuous layer of endophyte cells on the root epidermal surface (rhizoplane). To the best of my knowledge, the RHESt barrier represents a novel plant defence mechanism.

10.2. Significance and impact of the study 10.2.1 The ancient cereal, finger millet, is an untapped reservoir for antifungal microbes To the best of my knowledge, this is the first report of endophyte(s) from finger millet and the first report of them having anti-Fusarium activity. This study suggests that exploration of endophytes in the ancient, orphan crops grown by subsistence farmers may result in the identification of organisms or natural products with potential to control the diseases of mainstream, modern crops. Many orphan crops have been grown continuously without pesticides including fungicides. I hope the results presented here will encourage other researchers to explore the diversity of finger millet and other ancient, orphan crops which have been largely ignored by many Western scientists.

10.2.2 Wild plants are promising sources for endophytes with antifungal activities The potent anti-Fusarium bacterial endophytes reported from the library of 215 screened maize endophytes were originally isolated from the wild relatives of modern maize, namely the teosintes, Parviglumis and Z. diploperennis (4G12, 4G4, and 1D6). These endophytes showed an exceptionally broad spectrum of anti-fungal activities.

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10.2.3 Bacterial endophytes protect the host plant from pathogen invasion

10.2.3.1 Antifungal activity of finger millet bacterial endophyte (M6, Enterobacter sp.)

As noted above, in this thesis, I report that finger millet endophyte M6 has developed, in coordination with its host, a previously unreported plant defense mechanism. M6 forms a remarkable physical barrier on the rhizoplane as part of an unusual root hair-endophyte stacking (RHESt) structure that prevents entry and/or traps Fusarium hyphae. My data suggests that F. graminearum is being killed inside RHESt. I showed that a diverse arsenal of natural products is involved in the M6-killing machinery including phenazines, chitinase and colicin V. In particular, there was only one previous report of colicin V having anti-fungal activity, and hence my study suggests that this compound may have broader spectrum anti-microbial activity than previously thought. Further molecular data provides evidence of a previously unreported epistatic regulatory interaction between M6-genes required for the anti-fungal activity (phenazine biosynthesis) and those involved in promoting M6 resistance to a xenobiotic antibiotic produced by Fusarium (fusaric acid). These data, suggestive of an ongoing arms race, provide evidence for long-term co-evolution between finger millet, its microbial inhabitant(s) and the fungal pathogen F. graminearum, likely in Africa.

10.2.3.2 Antifungal activity of Paenibacillus and Citrobacter strains

In this study, three out of 215 candidate bacterial endophytes screened were reported as potent antagonists against F. graminearu. These endophytes are P. polymyxa (strain 1D6 and 4G4) and a Citrobacter sp. (strain 4G12). In combination with the published literature on these bacteria, these results suggest that P. polymyxa and Citrobacter sp. are promising candidates for biological control of F. graminearum. To the best of my knowledge, however, Citrobacter sp. were not previously reported to have anti-Fusarium activity, and my thesis suggests that this genus should be further explored for its anti-fungal biocontrol potential.

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10.3 Conclusions I conclude that orphan crops, and the ancient landraces and wild relatives of modern crops, may be valuable reservoirs for endophytes with potential to combat crop pathogens. These anti-fungal activities may have evolved as the result of long-term co- evolution between pathogens, endophytes and their host plants. Based on the discovery of RHESt in particular, I suggest that microbiome-host interactions should be further explored in ancient, orphan crops that have been continuously grown by subsistence farmers (e.g. fonio, tef, other millets, Bambara groundnut, quinoa, etc.) to discover novel types of plant-microbe symbiosis and defence mechanisms. The endophytes of wild, ancient and orphan crops may have potential to reduce pathogens and their mycotoxins in modern agriculture across plant host boundaries.

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Chapter 12: Appendices

Appendix Method A5.1: Methodological details of toxicity assays of the WF4 extract. For all toxicity assays described below, the original WF4 endophyte extract, which was dissolved in methanol, was replaced with water: 100 ml of the methanol extract was dried, and the residue was dissolved in 100 ml H20 (7 mg/ml).

Effect of WF4 endophyte extracts on leaf health – To generate the leaf materials for the toxicity assay, maize plants (hybrid 35F40) were grown in the Crop Science Greenhouse Facility (University of Guelph) using the following conditions: 16 h light (~600 µmol m-2 s-1) at 28˚C, 8 h dark at 23˚C, and 50% relative humidity. Plants were grown semi- hydroponically in pots containing Turface® clay, and irrigated with a nutrient solution containing: 0.4 g/L 28-14-14 fertilizer [28% total N (1.6% nitrate, 0.4% ammonium, 26% urea), 14% P2O5, 14% K2O], 0.4 g/L 15-15-30 fertilizer [15% total N (nitrate 8.8%, ammonium 2.95%, urea 3.25%), 15% P2O5, 30% K2O], 0.2 g/L NH4NO3, 0.4 g/L of

MgSO4•7H2O and 0.03 g/L of micronutrient mix (S, Co, Cu, Fe, Mn, Mo and Zn). Leaf punches were taken from leaf blades at the six-week stage. Punches (1/4 inch diameter) were taken from young leaves. To conduct the toxicity assay, 24-well plates (Costar #3526, Fisher Scientific, USA) were filled with either deionized water containing 0.05 % Triton X-100 (negative control) or endophyte extract with 0.05 % Triton X-100 (2.5 ml per well). In each well was placed one young leaf punch (0.6 mm, punctured by cushioned grip hand punch, # 923095, Fiskars Brands, Inc, China), placed with either the adaxial or abaxial surface facing up. The experiment was repeated using five aqueous dilutions of WF4 extract (100%, 80%, 50%, 20%, and 10%). The multi-well plates were kept in the dark at room temperature. Pictures were taken of leaves after 2, 4 and 6 days of incubation, and the percentage surface area that showed lesions was quantified using Assess Software (Version 2.0, American Phytopathological Society) in comparison to controls.

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Effect of WF4 endophyte extracts on seed germination - Two types of seeds were used including maize (hybrid 35F40, Ridgetown College, Canada) and spring wheat (cultivar Quantum, Ridgetown College, Canada). Whatman filter papers were soaked in WF4 extract or water control. For each toxicity assay, three water-soaked filter papers and three extract-soaked filter papers (3 replicates) were distributed into six Petri dishes (100 mm x 15 mm). Into each Petri dish, ten seeds were added which had been surface- sterilized as described earlier. The experiment was repeated using five dilutions of WF4 extract (100%, 80%, 50%, 20%, and 10%). The Petri dishes were kept in the dark for 1-2 weeks, and the percentage of germination (hypocotyl emergence) was compared to the respective control.

Effect of WF4 endophyte extracts on development of fruit flies (Drosophila melanogaster)- Sokolowski Lab Fly Food was first prepared by mixing two sterilized solutions together: the first solution contained 25 g yeast extract dissolved in 100 ml of distilled water, and the second solution was composed of 50 g sucrose, 14.5 g agar, 0.5 g potassium phosphate, 4 g potassium sodium tartrate, 0.25 g sodium chloride, 0.25 g calcium chloride, 0.25 g magnesium chloride and 0.25 g iron (III) sulphate dissolved in 400 ml distilled water. After thoroughly mixing, 2.5 ml of propionic acid was added. The effect of WF4 endophyte extract on fruit fly egg development was tested by mixing 500 µl of the extract (or water, as control) into 6 ml of Sokolowski Lab Fly Food medium; 1 ml of the resulting solution was pipetted into each well of a 12-well plate (1 ml volume/well). Into each well was added a 30 µl suspension of fruit fly eggs (in PBS, phosphate-buffered saline solution). A dissection stereomicroscope (Stemi 2000, Zeiss, Germany) was used to determine how many eggs were in each well, which was then adjusted to 30 eggs/well. The top of the 12-well plate was sealed with packing tape in which 4-5 holes were made using a syringe with a 20-22 gauge needle. The plates were incubated at 28˚C. Over a period of 23 days, fruit fly development was observed from larvae to pupae to adults. There were six wells used for the extract and the control.

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Appendix Table A5.1: 18S rDNA sequences and BLAST analysis of fungal endophytes isolated from finger millet in this study.

ID Sequence Tag WF1 ACNNNGACCTCTCGGCCAAGGTGATGTACTCGCTGGCCCTGTCAGTGTAGCG CGCGTGCGGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCGCGC ACTTCCATCGGCTTGAGCCGATAGTCCCCCTAAGAAGCCAGCGGCCCGCAA ATGCGGACCGGGCTATTTAAGGGCCGAGGTCTCGTTCGTTATCGCAATTAAG CAGACAAATCACTCCACCAACTAAGAACGGCCATGCACCACCATCCAAAAG ATCAAGAAAGAGCTCTCAATCTGTCAATCCTTATTTTGTCTGGACCTGGTGA GTTTCCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACGCCTGGTGGTGCC CTTCCGTCAATTTCTTTAAGTTTCAGCCTTGCGACCATACTCCCCCCAGAACC CAAAAACTTTGATTTCTCGTAAGGTGCCGAACGGGTCATAATAGAAACACC GTCCGATCCCTAGTCGGCATAGTTTATGGTTAAGACTACGACGGTATCTGAT CGTCTTCGATCCCCTAACTTTCGTTCCCTGATTAATGAAAACATCCTTGGCGA ATGCTTTCGCAGTAGTTAGTCTTCAGCAAATCCAAGAATTTCACCTCTGACA GCT WF3 CTNNGACGCCGTNNNGATTAATAGGGNNAGTCGGGGGCGTCAGTATTCAGC TGTCAGAGGTGAAATTCTTGGATTTGCTGAAGACTAACTACTGCGAAAGCAT TCGCCAAGGATGTTTTCATTAATCAGGGAACGAAAGTTAGGGGATCGAAGA CGATCAGATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGG ACGGGATTCTATAATGACCCGTTCGGCACCTTACGAGAAATCAAAGTTTTTG GGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAAATTGACGGA AGGGCACCACAAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGA AACTCACCAGGTCCAGACAAAATAAGGATTGACAGATTGAGAGCTCTTTCTT GATCTTTTGGATGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCT GCTTAATTGCGATAACGAACGAGACCTCGGCCCTTAAATAGCCCGGTCCGCA TTTGCGGGCCGCTGGCTTCTTAAGGGGACTATCGGCTCAAGCCGATGGAAGT GCGCGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGC GCTACACTGACAGGGCCAGCGAGTACATCACCTTAACCGAGAGGTTTGGGT AATCTTGTTAAACCCTGTCGTGCTGGGGATAGAGNATTGC WF4 GANNGCCNTAATGATTAATAGGGACAGTCGGGGGCATCAGTATTCAATTGT CAGAGGTGAAATTCTTGGATTTATTGAAGACTAACTACTGCGAAAGCATTTG CCAAGGATGTTTTCATTAATCAGTGAACGAAAGTTAGGGGATCGAAGACGA TCAGATACCGTCGTAGTCTTAACCGTAAACTATGCCGACTAGGGATCGGGCG ATGTTCTTTTTCTGACTCGCTCGGCACCTTACGAGAAATCAAAGTTTTTGGGT TCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAAATTGACGGAAGG GCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAAC TCACCAGGTCCAGATGAAATAAGGATTGACAGATTGAGAGCTCTTTCTTGAT TTTTCAGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGCT TAATTGCGATAACGAACGAGACCTTAACCTGCTAAATAGCCAGGCTAGCTTT GGCTGGTCGCCGGCTTCTTAGAGGGACTATCGGCTCAAGCCGATGGAAGTTT GAGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGC TACACTGACAGAGCCNACGAGTTATTCACCTTGGCCGGAAGGTCTGGGTAAT CTTGTTAAACTCTGTCGTGCTGGGGATAGAGNNTTGCAATAN WF6 GGACGCCGTATGATTAATAGGGATAGTCGGGGGCGTCAGTATTCAGCTGTC

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AGAGGTGAAATTCTTGGATTTGCTGAAGACTAACTACTGCGAAAGCATTCGC CAAGGATGTTTTCATTAATCAGGGAACGAAAGTTAGGGGATCGAAGACGAT CAGATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGACG GGATTCTATAATGACCCGTTCGGCACCTTACGAGAAATCAAAGTTTTTGGGT TCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAAATTGACGGAAGG GCACCACAAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAAC TCACCAGGTCCAGACAAAATAAGGATTGACAGATTGAGAGCTCTTTCTTGAT CTTTTGGATGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGCT TAATTGCGATAACGAACGAGACCTCGGCCCTTAAATAGCCCGGTCCGCATTT GCGGGCCGCTGGCTTCTTAGGGGGACTATCGGCTCAAGCCGATGGAAGTGC GCGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGC TACACTGACAGGGCCAGCGAGTACATCACCTTAACCGAGAGGTTTGGGTAA TCTTGTTAAACCCTGTCGTGCTGG WF7 AGGACCGCCGTANTGATTAATAGGGATAGTCGGGGGCGTCAGTATTCAGCT GTCAGAGGTGAAATTCTTGGATTTGCTGAAGACTAACTACTGCGAAAGCATT CGCCAAGGATGTTTTCATTAATCAGGGAACGAAAGTTAGGGGATCGAAGAC GATCAGATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGA CGGGATTCTATAATGACCCGTTCGGCACCTTACGAGAAATCAAAGTTTTTGG GTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGAAATTGACGGAA GGGCACCACAAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAA ACTCACCAGGTCCAGACAAAATAAGGATTGACAGATTGAGAGCTCTTTCTTG ATCTTTTGGATGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTG CTTAATTGCGATAACGAACGAGACCTCGGCCCTTAAATAGCCCGGTCCGCAT TTGCGGGCCGCTGGCTTCTTAAGGGGACTATCGGCTCAAGCCGATGGAAGTG CGCGGCAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCG CTACACTGACAGGGCCAGCGAGTACATCACCTTAACCGAGAGGTTTGGGTA ATCTTGTTAAACCCTGTCGTGCTGG

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Appendix Table A5.2. Taxonomic identification of the endophytic fungi isolated from finger millet.

