Molecular and physiological mechanisms underlying the antifungal and nutrient acquisition ...

274 Pages·2016·9.03 MB·English

by  Manish Raizada

Molecular and physiological mechanisms underlying the antifungal and
nutrient acquisition activities of beneficial microbes

by
Hanan R. Shehata

A Thesis
presented to
The University of Guelph

In partial fulfilment of requirements
for the degree of
Doctor of Philosophy
in
Plant Agriculture

Guelph, Ontario, Canada

© Hanan R. Shehata, January, 2016
ABSTRACT
Molecular and Physiological Mechanisms Underlying Antifungal and Nutrient
Acquisition Activities of Beneficial Microbes

Hanan R. Shehata Advisor: Dr. Manish N. Raizada
University of Guelph, 2016 Co-Advisor: Dr. Eric M. Lyons

Endophytes are microbes that inhabit host plants without causing disease. Wild
and ancient relatives of modern crops are grown without fungicides or synthetic
fertilizers. I hypothesized that these genotypes possess beneficial endophytes that help
their hosts to combat fungal pathogens and/or acquire nutrients. To test the first
hypothesis, 190 bacterial endophytes isolated from wild, ancient and modern genotypes
of Zea (corn grass family) were tested for their ability to combat fungal pathogens. For
high-throughput disease screening, a relative of Zea, creeping bentgrass (Agrostis
stolonifera L.) was used as a model system, as it can be grown rapidly in test tubes. Two
pathogens of creeping bentgrass were selected as targets: Sclerotinia homoeocarpa and
Rhizoctonia solani. Three endophyte strains (all Burkholderia gladioli) were found to
combat S. homoeocarpa, while four strains (3 Burkholderia gladioli and 1 Paenibacillus
polymyxa) could suppress R. solani. All the Burkholderia endophytes originated from
wild or ancient maize, while the single Paenibacillus strain originated from modern
maize. These results support the hypotheses that wild and ancient maize genotypes host
microbes that can control fungal pathogens. B. gladioli strain 3A12 was selected for
isolation of the underlying antifungal genes. Tn5-mutagenesis combined with whole
genome sequencing revealed that the antifungal genes encode: YajQ (a c-di-GMP
receptor), fatty acid desaturase, lysine-tRNA synthetase, tolR, arginine/ornithine/lysine
decarboxylase and succinate dehydrogenase. The candidate mutants showed similar
phenotypes (reduced motility, biofilm formation, attachment and microcolony formation
around target fungi), suggesting they may belong to the same genetic network.

To test the second hypothesis, 73 endophytes were screened for their ability to
promote growth of annual ryegrass on rock-phosphate as the sole P source. One
endophyte, isolated from a wild maize genotype, was able to promote root growth. This
endophyte was found to secrete acids to help solubilize rock P in vitro, and synthesize
auxin and promote lateral root and root hair growth, presumably to increase P-
scavenging. The endophyte can localize to epidermal root surfaces including on root
hairs, and remarkably, also near lateral root primordia. The results support the hypothesis
that wild plants may be a reservoir of microbes that assist their hosts with nutrient
acquisition.

KEYWORDS:
Endophyte, Zea, beneficial bacteria, Sclerotinia homoeocarpa, Rhizoctonia
solani, Burkholderia gladioli, Paenibacillus polymyxa, antifungal, phosphate
solubilization, transposon mutagenesis, antifungal genes
ACKNOWLEDGMENTS
I would like to thank and express my sincere gratitude to Dr. Manish N. Raizada
for his continuous support, encouragement, valuable advice, extended discussions and for
caring about my future. Thanks Dr. Raizada.

I would like to thank all members of my PhD advisory committee: Dr. Lyons, Dr.
Jordan and Dr. Griffiths for their advice and support and for allowing me to work in their
laboratories.

Thanks to all my labmates for help and support; Dr. Van Straaten and Peter
Marenghi (Agrium Inc); Jan Brazolot and Chris Grainger (University of Guelph); Ken
Carey and Alex Porter (GTI, University of Guelph) and undergraduate students: Sophia
Watts, Navneet Madhar, Marina Attalah and Emillie Philippe, Finlay Small and Sara
Wyngaarden for technical help.

I am thankful to the Egyptian government for funding my study, and to my
professors, colleagues and members of the Microbiology Department, Faculty of
Pharmacy, Mansoura University, for their great support.

Words are not enough to express all my gratitude to my mother, sisters and
brother with special thanks to my husband Amro Elhelaly and to my kids Mohammed
and Ahmed for continued love and support.

iv
TABLE OF CONTENTS
Abstract
Acknowledgements I
List of Tables X
List of Figures XI
List of Abbreviations XIV
Chapter 1: General overview 1

Chapter 2: Literature review 3
2.1. Endophytes 3
2.2. Endophytes can antagonize fungal pathogens 4
2.2.1. Mechanisms of biological control 5
2.2.1.1. Direct antagonism 5
2.2.1.2. Inducing systemic resistance (ISR) 6
2.2.1.3. Competition for nutrients or space 6
2.2.2. Bacterial virulence 6
2.3. Endophytes can help with nutrient acquisition 7
2.3.1. Phosphate solubilization 7
2.4. Turfgrass 9
2.5 Dollar spot disease 9
2.5.1 Life cycle of S. homoeocarpa 11
2.5.2 Dollar spot symptoms 11
2.5.3. Current management protocols 13
2.6 Brown patch disease 13
2.6.1 Life cycle of R. solani 15
2.6.2 Brown patch symptoms 15
2.6.3 Current management strategies 15
2.7 Annual ryegrass 15
2.8 Wild and ancient plants as sources of endophytes 16
2.9 The source of maize endophyte collection used in this thesis 17
2.10 Objectives and hypotheses 19

Chapter 3: Bacterial endophytes from wild and ancient maize suppress the
fungal pathogen Sclerotinia homoeocarpa 20
3.1 Abstract 21
3.2 Introduction 22
3.3 Materials and Methods 24
3.3.1 Source of endophytes 24
3.3.2 In vitro screen for endophytes with activity against S. homoeocarpa 26
3.3.3 Taxonomic identification of candidate endophytes 26
3.3.4 Greenhouse testing of antifungal endophyte candidates using 27
creeping bentgrass
3.3.5 Characterization of in vitro interactions between pathogen and
endophytes 29
3.3.6 Antifungal target spectrum of strain 3A12, 3C11 and 5C9 30
v
3.4 Results 31
3.4.1 In vitro screen for endophyte activity against S. homoeocarpa 31
3.4.2 Taxonomic identification of candidate endophytes 31
3.4.3 Greenhouse testing of antifungal endophyte candidates 34
3.4.4 Characterization of in vitro interactions between the pathogen and
endophytes 37
3.4.5 Antifungal target spectrum of candidate antifungal endophytes 39
3.5 Discussion 41
3.5.1 Ancient agroecological selection for endophytes? 43
3.5.2 The effect of host evolutionary transitions on endophytes 43
3.5.3 Antifungal Burkholderia endophytes may have co-evolved with Zea
plants 45
3.6 Future applications 48
3.7 Acknowledgements 49