Endophyte ID Best BLAST Match BLAST Percent Source E-value Maximum Identity Root WF1 Aspergillus niger 0.0 100% Root WF3 Penicillium griseofulvum 0.0 99% Root WF4 Phoma sp. 0.0 99% Root WF6 Penicillium 0.0 99% chrysogenum Root WF7 Penicillium expansum 0.0 99%

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Appendix Table A5.3: List of retention times and expected masses of compounds from extract of WF4 fungus fermented on rice. The antifungal compounds are highlighted in yellow.

RT Mass Height

19.04 310.1762 11379

18.62 615.2709 21728

18.09 50974

17.4 237.0778 92309

17.35 496.137 13106

17.02 34135

15.9 197.1041 55204

15.9 20229

15.86 241.2755 23456

15.81 197.1042 10361

15.76 394.2085 170659

15.4 225.1713 14270

15.3 642.2621 18533

14.8 17886

14.67 506.145 123545

14.65 528.1266 65754

14.58 267.0881 72341

14.54 253.0726 1200810

14.39 296.1716 47065

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Appendix Table A5.4: List of retention times and expected masses of compounds from extract of WF4 fungus fermented on millet. The antifungal compounds are highlighted in yellow.

RT Mass Height

19.2 329.2929 1121689

19.05 714.4097 298849

19.02 328.2038 208142

18.94 272.0682 615910

18.43 311.2824 233954

18.38 742.3683 341327

18.36 360.2114 237420

18.06 340.3195 389414

17.96 709.5118 498328

17.15 315.2769 311725

17.12 288.0631 455058

16.52 348.2294 241596

15.98 258.0527 198187

15.95 320.1623 266682

15.92 416.2422 206654

15.81 358.2829 307812

15.62 364.2247 514146

15.61 414.2264 1282090

15.18 288.0634 220648

15.09 272.0681 324955

15.07 290.0789 395867

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14.63 508.1538 310107

14.63 253.0736 795146

13.39 364.2239 201331

13.24 278.0785 196535

13.24 260.068 624325

12.64 234.0888 332494

12.44 402.0942 184080

12.22 234.0523 226496

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Appendix Table A5.5. NMR data for atomic assignments of compounds isolated from WF4. The table includes compounds from WF4 fungi fermented on both rice and finger millet.

Atom No. Carbon 1H-NMR 13C-NMR Compound 1

2 C ------158.3 3 C ------157.2 4 C ------142.2 5 C ------134.9 6 CH 7.34 129.3 7 CH 7.32 126.4 8 CH 7.29 124.4 9 CH 7.09 122.1 10 C ------133.1 3-OH 12.2 NH 9.1 Phenyl moiety 1’ C ------124.06 2’ CH 7.07 120.3 3’ C ------120.9 4’ CH 6.82 116.6 5’ CH 6.72 115.2 6’ CH 6.71 114.6 Ar-OH 9.5 Compound 2 2 C 178.9 3 C 105.6 4 C 198.8 5 CH 3.7 67.20 6 C 195.01 7 CH3 2.4 24.26 8 CH 1.89 38.3 9 CH2 1.34 27.06 10 CH3 0.95 12.42 11 CH3 1.01 16.65 Compound 3 1 C 139.8 2 C 99 3 C 166.2 4 CH 7.3 101.9 5 C 166.8 6 CH 6.4 104.3 7 C 167

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CH3 2.8 25.8 1’ C 109 2’ C 154.4 3’ CH 6.6 100.6 4’ C 159.8 5’ CH 6.4 118.56 6’ C 140 Compound 4 1 C 138.2 2 C 98.9 3 C 165.1 4 CH 7.3 99.6 5 C 166.6 6 CH 6.59 102 7 C 166.6 CH3 2.8 25.5 OCH3 3.9 56.3 1’ C 109 2’ C 153 3’ CH 6.7 103.8 4’ C 164.6 5’ CH 6.6 118.1 6’ C 138.8

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Appendix Figure A5.1. Flow chart illustrating the bio-guided purification of the active anti-Fusarium compounds from the extract of endophyte WF4 grown on rice culture.

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Appendix Figure A5.2: Flow chart illustrating the bio-guided purification of the active anti-Fusarium compounds from the extract of endophyte WF4 grown on millet culture.

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Appendix Figure A5.3

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Appendix Figure A5.3: Preliminary toxicity assays of the extract from endophyte WF4. (A) The methodology used to test the extract for its ability to cause lesions on maize leaf punches (n=4 per dilution). (B) Quantification of the leaf lesions caused by different dilutions of the extract (n=4). (C-F) Seed germination toxicity assay: (C) Representative pictures of corn seeds on filter paper exposed to the endophyte extract compared to the buffer. (D) Quantification of the effect of different extract aqueous dilutions or the buffer control, on the germination of corn seeds. (E) Representative pictures of wheat seeds on filter paper exposed to the endophyte extract compared to the buffer control. (F) Quantification of the effect of different extract aqueous dilutions (100%, 80%, 50% dilutions) or the buffer control on the germination of wheat seeds. (G,H) Fruit fly toxicity assay: (G) Picture showing the different developmental stages of the fly: larva, pupa and adult. (H) Quantification of the effect of the extract or the buffer control on the onset, duration and percentage (relative to starting eggs) of each developmental stage. For all of the toxicity assays, an asterisk indicates that the mean is significantly different than the control (p≤0.05, ann-Whitney, n=6 unless otherwise indicated). The error bars represent the standard error of the mean (SEM).

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Appendix Table A6.1. NMR data for atomic assignments of compounds isolated from finger millet fungi.

Compound 1 isolated from WF5 Atom No. Carbon 1H-NMR 13C-NMR 1 C ---- 153.9 2 CH2 3.83 33 3 C ----- 146.7 4 CH 6.75 114.2 5 C ----- 125.1 6 CH 6.65 111.8 7 CH 6.95 110 8 C ---- Ar-OH 9.32 Compound 2 isolated from WF6 Atom No. Carbon 1H-NMR 13C-NMR 1, 5, 7 CH 2.86 45.9, 52, 47.5 2 CH2 1.91 32.4 3 CH2 2.42 30.2 4 CH ---- 151.2 6 CH 3.95 85.2 8 α CH2 1.56 30.9 8 β, 9 β 9 CH2 2.24 36.2 10 C ---- 149.2 11 C ---- 139.7 12 C ---- 170.27 13 α CH2 6.2 120.19 13 β 5.46 14 α CH2 5.24 109.59 14 β 5.04 15 α CH2 4.8 112.6 15 β 4.7 Compound 3 isolated from WF7 Atom no. Carbon H1-NMR C13-NMR 1 CH 6.14 s 94 3 CH 5.6 d 143.4 4 CH 5.5 d 104.7 5 CH 71.3 6 CH 3. 3 d 77.1 7a CH2 1.76 d 45 7b 2.15 dd 8 C 87.3 9 CH 2.9 m 53.4 10 CH3 1.33 s 22.4

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Sugar moiety 1\\ CH 3.98 76.52 2\\ CH 3.7 75.99 3\\ CH 3.84 72.79 4\\ CH 3.51 69.9 5\\ CH 3.45 72.73 6\\ CH2 3.36 61.8 Acyl moiety 1\ C ----- 134.28 2\ CH 7.58 s 129 3\ C H 7.47 s 128.3 4\ CH 7.46 s 130.3 5\ CH 7.3 s 128.8 6\ CH 7.55 s 128.2 α CH 6.38 d 119 β CH 6.36 d 144.9 C=O C 167.32

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Appendix Methods A7

Appendix Method A7.1: Suppression of F. graminearum in wheat.

The candidate anti-Fusarium endophyte M6 was tested for their ability to suppress F. graminearum in Quantum spring wheat (C&M Seeds, Canada) in replicated greenhouse trials (Crop Science Greenhouse Facility, University of Guelph):

Seed treatment: Seeds of spring wheat were surface sterilized as follows: seeds were washed in 0.1% Triton X-100 detergent with shaking for 10 min; the detergent was poured off, 3% sodium hypochlorite was then added (twice for 10 min), which was followed by rinsing with distilled, autoclaved water, washing with 95% ethanol for 10 min; and finally the seeds were washed 5-6 times with distilled, autoclaved water. Complete surface sterilization was verified by inoculating the last wash on PDA and LB plates at 25°C and 37°C, respectively; none of these washes showed growth. The seeds were then coated with the endophyte. To prepare endophyte inoculant, M6 was incubated at 37˚C for 24 h in LB medium, centrifuged, washed and re-suspended in PBS buffer to an OD600 of 0.5. Afterwards, 500 μl of 6 was rocked for 2 h on a hori ontal shaker (Serial #980216M, National Labnet Company, USA) with 10 ml polyvinyl pyrrolidine (PVP, Catalog # 9003398, Sigma Aldrich, USA) which was used as a seed-coating agent. Endophyte- or buffer-coated seeds were germinated on wet paper towels in the dark for 7 days; uniformly sized seedlings were moved to pots containing Turface clay (Turface Athletics Inc, USA) in a greenhouse.

Pathogen treatment: Pathogen spores were prepared as follows: the liquid spore suspension medium was prepared using distilled water as follows (per L): 2 g KNO3, 2 g

KH2PO4, 1 g MgSO4, 1 g KCl, 1 g dextrose, 20 mg/100 ml (of each) of minor elements

(FeCl2, ZnSO4, MnSO4). Subsequently, 350 ml of the medium was added to 2 L flasks, then sterilized at 121°C for 10 min. Followed by cooling at room temperature, 3-4 PDA

385 plugs of F. graminearum or 10 ml liquid conidia suspensions were added to each flask under sterile conditions, and these were subsequently incubated on shaker tables under a 12:12 h UV light:dark cycle for ~two weeks at room temperature. Each spore solution was standardized to 20,000 spores/ml with a haemocytometer, then refrigerated or used to treat plants directly. Per plant, 1 ml of the F. graminearum spore suspension (20,000 spores/ml) was then sprayed directly onto wheat heads at the flowering stage with the emergence of anthers (flowers in anthesis, as anthers are the primary sites of infection). No surfactant or other formulation agents were used.

Endophyte treatment: The bacterium M6 was introduced twice more to ensure its high titre: the first endophyte spray was performed simultaneously with the pathogen (at the flowering stage with the emergence of anthers), while the second spray was the endophyte alone, 3 days later. M6 was grown overnight in LB medium then 1 ml of

OD600 0.4-0.6 was used for spraying. No surfactant or other formulation agents were used.

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Hoagland’s solution, until seed maturity (Gaudin et al., 2011). During the Fusarium or endophyte spray treatments, the greenhouse temperature was 28-30˚C. To promote Fusarium growth, the greenhouse was sprayed twice per week with water to achieve a typical relative humidity of ~60-80%.

Disease scoring: At full maturity, following seed drying, disease was scored as the percentage of grains per plant with visually observable disease symptoms relative to the total number of grains per plant. The infected seeds were defined as having a rough, shriveled surface and/or white hyphae (Schmale and Bergstrom 2003). Total dry grain weight at harvest per plant was also measured. Results were analyzed and compared using Mann-Whitney t-test (P≤0.05). Prism Software v5 (GraphPad Software, USA) was used for the statistical analysis.

Appendix Method A7.2: Testing the ability of M6 to produce IAA.

Endophytic IAA (auxin) production was measured as previously described (Bric et al., 1991). Strain M6 was cultured on LB agar plates supplemented with 5 mM L-tryptophan, then incubated at 30˚C for 3 days, with 3 replicates. A nitrocellulose membrane was placed on 6 colonies, and them the plates were incubated at 4˚C for 24 h. Whatman # 2 filter papers were placed into fresh Petri dishes, saturated with Salkowski reagent (0.01 M ferric chloride in 35% perchloric acid), and then the nitrocellulose membranes were shifted onto the filter paper. A change in color to pink or red surrounding the colonies indicated IAA production.

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Appendix Method A7.3: Identification of genes disrupted by Tn5 insertions of interest

Genomic DNA of the candidate Tn5 mutants was extracted using a Bacterial Genomic DNA Isolation Kit (#17900, Norgen Biotek, Canada). One microgram of the extracted DNA was digested with BamHI (#15201023, Invitrogen, USA) at 37˚C for 1 h, then purified using illustra GFX PCR DNA and Gel Band Purification Kits . The purified DNA fragments were allowed to self-ligate for 1 h at room temperature using ExpressLink™ T4 DNA Ligase (A13726, Invitrogen, USA) then purified using illustra GFX PCR DNA and Gel Band Purification Kits. Two μL of each ligation mixture were used to transform Transfor ax™ EC100D™ pir-116 Electrocompetent E.coli (EC6P095H, Epicentre, USA) by electroporation (as described above). Plasmid DNA was extracted from recovered colonies using a QIAprep Spin Miniprep Kit (#27106, Qiagen, USA). To identify the open reading frame of each disrupted gene, Tn5 transposon- specific read out- primers, KAN-2 FP-1 Forward Primer with sequence 5′ - ACCTACAACAAAGCTCTCATCAACC - 3′ and R6KAN-2 RP-1 Reverse Primer with sequence 5′ - CTACCCTGTGGAACACCTACATCT - 3′, were used for sequencing at the Genomics Facility, University of Guelph.

Appendix Method A7.4: Quantitative Real-time PCR to measure gene expression of the candidate anti-fungal genes

To test if the candidate genes are inducible by F. graminearum or constitutively expressed, real-time PCR analysis was conducted using gene specific primers designed using Primer Express software 3.0 (Table A7.1). Wild type strain M6 or mutant strain was grown overnight in LB medium (37 ˚C, 225 rpm). Thereafter, the culture was transferred to fresh sterilized LB medium (1:100) to a final OD600 of 0.02 then allowed to grow (37 ˚C, 225 rpm) until OD600= 0.14. The culture was divided under sterile conditions into three/four flasks, 100 ml each. To one flask, 3 ml of 48 hr old F.graminearum grown in potato dextrose broth was added. To the second flask, 0.1 g pre- sterilized chitin (Catalog # C7170, Sigma Aldrich, USA) and 3 ml potato dextrose broth

388 were added. To the third flask, the blank control, only 3 ml potato dextrose broth was added (blank control). To the fourth flask, sub-inhibitory concentration of fusaric acid (0.005) was added. The flasks were incubated under the same conditions (37˚C, 250 rpm). Following incubation, 1 ml samples were collected from each flask at regular time intervals [1 h (OD595=0.3), 2 h (OD595=1.1), 3 h (OD595= 1.4) and 4 h (OD595= 1.4-1.5)] until the bacterial growth reached the stationary phase. RNA was extracted following a standard protocol (E.Z.N.A Total RNA kit,Catalog #R6834-01, Omega Bio-tek, USA). To remove any DNA contamination from the extracted RNA, samples were treated with a DNA-free™ Kit (AM1906, Ambion) following the manufacturer’s protocol. RNA samples were analyzed by qPCR at the Genomics Facility, University of Guelph. RNA quality was checked using Bioanalyzer 2100 (Agilent). cDNA was made using a cDNA Reverse Transcription Kit (#4368814, Applied BioSystems, USA). The 20 µl reaction mixture for real time PCR consisted of: 1 µl of ultiScribe™ Reverse Transcriptase (50 U/µl), 2 µl of 10X reverse transcription buffer, 2 µl of 10X random primers, 0.8 µl of 25X (100 mM) dNTPs, 10 µl of RNA (90 ng/µl), and 4.2 µl of nuclease-free water. The reverse transcription conditions were: 10 min at 25°C, 120 min at 37°C, 5 min at 85°C, hold at 4°C. After completion of this cycle, 120 µl of water were added to 20 µl of cDNA, then 5 µl were used in each qPCR reaction. Each qPCR reaction mixture consisted of 5 µl of cDNA, 10 µl of 2X PerfeCTa SYBR Green FastMix with ROX (Quanta BioScience), 0.8 µl of forward and reverse Primer (5 µM), and 4.2 µl of nuclease-free water. 16S primers were used as a housekeeping gene to normalize gene expression data. For the housekeeping gene, a 400X dilution primer was used. The conditions for qPCR were 30 s at 95°C and 40 cycles of (3 s at 95°C and 30 s at 60°C). Gene expression was calculated using the Delta Ct (cycle threshold), where Delta Ct = Ct gene of interest-Ct housekeeping gene.

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Appendix Table A7.1: Gene-specific primers used in quantitative real-time PCR analysis.

Gene ID PCR primers ewpR 5D7F: 5′- GGCATAACTTCCTGCGCTAC - 3′ 5D7R: 5′: CAGTACGCCATCAATCATCG - 3′ ewpF1 Ph F1F: 5′- TTTTCTCACCGGGCGTTTC- 3′ Ph F1R: 5′- GTATGTGCGGAGCCGGTAA- 3′ ewpF2 Ph F2F: 5′- AAATGGCGCAGCAGCATAA- 3′ Ph F2R: 5′- GTCGGTGCGCACGAAAA- 3′ ewfR D5F : 5′- GGGGACAGTAACGACGAAAC - 3′ D5R : 5′- CGGCAATCTGTCGATATGAA - 3′ ewfD FusE/MFPF: 5′- TGGCCGTGCGGGATAAT- 3′ FusE/ FPR: 5′- GGATCGATGGTGTAAAGCACATC- 3′ ewfE FusEF: 5′- CGTCGAGCCCACCTTTAGC- 3′ FusER: 5′- TCCGGCAATCTGTCGATATG- 3′ ewgS A8F 5: GGAGTCAAAACACGGAATTTACG - 3′ A8R 5: ATCTGATAAGCAGGGAAGATCTCTTT - 3′ ewvC B9F 5: TGTTTTATGCTTAAACTGGCGATT - 3′ B9R 5: CGAATGCGGTGGGATATCA - 3′ 16SrDNA (housekeeping 799F: 5′- AACMGGATTAGATACCCKG- 3′ gene) 1492R: 5′- GGTTACCTTGTTACGACTT- 3′

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Appendix Table A7.2: Taxonomic classification of finger millet bacterial endophytes based on 16S rDNA sequences and BLAST analysis

ID Tissue source BLAST analysis % of max. identity M1 Roots Enterobacter sp. 99 M2 Seeds Pantoea sp. 97 M3 Seeds Burkholderia sp. 99 M4 Shoots Pantoea sp. 98 M 5 Shoots Burkholderia sp. 99 M6 Roots Enterobacter sp. 99 M7 Shoots Burkholderia sp. 100

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Appendix Table A7.3: Effect of endophyte strain M6 isolated from finger millet on the growth of diverse fungal pathogens in vitro.

Target fungal species Nystatin Amphotericin M6 (10.0 U/ml) (250 µg/ml) (average± (average± (average± SEM)* SEM)* SEM)* Alternaria alternata 0.0±0.0 0.0±0.0 0.0±0.0 Alternaria arborescens 0.0±0.0 0.0±0.0 0.0±0.0 Aspergillus flavus 2.0±0.2 0.0±0.0 3.6±0.2 Aspergillus niger 0.0±0.0 2.0±0.0 0.0±0.0 Bionectria ochroleuca 2.0±0.2 0.5±0.2 0.0±0.0 Davidiella tassiana 1.5±0.2 0.5±0.3 0.0±0.0 Diplodia pinea 2.5±0.2 3.0±0.2 0.0±0.0 Diplodia seriata 3.0±0.2 2.0±0.2 0.0±0.0 Epicoccum nigrum 0.0±0.0 0.0±0.0 0.0±0.0 Fusarium avenaceum (isolate 1) 2.5±0.3 3.0±0.6 1.8±0.2 Fusarium graminearum 1.5±1.6 0.0±0.0 5.0±0.3 Fusarium lateritium 0.0±0.0 1.0±0.2 0.0±0.0 Fusarium sporotrichioides 1.0±0.2 1.0±0.2 2.8±0.2 Fusarium avenaceum (isolate 2) 0.0±0.0 0.0±0.0 0.0±0.0 Nigrospora oryzae 0.0±0.0 0.0±0.0 1.83±0.2 Nigrospora sphaerica 0.0±0.0 0.0±0.0 0.0±0.0 Paraconiothyrium brasiliense 0.0±0.0 0.0±0.0 0.0±0.0 Penicillium afellutanum 3.0±0.2 3.0±0.2 0.0±0.0 Penicillium expansum 2.0±0.2 5.0±0.2 4.9±0.1 Penicillium olsonii 1.5±0.3 3.5±0.3 0.0±0.0 Rosellinia corticium 2.0±0.2 4.5±0.3 0.0±0.0 *SEM is the standard error of the mean (Mann-Whitney t-test).

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Appendix Table A7.4: Suppression of F. graminearum disease symptoms in maize and wheat by endophyte M6 in replicated greenhouse trials.

% % Disease % Yield increase Average Infection reduction relative relative to Treatment yield per (mean ± to Fusarium only Fusarium only plant* SEM)* treatment treatment Greenhouse trial 1 in maize Fusarium only 33.69±2.3 0.0 41.77±1.4 0.0 Proline 23.10±2.0 31.4 50.9±1.2 21.85 fungicide M6 10.13±0.9 69.93 39.58±2.6 -5.2 Greenhouse trial 2 in maize Fusarium only 85.11±4.5 0.0 7.8±1.1 0.0 Proline 41.42±4.5 51.33 40.8±1.2 423 fungicide M6 6.9±0.9 91.89 34.70±2.7 344 Greenhouse trial 1 in wheat Fusarium only 51.8±6.3 0.0 10.5±2.6 0.0 Proline 31.70±2.6 38.8 10.9±1.3 3.8 fungicide M6 41.1±2.6 20.6 10.6±0.8 0.95 Greenhouse trial 2 in wheat Fusarium only 54.0±2.6 0.0 17.7±1.5 0.0 Proline 31.5±4.1 41.6 20.1±2.1 13.5 fungicide M6 30.7±3.6 43.1 21.1±1.3 19.2% *SEM is the standard error of the mean (Mann-Whitney t-test).

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Appendix Table A7.5: Reduction of DON mycotoxin accumulation during prolonged seed storage following treatment with endophyte M6.

DON content (ppm) % DON reduction relative to Treatment (mean ± SEM)* Fusarium only treatment* Greenhouse trial 1 in maize Fusarium only 3.4±0.4 0.0 Proline fungicide 0.7±0.4 79.4 M6 0.1±0.0 97 Greenhouse trial 2 in maize Fusarium only 3.5±0.3 0.0 Proline fungicide 0.1±0.0 97.1 M6 0.1±0.0 97.1 Green house trial 1 in wheat Fusarium only 7.6±0.3 0.0 Proline fungicide 0.5±0.1 94.6 M6 1.5±0.6 81.33 Green house trial 2 in wheat Fusarium only 5.5±0.7 0.0 Proline fungicide 1.3±0.9 76.3 M6 2.0±0.4 63.6 *SEM is the standard error of the mean (Mann-Whitney t-test).

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Appendix Table A7.6: Complete list of strain M6 Tn5 insertion mutants showing loss of antifungal activity against F. graminearum in vitro.

ID Gene Swarming Motility Presence Biofilm prediction assay assay of flagella (mean ± (mean ± (%) SEM)* SEM)* Wild type +++ 4.2±0.3 70% 0.8 ewa-1B3::Tn5 Transcription --- 0.6±0.02 50% 0.11±0.0 ewa-4B8::Tn5 regulator, AraC 0 ewy-1C5::Tn5 YjbH outer- ++ 0.5±0.03 10% 0.3±0.08 ewy-5D2::Tn5 membrane protein ewm-2D7::Tn5 4- --- 0.8±0.05 20% 0.07±0.0 hydroxyphenyl 0 acetate 3- monoxygenase ewp-5D7::Tn5 Transcription --- 0.5±0.03 30% 0.18±0.0 regulator, LysR 1 ewb-9F12::Tn5 Fatty acid --- 0.6±0.05 30% 0.3±0.11 ewb-7C5::Tn5 biosynthesis ewv-4B9::Tn5 Colicin V + 1.8±0.30 60% 0.6±0.05 production ewt-15A12::Tn5 Transport + 1.5±0.00 30% 0.6±0.04 permease protein ews-1H3::Tn5 Sensor histidine + 0.8±0.16 50% 0.8±0.2 kinase ewc-3H2::Tn5 Chitinase --- 1.0±0.00 20% 0.4±0.15 ewf-7D5::Tn5 Transcription + 1.5±0.20 50% 0.3±0.08 regulator, LysR ewg-10A8::Tn5 Di-guanylate --- 0.8±0.30 40% 0.1±0.01 Cyclase ewh-5B1::Tn5 Hig A protein +++ 1.6±0.30 30% 0.6±0.10 ewa-8E1::Tn5 Hypothetical ++ 2.3±0.40 20% 0.5±0.09 protein *SEM is the standard error of the mean (Mann-Whitney t-test).

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Appendix Table A7.7: M6 wild type nucleotide coding sequences (Ettinger et al., 2015) corresponding to Tn5 insertion mutants that showed loss of antifungal activity against F. graminearum in vitro

ID Gene sequence ewa-1B3::Tn5 gtgaataccattggcataaacagcgagcccatcctgacgcacagtggctttagcattaccgccgataccactcttgccgc agacaggcactatgacgttatctatcttcctgccctgtggcgcaatcctcgtgcagtggtcagacaacagcctgaactcct ggcatggcttagcgaacaggcggcgcgagggacccgcatcgcggccgtcggaacgggctgctgtttcctggcgga atcgggattgctcaacgggaaacccgccaccacccactggcactacttcaaacagttctcgcgtgactaccccaacgta aaattacaaaccaaacattttctcacgcaggccgataatatttactgtgccgccagcgtcaaagccctctcagatctgacc atccatttcatcgaaacgatatacgggaaacgtgtagccacacatacccaacggacctttttccatgaaattcggagtcag tttgatcgccagtgttacagtgaagaaaacaaaccccatccggatgaagatattgttcaaattcaaatctggataaaagcc aactgcgcctcggatatatccatgcaaaatcttgccgatatggctggcatgagtttgcgcaactttaatcgccgctttaaaa atgccaccgatatatcccccctgcagtatttattaaccgccagaattgaatccgccatgacaatgctgcaatccaccaatc tgagcattcaggagattgcgaatgcggtgggatatcaggatattgcgcactttaatcgccagtttaagcataaaacaacg gtttcaccgggggattaccgtaagaccgtccgggcaaagatgtttagtgcataa