Chapter 4: The Antifungal mechanisms of a bacterial endophyte with ancient
crop origins 50
4.1 Abstract 51
4.2 Introduction 53
4.3 Materials and Methods 55
4.3.1 Sources of biological materials 55
4.3.2 Testing endophyte strains on creeping bentgrass in glass tubes 56
4.3.3 GFP-tagging and microscopic imaging of strain 3A12 58
4.3.4 Scanning electron microscopy (SEM) of endophytes 59
4.3.5 Characterization of in vitro interactions between endophyte 3A12 and
S. homoeocarpa 60
4.3.6 Identification of the gene(s) responsible for the antifungal activity 60
4.3.7 Bacterial swimming motility assay 63
4.3.8 Transmission electron microscopy (TEM) 63
4.3.9 Biofilm formation assay 64
4.3.10 Examination of swarming and colony formation around the fungal
target 64
4.3.11 Chitinase activity assay 65
4.3.12 Quantitative reverse transcription PCR 65
4.3.13 Statistical and graphical analysis 67
4.4 Results 67
4.4.1 Microscopic imaging of strain 3A12 67
4.4.2 In vitro interactions between endophyte 3A12 and S. homoeocarpa 68
4.4.3 Identification of the gene(s) responsible for the fungicidal activity 70
4.4.4 Effect of mutants on motility 75
4.4.5 Effect of mutants on flagella number 75
4.4.6 Effect of mutants on biofilm formation 75
4.4.7 Effect of mutants on swarming, adhesion and colony formation
around their fungal target 80
4.4.8 Candidate gene predictions and assay for chitinase activity 82
4.5 Discussion 84
vi
4.5.1 The ancient peoples of Mexico may have inadvertently selected for
an endophyte with potent antifungal genes 84
4.5.2 Genes required for antifungal activity 85
4.5.2.1 YajQ 86
4.5.2.2 Fatty acid desaturase 87
4.5.2.3 lysine-tRNA synthetase 88
4.5.2.4 TolR 89
4.5.2.5 Arginine decarboxylase/ornithine decarboxylase/lysine
decarboxylase 90
4.5.2.6 Succinate dehydrogenase 91
4.5.3 Other candidate antifungal genes 92
4.5.3.1 Chitinase(s) 93
4.5.3.2 Phenazine(s) 93
4.5.3.3 Colicin V 94
4.5.4 Summary model 94
4.6 Conclusions 98
4.7 Acknowledgements 98

Chapter 5: Bacterial endophytes from wild, ancient and modern maize
combat Rhizoctonia solani in creeping bentgrass 99
5.1 Abstract 100
5.2 Introduction 102
5.3 Materials and Methods 104
5.3.1 Sources of biological materials 104
5.3.2 In planta screen for endophyte antifungal activity against Rhizoctonia
solani 106
5.3.3 Taxonomic identification of candidate endophytes 107
5.3.4 Characterization of in vitro interactions between candidate
endophytes and R. solani 108
5.3.5 Identification of the gene(s) responsible for the antifungal activity in
strain 3A12 109
5.4 Results 110
5.4.1 In planta screen for endophyte antifungal activity against Rhizoctonia 110
solani
5.4.2 Taxonomic identification of candidate endophytes 110
5.4.3 Characterization of the in vitro interactions between candidate
endophytes and R. solani 112
5.4.4 Identification of the gene(s) responsible for the antifungal activity in
strain 3A12 114
5.5 Discussion 118
5.5.1 Did antifungal Burkholderia and Paenibacillus endophytes co-evolve
with Zea? 118
5.5.2 B. gladioli as friend or foe of plants 120
5.5.3 B. gladioli strain 3A12 and its antifungal mechanisms 120
5.5.3.1 lysine-tRNA synthetase 121
5.5.3.2 TolR 122
vii
5.5.3.3 Arginine/ornithine/lysine decarboxylase 122
5.5.3.4 Succinate dehydrogenase 123
5.5.4 Other antifungal genes 124
5.5.5 The antifungal mechanisms of P. polymyxa 127
5.6 Conclusions 127
5.7 Acknowledgements 128

Chapter 6: Relevance of in vitro agar based screens to characterize the
antifungal activities of bacterial endophyte communities 129
6.1 Abstract 130
6.2 Introduction 131
6.3 Materials and Methods 132
6.3.1 Biological Materials 132
6.3.2 In planta screen 132
6.3.3 In vitro screen 134
6.3.4 Statistical analysis 135
6.4 Results 135
6.4.1 Screening for inhibition of S. homoeocarpa 135
6.4.2 Screening for inhibition of R. solani 135
6.5 Discussion 140
6.6 Acknowledgements 144