ewy-1C5::Tn5 atgaaaagaacctatctctacagcatgctggcgctctgcgtgagtgccgcgtgccatgcagaaacgtatccggcaccc attggcccgtctcagtcagacttcggcggcgtcggtttgctgcaaacgcccaccgcgcggatggcgcgcgaagggga aattagccttaactaccgtgataacgatcagtatcgttactactcggcgtcggtgcagctgttcccgtggcttgaaaccac gctgcgctacaccgacgtgcgtacgaaacagtacagcagcgttgatgcgttctccggcgaccagacctacaaagataa agccttcgacgtcaagctgcgcctgtgggaagagagctactggatgccgcaggtgtccgtgggcgccaaagatatcg gtggtaccggtctgtttgatgctgaatacatcgtggccagtaaagcctgggggccgttcgacttctcgctcggcctggga tggggctacctgggcactggcggtaacgtgaaaaatccgttttgctcctacagcgataaatactgctaccgcgataaca gctataagaaagcgggttccatcaacggtgaccagatgttccacggtccggcatcgctgtttggcggcgtggagtatca aacgccctggcagccattacgcctgaagctggaatatgaagggaatgactactcgcaggacttcgccgggaagattga gcagaagagcaagtttaacgtcggcgccatttatcgcgtcaccgactgggccgacgttaacctcagctacgagcgcgg caacaccgtgatgtttggcttcacgctgcgcaccaactttaacgatatgcggccacactacaatgataacgcgcgccctg cataccagccggagccgcaggatgcgattctgcagcactccgtggtggcaaaccagctgacgctgctgaaatacaat gccggcctggcggatccgaaaattcaggtgaaaggcgatacgctgtacgtgaccggcgagcaggtgaaataccgcg actcgcgcgaagggatcgaacgcgctaaccggatcgtaatgaacgatctgccggaggggatccgcacgatccgcgt gacggaaaaccgccttaacctgccgcaggtgacgacggaaacggacgttgccagcctcaagcgccatctggaaggt gaaccgctcgggcatgaaaccgagctggtgcaaaaacgcgtagaaccgatcgtgccggagaccaccgagcagggc tggtatatcgacaaatcgcgcttcgatttccatatcgatccggtgctgaaccagtccgtcggcgggccggaaaacttcta catgtatcagctgggcgtcatggcgacggcggatctgtggcttaccgaccacctgctgaccaccggtagcctgttcgg caacatcgctaataactacgacaagttcaactacaccaacccgccaaaagactcacagctgccgcgcgtgcgtactcg cgtgcgtgaatacgtgcagaacgatgcttacgtgaataacctgcaggccaactatttccagtacttcggcaatggcttcta cggccaggtgtacggcgggtatctggaaaccatgtacggcggcgcgggggcggaagtgctttatcgtcctgtcgaca gcaactgggcgttcggggttgatgccaactacgtcaagcagcgtgactggcgcagcgcgcaggacatgatgaagttc accgactacagcgtcaaaacgggccatctgaccgcctactggacgccgtcgttcgcgcctgacgtgctggtgaaagc cagcgttggtcagtacctggcgggcgataagggcggtacgctggatatctctaaacacttcgacagcggcgtcgtggt gggcggctatgccaccatcaccaacgtttcgccggacgaatacggggaaggggacttcaccaaaggggtctacgtgt cgattccgctggatctgttctcgtcaggcccaacccgcagccgtgcggcagtaggctggacgccgctgacgcgtgac gggggtcaacagcttggacgtaagttccagctgtatgacatgacgagcgataagaacattaacttccgctga

ewm-2D7::Tn5 atgaagcctgaagagttccgcgctgatgccaaacgcccgttaaccggcgaagagtatttaaaaagcctgcaggacggt cgtgagatttatatctacggcgagcgcgtcaaagacgtcaccacccatccggcatttcgcaacgcggcggcctccatc gcgcagatgtacgacgcgctgcacaagcctgacatgcaggacgcgctctgctggggcaccgacaccggcagcggc ggctatacccacaagtttttccgcgtggcgaaaagtgccgacgacctgcgccagcagcgcgacgccatcgccgagtg gtcgcgcctgagctacggctggatgggccgcacgccggactacaaagccgcgttcggctgcgcgctcggcgccaac ccggccttctacggccagttcgaacagaacgcccgtaactggtacacgcgcattcaggaaaccggcctgtactttaacc

396

acgccatcgttaacccgccgatcgaccgtcacaagccggcggacgaggtgaaagacgtctacatcaagctggagaa agagaccgacgccgggattatcgtcagcggcgcgaaagtggtagccaccaactccgccctgacccactacaacatga tcggcttcggctccgcgcaggtgatgggcgaaaacccggacttcgcgctgatgttcgtcgcgccgatggatgccgaa ggcgtgaagctgatctcccgcgcctcttacgaaatggtggcaggcgcaaccggatccccgtacgactacccgctctcc agccgctttgatgagaacgacgcgatcctggtgatggatcacgtgctgatcccgtgggaaaatgtgctgatctaccgcg atttcgaccgctgccgtcgctggacgatggaaggcggtttcgcgcggatgtacccgctgcaggcctgcgtgcgtctgg cggtgaagctcgacttcatcaccgccctgctgaagaaatccctggagtgcaccggcaccctggagttccgcggcgtac aggcggatctcggcgaagtggtggcctggcgtaacatgttctgggcgctgagcgactccatgtgttccgaagccaccc cgtgggtcaacggcgcgtatctgccggatcacgccgcgctgcaaacctaccgcgtgatggcgccgatggcctacgcc aagatcaagaacatcatcgagcgtaacgtgacgtccggtctgatctacctgccgtccagcgcccgggatctgaacaac ccgcagatcgaccagtatctggcgaaatacgtgcgcggctccaacgggatggatcacgtcgaacgcatcaagattttg aagctgatgtgggatgccatcggcagcgagtttggcggtcgtcacgagctatacgaaatcaactactccggcagccag gatgagatccgcctgcagtgtctgcgccaggcgcagagctccggcaacatggacaaaatgatggcgatggtcgaccg ctgcatgtccgaatacgaccagcacggctggaccgtaccgcacctgcacaacaacagcgatatcaacatgctcgaca agctgctgaaatag ewp-5D7::Tn5 atggacttaacccagcttgaaatgtttaacgccgtcgcgctgacgggcagcatcacccaggcggcgcagaaggtgcat cgcgtgccgtccaacctgacgacccgcatccgccagctggaagccgatcttggcgttgagctgtttattcgtgagaacc agcgtttgcgcttatctcccgccgggcataacttcctgcgctacagcaggcagatcctcgccctggtggatgaagcgcg catggtcgtcgcgggtgatgagccgcaggggttatttgccctcggcgcgctggaaagcaccgccgcggtgcgcattc ccgaaacgctggcgcagtttaaccagcgctatccgcgcattcagtttgccctttctaccgggccttccgggacgatgatt gatggcgtactggagggcaccttaagcgccgcctttgtcgacgggccgctgtcgcacccggagctggagggcatgc cggtctaccgggaagagatgatgctagtcacgcctgccgggcacgccgaggttgcacgcgccacgcaggttagcgg cagcgacgtttacgcatttcgcgcgaactgctcgtaccgtcgacatctggaaagctggtttcatgcggacagagccacg cctggccgcattcatgagatggagtcctaccacggcatgctcgcctgcgtcattgcgggcgcgggcatcgcgctgatg ccgcgctcgatgctggagagtatgccgggacatcatcaggttgaagcctggccgctggcggaaaactggcgctggct tacaacctggctggtgtggcggcgcggggcgatgacccgccagctggaagcttttatagcgctgctaaacgaacgtct tcaaccagcgccttctccataa ewb-9F12::Tn5 atgcccgcaaaatcatcaggaagcgcgtgggagcgttttgccggagtattacgtaatgcgcaaacggaatgtattgtta cgacagcaaagggagcagaaacgctaggccagctatcacttccgttatccccgcttatttttaccttcgacaaaccagac acagctgcgctccctgcgggctatcgtctgcatcctcttgatcgcacgttctccggggcatttcaccccgttccggtagcg gaaaacgatctggcttttttacagtacacctcgggttcgacgggctctccgaagggcgtgatggtgacccatggcaacc tgtgggccaactcgcatgccattcaccgctttttcggccatcacagcgaaagccggggcacgatctggctgccgcatttt catgatatggggctgattggcgggctactgcagcccgtgtttggcgcattcccctgtcgggtgatgtcgcccatgatgtt aatgaaaaatccccttaactggctcaaacatatttctgactatcaggcgacgacctccggcggccctaatttcgcctacga tctgtgcgtgcgcaagattggcagagagcaagttgaggcattagatctttctcgctgggatgtggctttttgcggcgcag agccgattcggcccgccacgctacagcagtttagcgaacactttgcgcccgcagggtttcggcccggcgcgttcctgc cctgctacggcatggcggaaaccacgctcatcgtcaccgggatggagaaaggacaagggctccgcgtttccgacga ggccggtacggtgagctgcgggcaggctctgccggataccgaggtgcgtatcgtcgatcccgatcgccatcagccgc ttgctgatggtgagagcggggaaatatggctgcgtggtccgagcgttgccgcaggatactgggacaatgacgccgcc acacgcgaaaccttccaggcgtctctggctggccatccgcacccgtggctgcgcagcggcgatatgggttttctccagt ccggccatttgtacgtcaccggacgactcaaagagctgctgattatcaacggtcagaaccactacccgacggatattga agagacgatccgccaggccgatcccgcgctggcggaagcgacggtttgcgtgtttgccagtgaagacgagcgcccg gttgcgctgcttgagctgatgacgcgccataaaaacgatctcgatatggcgacgctggcaccgtccgtgaccgcggcg gtggcggagcggcacggcatcacgctggatgaactgctccttgtcgggcgaagggcaattccccgcaccaccagcg ggaaactacagcgcacccgcgcgaaagcgatgcaccagcagggaaccctggaagtagcctggcgcagctgccag gacgcgtcgaaacctgttgaactcgcgggggaaaccccacccgcgctggcggcgctgatagccgggataatcagca gcgcgatgaacacgacgatcggcgaatcccagtgggacgaggcgtttaccggctttggcatgagctctctgcaggcg gtgggcgtgattggcgagcttgaacagcggctgggccgcgagctctctcccgcgctgatttatgactaccccaccatca atcggctggcggccgcgctggggcaacccgctgcggcccggccggtcagctcagccgtcgcggagagcgccattg cggtgattggcatcggcgtggagctgccgggacatagcggcgtggaggcgctgtggtcgctgctgcagcagggcca cagcacgaccggcgagatcccggcgcaccgctggcgtacctcgtcccttgacggttttaaccgtaaaggcagtttcttc gacgaggtcgacgcgttcgacgcaggctacttcggcatctctccccgtgaggccgtctatatcgatccgcagcatcgtc tgctgttagaaaccgttcaacaggcgctaaccgatgccggccttaaggcgtcctccctgcgcggtagcgatacggcgg

397 tctttgttgggatcagcgccagcgactacgcgctggcctgcggcgataacgtctcggcctacagcggcttaggcaacg cgcacagtatcgcggccaaccgaatttcttatctttatgatttaaaaggtccaagcgtcgccgtcgacacggcctgttcttc ctcgctggtggcgatagagggggcaatgcagagcctgcgggccggacgttgcgctctggccattgccggaggcgtt aatctggcgctgacgccacatttgcaaaaagtcttcaccgaagcccagatgctggcccccgacggccggtgtaaaacc ttcgacgcccgcgcggatggctatgttcgtggcgaagggtgtggcgtcgtggtgcttaagccgctttcacaggcgctg gcggatggcgatcgggtttatgccacgctggtggcgagcgccgtgaatcaggacggccgcagcaacggcattaccg cgccaaatggcccatcgcagcaggcggtcatcctgcaggcgatggcggacgccgggttggacagcgacagcattga ctatatcgaagcgcacggtacgggaaccgcgcttggcgatctgattgaatatcaggcgctggaagcggtgtttgcgga ccggaaaaagaccgcacctgtccaggtgggttcgatcaaaaccaacattggccaccttgaggcggcggcgggcgtg ctgggcgtggtgaaaacgtctctgatgctgcacttccggcaatacgtacctcacctcaattttcagcagaaaaacccgca tattgcggcgattagccgtcatgttgaggtgagcggcgcgcagcctgcctcatggcatgccgatggcgaagcgcgcta tgcgggcgtaagcagctttggcttcggcggtaccaacggtcatgtgattttgcgcagcgcgccagcggtggaaaaacg ccaggagcccgctgcgccgcacggcctgcttctggtcggttcacatgataaaggggcgtttacccttcagcgggaggc ggtcaaaaaagggttatcgacgtgccaggagagcgatattgccacctggtgtcggctggtgaacacccgctacgacg cggcccgctatcgcggcgtggcgtatggcgcggatcgctcccagctggcggaaagccttgcgcagctcaccgtctgc aaggtgggtaaagcccagccccaggtctggctcttcccggggcagggcacccagcaaatcggcatgggtgccgagc tgtatcaccatctgccgcactatcgcacccagtttgacgcgctggcgacgactattcagcagcgctatcagattgatatta cgcaggcgctgtttgcccgtgacgacagctggcagcgctgcgccagaacgtgtcagctctcattatttgcctgtagcta cgcgcttgctcagagcgtgatgcagttcggcccgcgtccggctgccgtaatggggcacagcctgggagagtactgcg cggcggttatcgctggctatctctcgctggacgacgggctggcaatggttcatcagcgcgcgctgttgatgtcagccct gacgcaggaaggggcgatggctgtcgtcttcagcggcgaagccgacgtccgtcagatgatttccccctggacgggc gacattgatattgccgcattcaacacgccgacattgaccaccatcgcaggcagtcgggcggcgattgacgcctgccttc aggccatatcttcaaaaggcggtcacgccagaaaaattaaaactgccagcgcatttcactcctcgatgatggatccgatc ctcggcgcctggcgcgagtggctggtcaacaacgtcaccttcacccgcgggacgatcccgttttacagcaacctgaac ggtgaggcgtgcgaccgcaccgacgccgactactggacccggcaaattcgccagcccgtgagtttccttcagggcgt gcaaaacgtgctggcacagggtgagttcacctttatcgatctgagcgcggacggttcgctgggcaaatttgtgaccgca actgaccgccgtcaccgggtgctggccgcaggcgaccggcgacatgagtacaaatcactgctgacgctgctgggtac gctgtggcagcaagggcacgacatcaactggagcgggctgtaccacgcgaccacgcgggaggcgctaaccctgcc cgccatccagttctgccgcaaacgttactggctggcgggtgagacgccagcgcagaccccatctgcaaaagaggacg ctatgtcaaatcaacaccatttagccgctgaaataaaagcgattattgccggttttcttgaggcggatcccgccgcgcttg acgactctctgccgttcctggaaatgggggcggactcgctggtgctgctggatgccatcaataccattaaagaccgcttt ggcgtagccatcccggtgcgggcgctgtttgaagagctcaatacgctggacgcggtgatcggatatgtggtggagca cgcgcagccggcggcttcgctcaccaccccggaaaccgccggcctggcggcacagcctgtcgcggcaccgcagg gtaccagcaggccggtgcctgatacggttcaggatctgattgcccgccagctggagctcatgtcccagcagctaaattt gcttaacggcacggcgcaggctctcccgatgccagccgcacccgcgacgccggacgttatcgcgcctgcgcccgtc gtggccccgaccgcaccggtgaaggccagcgcgcacaacagctggtttaaaaaagagaccaaaaaggtctccctcg gcgctgagcgcgaccagcatctggcgcaactcaccgaacggtttgtcgataaaacgggcgggtcaaaacgcaatgcc cagcaataccgcgccgtgctcgcggataaccgggcctctgcgggctttcgtttatccaccaaagagatgctttacccctt agtgggagagcgctctcagggttcgcgcatctgggatgtcgatggcaacgagtatattgattttacgatggggtttggcg ctaacctgctgggacatgcgccggactgcgtgcagcaggccgttgccgaccagctggcgcggggcatgcagattgg tccgcaaagcgccctggcgggcgaggtggcaacgcttatcagcgagctgaccggccagcagcgcgtggcattttgta actccggctccgaagcggtaatgagcgccgtgcgcctggcgcgggccgtgacggggaaaaataaagtcgcgctgttt agcggctcgtatcatggcgtgtttgacggcattctggggcgacagcaggggggagaaacccccgagcgcgcgacgc cgattgccgcaggcacgccgccatcgctggtggacgacctgctggtgctcgactacggcagcgaagagagcctggc gcttatcgcccgctatgccgccgagttggcggtggtgatcgtcgaaccggtgcagagccgttatcccgatcatcagcca cgcgactatctgcataccctgcgtgaactgacgacaacccacaacattgcgctgatgtttgacgaggtgatcaccggttt ccgtctggctgccggcggcgcgcaggcgtattacggcgttcaggcggatatcgcctcctacggaaagattgtcggcg gcggtatgccgatcggcgtgattgcaggctctgcgcgctttatggacagtatcgacggcggattctggcagtatggcga tgactcctggccgcaggctgaactgatcttctttgccggtacgttctccaagcatccgctgaccatggcggcaagcaaa gccgtgctggagtacatcaaaacgcatccggcgctgtatgacgacatcaaccaaaaaaccgcgcgtctggcgatgtcg ctaaatacctggttctcagcgaccggcacgccgattgagatcgtctcagccggcagcctgttccgtttcaaattcaacgg taactacgacatcctcttccaccacctgatgctgcgcggcattttcatctgggaagggcgtaactgttttgtctcggtcgcg cacgcggatgaggatatcgatcggtttattgcggcggtgaaagagagcgttaacgccatgcgcgtggacggcttctttg gggaggcgggattgcgccccgacacgcgttatgccgttgcagacagccagcagcgttttctgcagctggccgcgcag gatgaaagcgggctgttggccgggacgatcggcggcgtgatcgaaacgccgcgcaatgtagatggcgacatcatgc gcgccgcctggcaactgctgtgcgagcgccatgacgcgctgcgtatgcaattcaccgacgcgggtgaccttcaggtg

398

gccctggcgcccatcgtcgatatctgcgaggaaaacgctgcgcctgccgcctgtctcgctgaatttgctgcgcgtccttt tgatcttaccgccacgccgctggctcgcctgctgctggtccgccacgaggggaaaaccacgctcgccatctcggcaca tcatacggtggcggacggctggtcgttcatggtgatgctgcgcgagctgttgcatctttacgatgccctggcgtcgggta aacgtcctgacctggcggcagcagcaagctatctgcaggctatccgcgcgcagaacatcgtcagcgaggctgagcta cctgcgcgtcttgcggcgcttccagcgcgccgggtaacgccggaagttctgagcgtgcaagatccctccctgacgtat caggggcagcggctggtacagcgcctgtcgtatccgggcctgaccgggcagcttcgcaaagcctccgccgaactgc gggtgacccgcttcgcgatgctgaatgcgctctttaccctgacgcttgagaaggcgttcggccattccccggtgccggt cggcgtgccggatgccgggcgggacttcgggcagggggacgcgcttgtcgggcagtgcgtcaggctgttgccgcta tgtatcgacagcgctgcctgcgcgtcggtgtccggggtcgccagggcaatccatgacggtatcctcgcgcagcgcgg cgagcccgcactgccgtcacgctgcttccacggtcaggatgcgccgcttccgctgctggcgacctttaacgtcgagcc gcatgctccgctggctgagatgcgtcagtgggaagccagcctgtcgctgctcccgatcggcgccgtcgaattcccgct gatggttaacattctcgagacgaaagaggggctgtccgtcgagctggattaccagatgcgctatttcaccgaggaacga gcccgcgcgctactggagcacttcctgaaggcgataaccgtgctggccgagcagggagaagaggctgttgaagcatt attctcgtcgtcagaggcgctggccgcatcatga ewv-4B9::Tn5 atggtctggattgattacgccatcattgcggtgattggtttttcctgtctggttagcctgatccgtggctttgttcgtgaagcg ttatcgctggtgacttggggttgtgctttctttgtcgccagtcattactacacttacctgtctgtctggttcacgggctttgaag atgaactggtccgaaatggaatcgctatcgcggtgctgtttatcgcaacgctgattgtcggcgctatcgtgaattacgtga taggtcagctggtcgagaaaaccggtctgtcaggaacggacagggtactcgggatctgtttcggggcgttgcgaggc gtgctgattgtggccgcgatcctgttcttcctggatacctttaccgggttctccaaaagcgaagactggcagaaatcgca gctcattccagagttcagcttcatcatcagatggttctttgactatctgcaaagctcgtcgagttttttgcccagggcataa ewt-15A12::Tn5 atgtcatggcaatacttcaaacagacttacctggttaagttctggtcacctgtcccggccgttatcgcggcaggcattctct ctacctactatttcggcatcaccggcaccttctgggccgtgaccggcgagttcacccgctggggtggtcaattgctacag ctcgcaggcgtacatgccgaggagtggggttactttaaactcatccatctggacggcacaccgctcacccgcatcgac gggatgatgatcgtcggcatgttcggcggctgtttcgcggcggcgctgtgggcaaacaacgttaagttgcggatgccc aaaagccgcattcgtataatgcaggcggtgggcggcggtatcattgccgggtttggcgcccgtctggcgatgggctgt aacctggccgctttctttacggggattcctcagttttcgcttcacgcctggttctttgctgtcgccacggccatcggctctta cttcggtgcaaaattcaccctgctaccgctgttccgcattccggtgaaaatgacaaaagtcagcgcagcgtctccgttaa cccagaagccggaccaggcgcgtcgtcgtttccgcctcggtatgctggtctttttggctatggtcgcctgggcgctttgc actgcgatgaatcagcccaaactcggcctggcgatgctgttcggcgtgggttttggtctgctcattgaacgcgcgcagat ctgctttacctccgcgtttcgcgatatgtggatcaccggccgtacgatgatggcaaaggcgattatcgccgggatggcg gtcagcgccatcggcatcttcagctatgttcaactgggcgtcgaaccgaaaatcatgtgggctggccctaacgccgtga tcggcggcctgctgtttggcttcgggatcgtgctggctggcgggtgtgaaaccggctggatgtaccgcgccgtcgaag gccaggtacactactggtgggtgggtctgggaaatgttatcggctcgacgatcctggcgtacttctgggacgatctctcc ccggcgctggccacgagctgggataaggtcaacctgctgagcaccttcggccccctcggcggcctgctggtcaccta cgccctgctgctggtggcttttttactggttgtcgcacaggagaaacgcttcttccgccgcgcaagtgttaaaacagaaac ccaggagaatgctgcatga ews-1H3::Tn5 atgacgagccgtaaacctgcccatcttttactggtggatgacgatcccgggctgttaaagctgctggggatgcgtctggt gagtgaaggctacagcgtcgtgaccgccgaaagcgggctggaggggctgaagatcctcacccgcgagaaaatcgat ctggtgataagcgacctgcggatggacgaaatggatggcctgcagctgttcgcggagatccagaggcagcagccgg gtatgcccgtgatcattctgacggcgcacgggtcgatcccggatgcggttgccgcgacgcagcagggggtcttcagct tcctgaccaagccggtggacaaagacgcgctgtataaggctatcgacagcgcgctggaacatgccgccccggcggg ggatgaagcgtggcgggagtccatcgtcacccgcagccccattatgctgcgtctccttgaacaggcccggatggtggc gcagtccgacgtcagcgtgctcatcaacggccagagcggaaccgggaaagagatcctggcccaggcgatccataac gccagcccgcgcagtaaaaatgcctttatcgccattaactgcggcgcgcttccggaacagcttctcgaatctgaactgttt ggtcatgcccgcggcgcattcaccggcgcggtgagcagtcgggaagggctgttccaggcggcggaaggcggcac gctgttcctggacgagattggcgacatgcccgcgccgctgcaggtcaaactgctgcgcgtattgcaggagcgaaaagt tcgcccgctgggcagcaaccgcgatatcgatattaacgtgcgcattatttccgccacccaccgcgacttgccaaaagtg atggcccgcaacgagtttcgcgaagatctctactaccgtctgaacgtggtgaatctgaagatccctgcgctggcggagc gcgcggaagacattccgctgctggcgaatcatcttctgcgccaggcggccgatcgtcataaaccgtttgtgcgcgcgtt ttccaccgacgcgatgaagcggctgatggccgcaggctggccgggtaacgtgcgccagctggtgaacgtgattgag cagtgcgtggcgctgacctcctcaccggtaatcagcgatgcgcttgtagagcaggcgctggaaggagaaaacacggc gctgccgacgtttgcggaagcgcggaatcagttcgagctgaactatctgcgcaagctattgcagatcaccaaaggcaa

399

cgtgacccacgcggcgcgcatggccggacgcaaccgcaccgagttctacaagctgctgtcgcgccacgagctggaa gcaaacgattttaaagagtaa ewc-3H2::Tn5 atgaacaaaagaacattactcagtgttcttattgcgggcgcatgtgttgcaccgtttatggctcaggcaaccctgctgcag gccagcagcgaaccttacacccttaaggccagcgatctgcagaagaaagagcaggagttaaccaacttcccgctgat ggcttcggtgaagtcaaccatccgcacgctggacaacagcctggtggaacaaattgagcctggtaaatcgacaaaccc ggaaaacgttaagcgcgtggaagggattatcaaggccagcgactgggattatctcttcccactgcgtgcgccggaatat acgtacagcaacttcctgaaagcggtcggtaaattcccggcactgtgccagacctataccgatggtcgtaacagcgatg ccatctgccgtaagtctctggcaaccatgttcgcgcacttcgcgcaggaaactggcggccacgagtcctggcgtccgg aagccgagtggcgtcaggcgctggtttacgtgcgtgagatgggctggagcgaaggtcagaagggcggatataacgg cgaatgtaacccggatgtctggcaggggcagacctggccgtgcggtaaagacaaagatggcgatttcgttagctatttt ggccgcggtgcgaaacagctctcctataactacaactacggcccgttctctgaagcgatgtacggcgacgtccgcgta ctgctggacaaacctgagctggtggcggacacctggctgaaccttgcttccgcgatcttcttcttcgcctacccgcagcc gccaaaaccaagcatgctgcaggttatcgacggcacatggcagccgaacgatcacgataaggcgaatggtctggtac cgggcttcggagtgaccacgcagatcatcaacggcggcgtggagtgtggcggcccgactgagatcgcccagtctca aaaccgtatcaaatactacaaagagttcgccaactacctgaaagtgcctgttccgtctaacgaagtgctgggctgcgcca acatgaagcagttcgacgaaggcggtgctggcgcgctgaagatctactgggaacaggactggggatggagcgcgg ataccccgtcaggccagacctactcttgccagctggtgggttaccagacgccattcagcgcctttaaagagggtgacta caccaaatgcgtgaagcatttcttcaatgtgaacgtggtgggtgaagacgggacttccgacggcggtagcgtcacgcc agccccgacgccgacccctgtcgacccaacggacgaaggcaacaccacgccggtgccagacgataacaccccgg ctccggacgataatacgccagcgccggtaaaccatgcgccggtagcaaaaatcgccggtccggtgggtgcggttgaa gcgggtaaatcagtttctctgaacgcgtctggctcgaccgacgaagacggtaaccacctgacttatacctggacggctc cgaacggccagaccgtaagcggcgaagataaagcgattattaccttcaatgcgccggaagtggcagcggcgacgca gtacccgatcaaccttaccgtcagtgacggcgagctgagcagcaccaccagctatacgctgaacgtacaggctaagc agaccaacggcggccagaccgggacttacccgacctggacctccaaaaccaaatggaaagcaggcgatatcgttaa caaccgcggccagctgttccagtgcaaaccttatccgtacagcggctggtgcaataacgcgccatcctactacgagcc aggtaaagggattgcatggcaggacgcgtggactgcgctgtaa ewf-7D5::Tn5 atgatgaatcttttacactggcgcttgctggttgccgtggccgatgccagtaatatttcccgtgccgctgaagaggtcggt atgacgcagtcaggtgccagccaggcgatcgctcagttagaggcctcattaggatttgcggtattcacgcgagagcga cgccacatcggcgtgactgccctgggcgagcgggttgtcaaagaagcaaggagtatgctggcgcggctggatgcca tccgtgcactggcggatgagagccgggggctgagcggcggacgcattcgtcttgccagttttccttcggtgacatccac gtttttgcccgggctgctgcgcgacttaaagcgcctgcatcctgaaatcgaggtggtcgtcctcgaaggcacggacca ggaggtcgaagagtggctggcggcagatacggttgatctgggcgtggtcatgaaccctgttcccgggcgtgctgaag cgatcttagggcaggatgcatgggtcgtggtgttgccagccagtcaccgtcaggctcgttatacacgtacgcgcggcat cacccttgaggaactggcagaccagcccttcgtgctcgcaaccggcggttgcgccgtcaatggaaaaagcctgatgg aacaggctggcctgcagctgcctgacgtgcgggtgaccgtccgcgactggatcagcgcctgcctgctggtgcgcgaa ggcatgggcgtcgcgctgatacctgaatccgccctgccggatgaattacgcggtttgtgcgtcgtacccgtgacgcctt cgctgtgccgggaatttgggctggtatgttcgcaggtgggcaaatattcccgcgcgacgcaggcgttattagaagggtt gctcaaaatgaaaaggtctgcgtga ewg-10A8::Tn5 atgacccacaacatttctctcagaaatagggtgtgcgatgaacaaaagtttgtcatctcagcctgcggttttacgttccagg gatatcgtctgttccttaaccagtacggaatacaggcttcgcatattcattttgatggggatgaggcatcgcaacaggatat gaaaaatatccttataaatcagaatgctcatgttgtggtgtttctcggaaaaggtatcttaagtctcctggagagcctgaag cgactggcgtccgtgctcaatgcgttgcccgttattcgacgcgtcacgctgtatggcgacataccggatggctggctata tcgcaccctgggcagtcttttaaataatagttatcaattatcattgattcgactagcccgcgtttcggatgtcgtcactggttc tcatacgcaccatcatgtatttaaggaacgttcgtacttattacgcgatcgctacagggataattcttcgcaagacaacgtc aagtggctaacaaaaagagagattgacgttttattaaatttctaccgcggcatgtccgtaaaagaaatgtgcgatgaaatg ggactatctaataaaacggtttatacccaccgtaaggaaggcgtgctgaaattacgcttaattaagcggtggctacacga ttcgcacaatatcaatgcggaaagaagtatcaagcggcggagtcaaaacacggaatttacggataaggaagcagagat ttttaatgcattatataaaaaagagatcttccctgcttatcagatcattaccgatcgtgacaaaaaaggcgtaggctttgaga tactgatccgctggaacaaaaacggtaaaattgtcaagccgaccagttttctcacggatatttcaaatcatgagatatggtt gaaaattaccgcgctagttattcatgccgccgtgtcgggaattaataagtataatggtaaatactatttttctgtgaatattcc acctcgcctggcctccggaaatgcattgcctgatatggcaaggaaagctatcgacatgctgctcaaaccgcagtgggc cgagaagctggtctttgaattcgcagaagatattgacgtgacgaaggacaaagggatcccagaaaccatgcggcattt