Chapter 7: A swamp maize endophyte solubilizes rock-phosphate, promotes
root growth and localizes near stem cell niches 145
7.1 Abstract 146
7.2 Introduction 147
7.3 Materials and Methods 150
7.3.1 Seed and Endophyte Materials 150
7.3.2 Testing maize endophytes for ability to promote growth of annual
ryegrass grown on rock phosphate containing medium 152
7.3.3 Taxonomic identification of candidate endophyte based on 16S rRNA 154
7.3.4 Testing the ability of the candidate endophyte to solubilize rock
phosphate in vitro using Pikovskaya’s agar 155
7.3.5 Quantification of the amount of phosphate solubilized by the
candidate endophyte in vitro colorimetrically 155
7.3.6 Studying the effect of the candidate endophyte on annual ryegrass
root architecture 157
7.3.7 GFP tagging and confocal microscopy 157
7.3.8 Testing for endophytic production of auxin (indole acetic acid, IAA) 159
7.3.9 Testing for 1-aminocyclopropanecarboxylic acid (ACC) deaminase
production 160
7.3.10 Testing for acid production from endophyte colonies using NBRIP
agar medium 161
7.3.11 Testing for the ability of the candidate endophyte to produce acids 161
in planta
7.3.12 Statistical and graphical analysis 162
viii
7.4 Results 162
7.4.1 Testing maize endophytes for ability to promote growth of annual
ryegrass on rock phosphate 162
7.4.2 Taxonomic identification of candidate endophyte E10 164
7.4.3 Testing endophyte E10 for its ability to solubilize phosphate in vitro 164
7.4.4 The effect of endophyte E10 on root architecture 164
7.4.5 Studying the localization of GFP tagged strain 3E10 using confocal
microscopy 169
7.4.6 Testing for endophytic production of auxin (indole acetic acid, IAA) 169
7.4.7 Testing for 1-aminocyclopropanecarboxylic acid (ACC) deaminase
production 169
7.4.7 Testing for acid production in vitro using NBRIP agar medium 170
7.4.8 Testing for the ability of endophyte E10 to acidify the rhizosphere 170
7.5 Discussion 170
7.6 Study Implications and Conclusions 179
7.7 Acknowledgements 179

Chapter 8: General discussion 180
8.1: Summary 180
8.2 The host genotypes of antifungal endophytes may have selected for this
activity 183
8.3 Antifungal activity of Burkholderia species 183
8.4 The antifungal mechanisms of Burkholderia gladioli strain 3A12 184
8.5 Zea nicaraguensis may have selected for endophyte E10 185
8.6 Conclusions 186
8.7 Future experiments 186

Chapter 9: References 188
Chapter 10: Appendices 221

ix
LIST OF TABLES
Table 4.1: Summary of the genes which are essential for the antifungal activity 73
of strain 3A12 against S. homoeocarpa
Table 4.2: Summary of functions of candidate mutants. 79
Table 5.1: Summary of the genes which are essential for the antifungal activity 117
of strain 3A12 against R. solani
Table 7.1: Effect of Zea nicaraguensis endophyte E10 on crown root 166
measurements when plants are grown on media containing rock P
as the sole P source.
Table A3.1: List of Zea endophytes used in this study and their corresponding 221
zones of inhibition of S. homoeocarpa growth.
Table A4.5: Sequences of candidate antifungal genes. 235
Table A4.6: PCR primers used for gene expression analysis. 243
Table A5.1: List of Zea endophytes used in this study and in planta results for 244
antifungal activity against Rhizoctonia solani.
Table A6.1: List of all Zea endophytes used in this study and antifungal 248
screening results.
Table A6.2: Retesting of Zea endophytes that showed in planta antifungal 253
activity in Trial 1.
Table A7.1: List of Zea seed endophytes used in this study. 254

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