400

gcgtaatacagggtgtcgattgtttctggatgactgcttctctaatcaccaaaccatgttcccggtgcggcaggtgcatttt gatggactcaagctggatcgggatatcgttgagcattttgtggcaaacgacaatgactataacctgatcaaagcgataca gatttatagcgacatgaccggaacggactgtattgcagagggagtagacagtgaggaaaaatttgaaaaattagtcgcg ctgggcgtcaaaaactttcagggatattatttatcgcgagccgtgaaagaggacgagttggatcgcatggtcagaattttt agttaa ewh-5B1::Tn5 Atgacacttcaacaggcactccgcaaacccaccacgccgggcgacgtgttgcagtatgagtatcttgaaccgctcaat ctgaaaatcagcgatctggcggagatgctcaatgttcaccgcaacaccatcagcgcgctggtcaataacaatcgcaaa cttactgccgatatggcgatcaaactggcaaaagcctttgataccacgattgaattttggctgaacttacagctgaacgtc gatatctgggaagcgcaatctaactccaggactcaggaggagctaagccgtataaagaccgttgcggaagttatggct aagcgaaaatctggccagccggatgttgcctga ewa-8E1::Tn5 gtgatccgcaaagagaaagggcgcgtggatgtctatggcgaccgctttcgcgcacgtgcgcatcagctgacgccgcg tctgctgcaggttgcacgctatatccatgacaaccgcgaagcggtgatggaacagaccgcaatggagattgcgacgct gttgaaaacgtcggatgccacggtggtccgcgccattcaggcgctgggtttcgccgggctgcgcgatcttaagcaaac gctggagcagtggtttggtccggtggtcacctccagcgaaaagatgtccactacggtgaatacgctcgccagcgacgt caacgcgagcattgatttcgtgctggaggggcatcagcacacctgcgaggtgttatccgagccccacaaccgctacgc aatggcccaggcggtctctctgctggcgcaggcgaggcaggtcgccatatttggcattggcgcctccggcattctggc cgagtataccgccaggctgttcagccgtatggggttacccgccacgccgcttaaccgtaccggaatagggcttgccga gcagctgattgcgcttcagcgcggcgacgtgctggtgatgatggcgcagaaatccgcgcaccgggaggggcaaacc acgctgcgtgaagcaaaacggctgggcattcccaccatcctgctgaccaacgccctcgattcgcgtttcagcaaggag gccagcgtggtgatccacgttccgcgcggcggcgaaaaaggcaaaattccgctccacggcacggtgctgctgtgtct ggagatgatcattttatccgtcgcctccaccgagccgcagcgcaccatcaagtcgatgaagcgcatcaacgagttacac cgtgggttaaagccggggaagaaaagtacctaa

401

Appendix Figure A7.1: Image of Finger Millet Seedlings Previously Seed-coated with GFP-tagged M6 Showing No Pathogenic Symptoms, Consistent with the Strain Behaving as an Endophyte

402

Appendix Figure A7.2: Suppression of F. graminearum (Fg) by M6 and its Effect on Grain Yield in Greenhouse Trials. (A-B) Effect of endophyte M6 on grain yield per plant in two greenhouse trials for (A) maize and (B) wheat. (C) Effect of treatment with endophyte M6 on Fg disease symptoms in maize ears when the endophyte was applied as a seed coat or foliar spray compared to a Fusarium only control treatment. Letters that are different from one another within a trial indicate that their means are statistically different (P≤0.05). The whiskers indicate the range of data.

403

Appendix Figure A7.3: Assay for Production of Indole-3-acetic acid (IAA, auxin) by Wild Type Strain M6. Production of indole-3-acetic acid (IAA) in vitro by wild type strain M6 compared to a positive control (bacterial endophyte strain E10) (Shehata, 2016) using the Salkowski reagent colorimetric assay.

404

Appendix Figure A7.4

405

Appendix Figure A7.4: Tn5 Mutagenesis-Mediated Discovery, Validation and Complementation of Genes Required for the Anti- Fusarium Activity of Strain M6. (A) Loss of anti- F.graminearum activity associated with each Tn5 insertion mutant using the in vitro dual culture diffusion assay, along with a representative image (inset) of the mutant screen. (B- C) In planta validation of loss of anti-fungal activity of M6 mutant strains based on quantification of F. graminearum disease symptoms on maize ears, in (B) greenhouse trial 1, and (C) greenhouse trial 2. Only mutant strains that completely lost anti-fungal activity in vitro were selected for in planta validation. The whiskers indicate the range of data points. Letters that are different from one another indicate that their means are statistically different (P≤0.05). (D) Genetic complementation of Tn5 mutants with the predicted, corresponding wild type coding sequences. Shown are representative images.

406

Appendix Figure A7.5

407

Appendix Figure A7.5: Model to Illustrate the Interaction Between Strain M6, the Host Plant and F. graminearum pathogen. Following pathogen sensing, M6 swarm towards Fusarium hyphae and induces local hair growth, perhaps mediated by M6-IAA production. M6 then forms microcolony stacks between the elongated and bent root hairs resulting root hair-endophyte stack (RHESt) formation, likely mediated by biofilms. The RHEST formation prevents entry and/or traps F. graminearum for subsequent killing. M6 killing requires diverse antimicrobial compounds including phenazines. Fusarium will produce fusaric acid which interferes with phenazine biosynthesis. However, M6 has a specialized fusaric acid-resistance pump system which is predicted to pump the mycotoxin outside the endophyte cell. This illustration was created by Lisa Smith.

408

Appendix Figure A7.6: Assays for Production of Butanediol and Chitinase by Strain M6. (A) Entire GC chromatogram showing an arrow pointing to a peak with RT 11.13 with a molecular weight and fragmentation pattern (inset) that matches 2, 3 butanediol when searched against the NIST 2008 database. (B) Quantification of chitinase production by an M6 mutant strain carrying a Tn5 insertion in a chitinase encoding gene (ewc-3H2::Tn5) compared to wild type M6 (see Appendix Table A7.6).

409

Appendix Table A8.1: Taxonomic classification of maize bacterial endophytes based on 16S rDNA and 23S rDNA sequences and BLAST analysis.

ID 16S rRNA sequence 23S rRNA Sequence 1D6 GTTAGGGGTNTCGATACCCTNGNNNNNNAAGTT AGCCTTGGCAGANGGTCCTGCCGGATTCA AACACATTAAGCATTCCGCCTGGGGAGTACGGT TACGGGGTTTCACGTGCCCCGCACTACTC CGCAAGACTGAAACTCAAAGGAATTGACGGGG GGGATCCGTCTCGGAGGGAACANGTTTTG ACCCGCACAAGCAGTGGAGTATGTGGTTTAATT AACTACAGGGCTTTTACCTTCTCTGGCGG CGAAGCAACGCGAAGAACCTTACCAGGTCTTGA GCCTTTCCAGACCTCTTCATCTAACCGGTT CATCCCTNTGACCGGTCTAGAGATAGANCTTTCC CCTTTGTAACTCCATGTGAGACGTCCCAC TTCGGGACAGAGGAGACAGGTGGTGCATGGTTG AACCCCAAGGAGCAAGCTCCTTGGTTTGG TCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGT GCTAATCCGCGTTCGCTCGCCGCTACTGA CCCGCAACGAGCGCAACCCTTATGCTTAGTTGC CGGAATCACTATTGTTTTCTCTTCCTCAGG CAGCAGGTCAAGCTGGGCACTCTAAGCAGACTG GTACTTAGATGTTTCAGTTCCCCTGGTCTG CCGGTGACAAACCGGAGGAAGGTGGGGATGAC CCTCTCACTCACCTATGAATTCAGTGAGT GTCAAATCATCATGCCCCTTATGACCTGGGCTAC AGTGACTGTCGATGAAGACAGCCGGGTTC ACACGTACTACAATGGCCGGTACAACGGGAAGC CCCCGTTC GAANNNNNNANNNGGAGCCAATCCTAGAAAAG CCGGTCTCAGTTCGGATTGTAGGCTGCAACTCGC CTACATGAAGTCGGAATTGCTAGTAATCGCGGA TCAGCATGCCGCGGTGAATACGTTCCCGGGTCTT GTACACACCGCCCGTCACACCACGAGAGTTTAC AACACCCGAAGTCGGTGAGGTAACCGCAAGGN NCCAGCCGCCGAAGGTGGGGTAGATGATTGGGG TGAAGTCGTAACNAGGTANNNTTGNCCTAAATC TATGGGACCATCAGGTGCGGCATTTGCTACTTAC GATCCACAATCCCCAAAGCTATGGGAACCACGG CTTCAATTGTGTAATTCGTAAGGCGGACCCCTCA TGCCAGCGTTCTGACTTTGAGTTTCCTTAACTGC CCCTGGGCGTGTTCGTCACCTCATACACCAAATT AAGCTTCGTTGCGCTTCTTGGAATGGTCCAGAAC TGTAGGGAAACTGGCCAGATCTCTANNNNNAAA GGAAGCCCTGTCTCCTCTGTCCACCACGTACCAA CAGCAGTCGAGCACAGCACTCTACAACCCAATT CAGGGCGTTGCTCGCGTTGGGAATACGAATCAA CGGTCGTCCAGTTCGACCCGTGAGATTCGTCTGA CGGCCACTGTTTGGCCTCCTTCCACCCCTACTGC AGTTTAGTAGTACGGGGAATACTGGACCCGATG TGTGCATGATGTTACCGGCCATGTTGCCCTTCGC TTCCTCGCTAGACCTCGGTTAGGATCTTTTCGGC CAGAGTCAAGCCTAACATCCGACGTTGAGCGGA TGTACTTCAGCCTTAACGATCATTAGCGCCTAGT CGTACGGCGCCACTTATGCAAGGGCCCAGAACA TGTGTGGCGGGCAGTGTGGTGCTCTCAAATGTT GTGGGCTTCAGCCACTCCATTGGCGTTCNNNGG TCG 3H9 GTGNNAGGGNNNNNNATACCCTTGGNTGCCGNA ACTCNCTGNTTCNTAGGTGAGTGAGAGGC AGTTAACACATTAAGCATTCCGCCTGGGGAGTA AGACCAGGGGAACTGAAACATCTAAGTA CGGTCGCAAGACTGAAACTCAAAGGAATTGACG CCCTGAGGAAGAGAAAACAATAGTGATT GGGACCCGCACAAGCAGTGGAGTATGTGGTTTA CCGTCAGTAGCGGCGAGCGAACGCGGAT ATTCGAAGCAACGCGAAGAACCTTACCAGGTCT TAGCCCAAACCAAGGAGCTTGCTCCTTGG TGACATCCCTNTGACCGGTCTAGAGATAGANCT GGTTGTGGGACGTCTCACATGGAGTTACA TTCCTTCGGGACAGAGGAGACAGGTGGTGCATG AAGGAACCGGTTAGATGAAGAGGTCTGG GTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTT AAAGGCCCGCCAGAGAAGGTAAAAGCCC AAGTCCCGCAACGAGCGCAACCCTTATGCTTAG TGTAGTTCAAAACTTGTTCCCTCCGAGAC

410

TTGCCAGCAGGTCAAGCTGGGCACTCTAAGCAG GGATCCCGAGTAGTGCGGGGCACGTGAA ACTGCCGGTGACAAACCGGAGGAAGGTGGGGA ACCCCGTATGAATCCGGCAGGACCATCTG TGACGTCAAATCATCATGCCCCTTATGACCTGGG CCAAGGCTAAATACTCCCTGGCGACCGAT CTACACACGTACTACAATGGCCGGTACAACGGG AGTGAAGCANTACCGTGAGGGAAAGGCG AAGCGAANGAGCNANNNGGAGCCAATCCTAGA AAAGCCGGTCTCAGTTCGGATTGTAGGCTGCAA CTCGCCTACATGAAGTCGGAATTGCTAGTAATC GCGGATCAGCATGCCGCGGTGAATACGTTCCCG GGTCTTGTACACACCGCCCGTCACACCACGAGA GTTTACAACACCCGAAGTCGGTGAGGTAACCGC AAGGNGCCAGCCGCCGAAGGTGGGGTAGATGA TTGGGGTGAAGTCGTAACTATGGGACCATCAGG TGCGGCATTTGCTACTTACGATCCACAATCCCCA AAGCTATGGGAACCACGGCTTCAATTGTGTAAT TCGTAAGGCGGACCCCTCATGCCAGCGTTCTGA CTTTGAGTTTCCTTAACTGCCCCTGGGCGTGTTC GTCACCTCATACACCAAATTAAGCTTCGTTGCGC TTCTTGGAATGGTCCAGAACTGTAGGGAAACTG GCCAGATCTCTANNNNGAAAGGAAGCCCTGTCT CCTCTGTCCACCACGTACCAACAGCAGTCGAGC ACAGCACTCTACAACCCAATTCAGGGCGTTGCT CGCGTTGGGAATACGAATCAACGGTCGTCCAGT TCGACCCGTGAGATTCGTCTGACGGCCACTGTTT GGCCTCCTTCCACCCCTACTGCAGTTTAGTAGTA CGGGGAATACTGGACCCGATGTGTGCAAGTGTG GTGCTCTCAAATGTTGTGGGCTTCAGCCACTCCA TTGGCGTTNNNGGTCGTGTTACCGGCCATGTTGC CCTTCGCTTCCTCNNNNNNCCTCGGTTAGGATCT TTTCGGCCAGAGTCAAGCCTAACATCCGACGTT GAGCGGATGTACTTCAGCCTTAACGATCATTAG CGCCTAGTCGTACGGCGCCACTTATGCAAGGGC CCAGAACATGTGTGGCGGGC

4G12 TGTGCCNTTGAGGCGTGGCTTCCGGAGCTAACG AGGTTAACGAGGCGAACCGGGGGAACTG CGTTAAGTCGACCGCCTGGGGAGTACGGCCGCA AAACATCTAAGTACCCCGAGGAAAAGAA AGGTTAAAACTCAAATGAATTGACGGGGGCCCG ATCAACCGAGATTCCCCCAGTAGCGGCGA CACAAGCGGTGGAGCATGTGGTTTAATTCGATG GCGAACGGGGAGCAGCCCAGAGTCTGAA CAACGCGAAGAACCTTACCTACTCTTGACATCC TCAGCTTGTGTGTTAGTGGAAGCGTCTGG AGAGAACTTANCAGAGATGNNTTGGTGCCTTCG AAAGTCGCACGGTACAGGGTGACAGTCC GGAACTCTGAGACAGGTGCTGCATGGCTGTCGT CGTACACGAAAGCACACAGGCTGTGAAC CAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCG TCGAAGAGTAGGGCGGGACACGTGGTAT CAACGAGCGCAACCCTTATCCTTTGTTGCCAGCG CCTGTCTGAATATGGGGGGACCATCCTCC GTNNGGCCGGGAACTCAAAGGAGACTGCCAGTG AAGGCTAAATACTCCTGACTGACCGATAG ATAAACTGGAGGAAGGTGGGGATGACGTCAAGT TGAACCAGTACCGTGAGGGAAAGGCGAA CATCATGGCCCTTACGAGTAGGGCTACACACGT GCTACAATGGCGCATACAAAGAGAAGCGACCTC GCGAGAGCAAGCGGACCTCATAAAGTGCGTCGT AGTCCGGATTGGAGTCTGCAACTCGACTCCATG AAGTCGGAATCGCTAGTAATCGTAGATCAGAAT GCTACGGTGAATACGTTCCCGGGCCTTGTACAC ACCGCCCGTCACACCATGGGAGTGGGTTGCAAA AGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCT TACCACTTTGTGATTCATGACTGGGGTGAAGTCG TAACNAAGGTAAGGGGCGTTGCTCGCGTTGGGA ATAGGAAAACAANCGGTCGNCANNCCGGCCCTT GAGTTTCCTCTGACGGTCACTATTTGACCTCCTT

411

CCACCCCTACTGCAGTTCAGTAGTACCGGGAAT GCTCATCCCGATGTGTGCACGATGTTACCGCGTA TGTTTCTCTTCGCTGGAGCGCTCTCGTTCGCCTG GAGTATTTCACGCAGCATCAGGCCTAACCTCAG ACGTTGAGCTGAGGTACTTCAGCCTTAGCGATC ATTAGCATCTAGTCTTACGATGCCACTTATGCAA GGGCCCGGAACATGTGTGGCGGGCAGTGTGGTA CCCTCACCCAACGTTTTCTTCATCCATCGAATTG GAAGCCCTCCCGCNNATGGTG 4G4 GGGTTTCGATACCCTTGGTGCCGAAGTTAACAC TAGGTGAGTGAGAGGCAGACCAGGGGAA ATTAAGCATTCCGCCTGGGGAGTACGGTCGCAA CTGAAACATCTAAGTACCCTGAGGAAGA GACTGAAACTCAAAGGAATTGACGGGGACCCGC GAAAACAATAGTGATTCCGTCAGTAGCGG ACAAGCAGTGGAGTATGTGGTTTAATTCGAAGC CGAGCGAACGCGGATTAGCCCAAACCAA AACGCGAAGAACCTTACCAGGTCTTGACATCCC GGAGCTTGCTCCTTGGGGTTGTGGGACGT TNTGACCGGTCTAGAGATAGANCTTTCCTTCGG CTCACATGGAGTTACAAAGGAACCGGTTA GACAGAGGAGACAGGTGGTGCATGGTTGTCGTC GATGAAGAGGTCTGGAAAGGCCCGCCAG AGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCG AGAAGGTAAAAGCCCTGTAGTTCAAAAC CAACGAGCGCAACCCTTATGCTTAGTTGCCAGC NTGTTCCCTCCGAGACGGATCCCGAGTAG AGGTCAAGCTGGGCACTCTAAGCAGACTGCCGG TGCGGGGCACGTGAAACCCCGTATGAATC TGACAAACCGGAGGAAGGTGGGGATGACGTCA CGGCAGGACCATCTGCCAAGGCTAAATAC AATCATCATGCCCCTTATGACCTGGGCTACACAC TCCCTGGCGACCGATAGTGAAGCA GTACTACAATGGCCGGTACAACGGGAAGCGAAG GAGCNANNTGGAGCCAATCCTAGAAAAGCCGGT CTCAGTTCGGATTGTAGGCTGCAACTCGCCTACA TGAAGTCGGAATTGCTAGTAATCGCGGATCAGC ATGCCGCGGTGAATACGTTCCCGGGTCTTGTAC ACACCGCCCGTCACACCACGAGAGTTTACAACA CCCGAAGTCGGTGAGGTAACCGCAAGGAGCCAG CCGCCGAAGGTGGGGTAGATGATTGGGGTGAAG TCGTAACGGGACCATCAGGTGCGGCATTTGCTA CTTACGATCCACAATCCCCAAAGCTATGGGAAC CACGGCTTCAATTGTGTAATTCGTAAGGCGGAC CCCTCATGCCAGCGTTCTGACTTTGAGTTTCCTT AACTGCCCCTGGGCGTGTTCGTCACCTCATACAC CAAATTAAGCTTCGTTGCGCTTCTTGGAATGGTC CAGAACTGTAGGGANACTGGCCAGATCTCTANN NNNAAAGGAAGCCCTGTCTCCTCTGTCCACCAC GTACCAACAGCAGTCGAGCACAGCACTCTACAA CCCAATTCAGGGCGTTGCTCGCGTTGGGAATAC GAATCAACGGTCGTCCAGTTCGACCCGTGAGAT TCGTCTGACGGCCACTGTTTGGCCTCCTTCCACC CCTACTGCAGTTTAGTAGTACGGGGAATACTGG ACCCGATGTGTGCATTTACCGGCCATGTTGCCCT TCGCTTCCTCGCTAGACCTCGGTTAGGATCTTTT CGGCCAGAGTCAAGCCTAACATCCGACGTTGAG CGGATGTACTTCAGCCTTAACGATCATTAGCGCC TAGTCGTACGGCGCCACTTATGCAAGGGCCCAG AACATGTGTGGCGGGCAGTGTGGTGCTCTCAAA TGTTGTGGGCTTCAGCCACTCCATTGGCGTTCNN NGG

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Appendix Table A8.2: Sequences of fusA orthologs from Paenibacillus endophytic species identified in this study.

Strain ID Sequence 1D6 TCCGGCCCGGCAGAAGCGATCAATATAGCGCTGCAAGATGGGGGCTTAAC ACCGCAGGAGATTGACTATGTCAATGCCCACGGTACGAGTACGCCATTTAA TGATCGCATGGAGATTAATGCTATTCAAAAAGTATTTGGAGAGCATGCACC CCGGTTGGCAATATCGTCTATTAAATCCATGACAGGCCATTNAATGGGCGG TGCTGGGGCAGTCGAATTAATTGCAACGGTACAGANCATGATCCACANCA AAGTACCACCGACNCTCAATTGTGACAATCCCGAAGCCCCTGAACTGAATT TTGTACCGC

3H9 GGTAGCCCGGCCACTTCCGAGAATATAACCACGCCGACGCTTGACCACTTC AAAATAGAGATCTATTCGGTCCCCAGGCTTCGCAAGGCTATGAAAGCGAAC GCCATTGAGGTTGGTTAGGTATGAAATACCTCTTTTGTCAATCGCAGCGAA TGCCGCTAATTGTGCAAGCGACTCCACGATCAGCATAGGAGGCATGTATGG ATTAGCTTCTGTAATAAACCATTCGCTTGAGGTAACAAGCTTGTACCCCTTT GCCCATTGCGACGTTTCGTACCCGGTCACACCATCGAGCAGAAGAAATGGG TACCTGTGCGGAAGCACATC

4G4 GTGTCCTCCAAAACCAAAAGAATTACTAATCGCCGCACGGACTTCACGTTC CTGATATACATGCGGTACAAAATTCAGTTCAGGGGCTTCGGGATTGTCACA ATTGAGTGTCGGTGGTACTTTGCTGTGGATCATGCTCTGTACCGTTGCAATT AATTCGACTGCCCCAGCACCGCCCATTAAATGGCCTGTCATGGATTTAATA GACGATATTGCCAACCGGGGTGCATGCTCTCCAAATACTTTTTGAATAGCA TTAATCTCCATGCGATCATTAAATGGCGTACTCGTACCGTGGGCATTGACA TAGTCAATCTCCTGCGGTGTTAAGCCCCCATCTTGCAGCGCTATATTGATCG CTTCTGCCGGGCCGGAGCCCGAGGGATCTGG

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Appendix Table A8.3: Origin of the endophytes screened in this study including the maize genotypes and tissues from which they were isolated, and the best BLAST matches to the GenBank database.

Maize host class Maize host Tissue Plate ID GenBank best BLAST match Percent of identity

Modern B73 Seed 3A1 Pantoea sp. 99

Modern B73 Seed 3A2 Enterobacter sp. 99

Modern B73 Seed 3A3 Rhodococcus sp. 95

Modern B73 Seed 3A4 Pseudomonas sp. 99

Traditional Bolita Seed 3A5 Pantoea sp. 99

Traditional Bolita Seed 3A7 Enterobacter sp. 99

Traditional Bolita Seed 3A8 Enterobacter sp. 99

Traditional Chapalote Seed 3A9 Enterobacter sp. 98

Traditional Chapalote Seed 3A10 Hafnia sp. 100

Traditional Chapalote Seed 3A11 Escherichia sp. 90

Traditional Chapalote Seed 3A12 Burkholderia sp. 98

Traditional Chapalote Seed 3B1 Enterobacter sp. 99

Traditional Chapalote Seed 3B2 Methylobacterium sp. 99

Traditional Chapalote Seed 3B3 Micrococcus sp. 99

Traditional Chapalote Seed 3B4 Pantoea sp. 9

Traditional Chapalote Seed 3B5 Enterobacter sp. 99

Traditional Chapalote Seed 3B6 Pseudomonas sp. 99

Traditional Chapalote Seed 3B7 Enterobacter sp. 99

Traditional Chapalote Seed 3B8 Methylobacterium sp. 100

Traditional Cristalino Seed 3B9 Escherichia sp. 99

Traditional Cristalino Seed 3B10 Staphylococcus sp. 99

Traditional Cristalino Seed 3B11 Pseudomonas sp. 97

Traditional Cristalino Seed 3B12 Pseudomonas sp. 97

Wild Diploperennis Seed 3C1 Enterobacter sp. 84

Wild Diploperennis Seed 3C2 Pseudomonas sp. 100

Wild Diploperennis Seed 3C3 Pseudomonas sp. 100

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Wild Diploperennis Seed 3C4 Pantoea sp. 98

Wild Diploperennis Seed 3C5 Pantoea sp. 98

Wild Diploperennis Seed 3C6 Pantoea sp. 99

Wild Diploperennis Seed 3C7 (1D6) Paenibacillus sp. 97

Wild Diploperennis Seed 3C8 Pseudomonas sp. 99

Wild Diploperennis Seed 3C9 Pseudomonas sp. 99

Wild Diploperennis Seed 3C10 Enterobacter sp. 98

Wild Diploperennis Seed 3C11 Burkholderia sp. 99

Wild Diploperennis Seed 3C12 Enterobacter sp. 100

Wild Diploperennis Seed 3D1 Enterobacter sp. 99

Traditional Gaspe Flint Seed 3D2 Pantoea sp. 99

Traditional Gaspe Flint Seed 3D3 Pseudomonas sp. 99

Modern B73 Seed 3D4 Pantoea sp. 99

Traditional Gaspe Flint Seed 3D5 Arthrobacter sp. 100

Traditional Gaspe Flint Seed 3D6 Cellulomonas sp. 98

Traditional Jala Seed 3D7 Pantoea sp. 99

Wild Nicaraguensis Seed 3D8 Arthrobacter sp. 99

Wild Nicaraguensis Seed 3D9 Enterobacter sp. 93

Wild Nicaraguensis Seed 3D10 Enterobacter sp. 100

Wild Nicaraguensis Seed 3D11 Pantoea sp. 99

Wild Nicaraguensis Seed 3D12 Enterobacter sp. 99

Wild Nicaraguensis Seed 3 E1 Enterobacter sp. 97

Wild Nicaraguensis Seed 3 E2 Stenotrophomonas sp. 97

Wild Nicaraguensis Seed 3 E3 Stenotrophomonas sp. 100

Wild Nicaraguensis Seed 3 E4 Klebsiella sp. 100

Wild Nicaraguensis Seed 3 E5 Citrobacter sp. 100

Wild Nicaraguensis Seed 3 E6 Pantoea sp. 100

Wild Nicaraguensis Seed 3 E7 Paenibacillus sp. 99

Wild Nicaraguensis Seed 3 E8 Cellulomonas sp. 100

Wild Nicaraguensis Seed 3 E9 Microbacterium sp. 98

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Wild Nicaraguensis Seed 3 E10 Paenibacillus sp. 98

Wild Nicaraguensis Seed 3 E11 Pantoea sp. 100

Wild Mexicana Seed 3 E12 Pantoea sp. 99

Wild Mexicana Seed 3F1 Pseudomonas sp. 99

Wild Mexicana Seed 3F2 Pseudomonas sp. 98

Wild Mexicana Seed 3F3 Pantoea sp. 99

Wild Mexicana Seed 3F4 Pseudomonas sp. 86

Wild Mexicana Seed 3F5 Pseudomonas sp. 99

Wild Mexicana Seed 3F6 Pseudomonas sp. 86

Wild Mexicana Seed 3F7 Pantoea sp. 100

Wild Mexicana Seed 3F8 Stenotrophomonas sp. 100

Traditional Mixteco Seed 3F9 Burkholderia sp. 100

Traditional Mixteco Seed 3F10 Burkholderia sp. 100

Wild Nicaraguensis Seed 3F11 Enterobacter sp. 99

Traditional Nal-Tel Seed 3F12 Streptomyces sp. 100

Traditional Nal-Tel Seed 3G1 Methylobacterium sp. 100

Traditional Nal-Tel Seed 3G2 Paenibacillus sp. 98

Traditional Nal-Tel Seed 3G3 Stenotrophomonas sp. 100

Wild Parviglumis Seed 3G4 Pantoea sp. 99

Wild Parviglumis Seed 3G5 Luteibacter sp. 99

Wild Parviglumis Seed 3G6 Stenotrophomonas sp. 100

Wild Parviglumis Seed 3G7 Pantoea sp. 99

Wild Parviglumis Seed 3G8 Klebsiella sp. 100

Wild Nicaraguensis Seed 3G9 Paenibacillus sp. 100

Wild Parviglumis Seed 3G10 Klebsiella sp. 100

Wild Parviglumis Seed 3G11 Enterobacter sp. 99

Modern Pioneer 3751 Seed 3H1 Bacillus sp. 98

Modern Pioneer 3751 Seed 3H2 Deinococcus sp. 99

Modern Pioneer 3751 Seed 3H3 Deinococcus sp. 99

Modern Pioneer 3751 Seed 3H4 Rhodococcus sp. 99

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Modern Pioneer 3751 Seed 3H5 Microbacterium sp. 99

Modern Pioneer 3751 Seed 3H8 Bacillus sp. 100

Modern Pioneer 3751 Seed 3H9 Paenibacillus sp. 98

Traditional Tuxpeno Seed 3H10 Bradyrhizobium sp. 100

Modern Pioneer 3751 Seed 3H11 Rhodococcus sp. 99

Traditional Mixteco Shoot 4A1 Microbacterium sp. 84

Traditional Mixteco Shoot 4A2 Micrococcus sp. 90

Traditional Mixteco Shoot 4A3 Pantoea sp. 97

Traditional Mixteco Shoot 4A4 Staphylococcus sp. 98

Traditional Mixteco Shoot 4A5 Pantoea sp. 100

Traditional Mixteco Shoot 4A6 Paenibacillus sp. 98

Traditional Mixteco Shoot 4A7 Methylobacterium sp. 97

Traditional Mixteco Shoot 4A8 Pantoea sp. 93

Traditional Mixteco Shoot 4A9 Stenotrophomonas sp. 100

Traditional Mixteco Root 4A10 Bacillus sp. 84

Traditional Mixteco Root 4A11 Methylobacterium sp. 97

Traditional Mixteco Root 4A12 Pantoea sp. 100

Traditional Mixteco Root 4B1 Paenibacillus sp. 97

Traditional Mixteco Root 4B2 Staphylococcus sp. 100

Traditional Mixteco Root 4B3 Enterobacter sp. 94

Traditional Mixteco Root 4B4 Flexibacter sp. 92

Traditional Mixteco Root 4B5 Microbacterium sp. 92

Traditional Mixteco Root 4B6 Enterobacter sp. 97

Traditional Mixteco Root 4B7 Stenotrophomonas sp. 93

Traditional Mixteco Root 4B8 Enterobacter sp. 100

Traditional Mixteco Shoot 4B9 Stenotrophomonas sp. 98

Traditional Mixteco Shoot 4B10 Paenibacillussp. 98

Traditional Mixteco Shoot 4B11 Microbacterium sp. 98

Traditional Mixteco Root 4B12 Stenotrophomonas sp. 99

Traditional Mixteco Root 4C2 Pseudomonas sp. 100

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Traditional Mixteco Shoot 4C3 Micrococcus sp. 77

Traditional Mixteco Root 4C4 Pedobacter sp. 97

Traditional Mixteco Root 4C5 Enterobacter sp. 98

Traditional Mixteco Root 4C6 Enterobacter sp. 99

Traditional Mixteco Root 4C7 Agrobacterium sp. 95

Traditional Mixteco Root 4C8 Stenotrophomonas sp. 99

Traditional Mixteco Root 4C9 Xanthomonas sp. 99

Traditional Mixteco Root 4C10 Sphingomonas sp. 98

Traditional Mixteco Root 4C11 Lysobacter sp. 98

Traditional Mixteco Root 4C12 Enterobacter sp. 97

Traditional Mixteco Root 4D1 Bacillus sp. 99

Traditional Mixteco Root 4D2 Pseudomonas sp. 100

Traditional Mixteco Root 4D3 Rhizobium sp. 83

Traditional Mixteco Root 4D4 Paenibacillus sp. 91

Traditional Mixteco Root 4D5 Lysobacter sp. 95

Wild Parviglumis Shoot 4D6 Rhizobium sp. 96

Wild Parviglumis Shoot 4D7 Xanthomonas sp. 98

Wild Parviglumis Shoot 4D8 Stenotrophomonas sp. 100

Wild Parviglumis Root 4D9 Xanthomonas sp. 96

Wild Parviglumis Root 4D10 Pantoea sp. 97

Wild Parviglumis Root 4D11 Pantoea sp. 99

Wild Parviglumis Root 4D12 Xanthomonas sp. 98

Wild Parviglumis Root 4 E1 Klebsiella sp. 99

Wild Parviglumis Root 4 E2 Herbaspirillum sp. 99

Wild Parviglumis Root 4 E3 Pantoea sp. 99

Wild Parviglumis Root 4 E4 Enterobacter sp. 99

Wild Parviglumis Root 4 E5 Delftia sp. 100

Wild Parviglumis Root 4 E6 Microbacterium sp. 98

Wild Parviglumis Root 4 E7 Enterobacter sp. 98

Wild Parviglumis Root 4 E8 Pantoea sp. 98

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Wild Parviglumis Root 4 E9 Xanthomonas sp. 99

Wild Parviglumis Shoot 4 E10 Paenibacillus sp. 99

Wild Parviglumis Shoot 4 E11 Paenibacillus sp. 98

Wild Parviglumis Shoot 4 E12 Pantoea sp. 97

Wild Parviglumis Root 4F1 Xanthomonas sp. 99

Wild Parviglumis Root 4F2 Pseudomonas sp. 94

Wild Parviglumis Root 4F3 Enterobacter sp. 97

Wild Parviglumis Root 4F4 Pantoea sp. 100

Wild Parviglumis Root 4F5 Microbacterium sp. 95

Wild Parviglumis Root 4F6 Xanthomonas sp. 98

Wild Parviglumis Root 4F7 Enterobacter sp. 99

Wild Parviglumis Root 4F8 Enterobacter sp. 100

Wild Parviglumis Root 4F9 Pantoea sp. 97

Wild Parviglumis Root 4F10 Enterobacter sp. 98

Wild Parviglumis Root 4F11 Microbacterium sp. 94

Wild Parviglumis Root 4F12 Stenotrophomonas sp. 99

Wild Parviglumis Root 4G1 Cupriavidus sp. 98

Wild Parviglumis Root 4G2 Mesorhizobium sp. 97

Wild Parviglumis Root 4G3 Delftia sp. 95

Wild Parviglumis Root 4G4 Paenibacillus sp. 99

Wild Parviglumis Shoot 4G5 Bacillus sp. 99

Wild Parviglumis Shoot 4G6 Microbacterium sp. 99

Wild Parviglumis Shoot 4G7 Paenibacillus sp. 98

Wild Parviglumis Root 4G8 Enterobacter sp. 97

Wild Parviglumis Root 4G9 Pantoea sp. 99

Wild Parviglumis Root 4G10 Microbacterium sp. 100

Wild Parviglumis Root 4G11 Kytococcus sp. 84

Wild Parviglumis Root 4G12 Citrobacter sp. 99

Wild Parviglumis Root 4H1 Pantoea sp. 97

Wild Parviglumis Root 4H2 Agrobacterium sp. 98

419

Wild Parviglumis Root 4H3 Pedobacter sp. 90

Wild Parviglumis Root 4H4 Curtobacterium sp. 99

Wild Parviglumis Root 4H5 Stenotrophomonas sp. 99

Wild Parviglumis Root 4H6 Cohnella sp. 99

Wild Parviglumis Root 4H7 Enterobacter sp. 100

Wild Parviglumis Root 4H8 Enterobacter sp. 97

Modern Pioneer 3751 Shoot 4H9 Microbacterium sp. 97

Modern Pioneer 3751 Root 4H12 Paenibacillus sp. 99

Modern Pioneer 3751 Root 5A2 Pantoea sp. 98

Modern Pioneer 3751 Root 5A3 Enterobacter sp. 99

Modern Pioneer 3751 Root 5A4 Enterobacter sp. 97

Modern Pioneer 3751 Root 5A5 Enterobacter sp. 97

Modern Pioneer 3751 Root 5A6 Paenibacillus sp. 98

Modern Pioneer 3751 Root 5A7 Paenibacillus sp. 99

Modern Pioneer 3751 Root 5A8 Pandoraea sp. 99

Modern Pioneer 3751 Root 5A9 Sphingobium sp. 95

Modern Pioneer 3751 Root 5A10 Paenibacillus sp. 97

Modern Pioneer 3751 Shoot 5A11 Pseudomonas sp. 100

Modern Pioneer 3751 Shoot 5A12 Janthinobacterium sp. 98

Modern Pioneer 3751 Root 5B1 Sphingomonas melonis 80

Modern Pioneer 3751 Root 5B2 Microbacterium sp. 95

Modern Pioneer 3751 Root 5B3 Enterobacter sp. 99

Modern Pioneer 3751 Root 5B4 Enterobacter sp. 97

Modern Pioneer 3751 Root 5B5 Enterobacter sp. 99

Modern Pioneer 3751 Root 5B6 Bacillus sp. 83

Modern Pioneer 3751 Root 5B7 Pedobacter sp. 95

Modern Pioneer 3751 Root 5B8 Burkholderia sp. 100

Modern Pioneer 3751 Root 5B9 Pseudomonas sp. 95

Modern Pioneer 3751 Root 5B10 Agrobacterium sp. 98

Modern Pioneer 3751 Root 5B11 Agrobacterium sp. 94

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Modern Pioneer 3751 Root 5B12 Enterobacter sp. 96

Modern Pioneer 3751 Shoot 5C2 Agrobacterium sp. 97

Modern Pioneer 3751 Root 5C3 Agrobacterium sp. 95

Modern Pioneer 3751 Root 5C4 Mycobacterium sp. 100

Modern Pioneer 3751 Root 5C5 Paenibacillus sp. 97

Modern Pioneer 3751 Root 5C6 Microbacterium sp. 99

Modern Pioneer 3751 Root 5C7 Microbacterium sp. 89

Modern Pioneer 3751 Root 5C8 Paenibacillus sp. 93

Wild Parviglumis Root 5C9 Burkholderia sp. 100

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Appendix Figure A8.1: Test for the ability of the candidate endophytes to detoxify DON in vitro. Shown are representative HPLC chromatograms showing that none of the endophyte treatments caused DON detoxification. Shown are treatments with: (A) the buffer control (LB media), (B) endophyte 1D6, (C) endophyte 3H9, (D) endophyte 4G12 and (E) endophyte 4G4.

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