Identification of Plant Growth-Promoting Bacterial Endophytes Using Culture-Dependent In Vitro and Laboratory In Planta Methods by Evan Mayer A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Cell and Systems Biology University of Toronto ©Copyright by Evan Mayer 2019 Identification of Plant Growth-Promoting Bacterial Endophytes Using Culture-Dependent In Vitro and Laboratory In Planta Methods Evan Mayer Master of Science Cell and Systems Biology University of Toronto 2019 Abstract This study examined a collection of 220 bacterial endophytes isolated from herbaceous plants growing in petroleum-contaminated soils for plant growth promotion abilities. 12% of endophytes tested induced statistically significant growth benefits in Arabidopsis. The strongest plant growth promoting endophyte, Plantibacter flavus strain 251, increased A. thaliana fresh biomass and total root length by 4.7 and 5.8 times respectively over control plants and contained genes for auxin and ACC deaminase production. In vitro tests showed that 26% of endophytes from 19 different genera were potentially capable of nitrogen fixation, while 42% of endophytes from 16 different genera were capable of phosphate solubilization. Of the potential nitrogen fixers, 80% demonstrated free-living nitrogen fixation capability when grown in nitrogen-free media. None of the three endophytes tested improved Arabidopsis tolerance to drought stress. Overall, this study showed that a large variety of bacterial endophytes are capable of direct plant growth promotion. ii Acknowledgements I would like to thank my supervisor, Roberta Fulthorpe, for giving me the opportunity to not just get a Master’s degree, but to also grow as a person. I feel like I have become more independent, creative, and passionate about science as a result of working with you for two years at U of T Scarborough, and I’m very appreciative of that. I would like to thank my committee members, Marney Isaac and Keiko Yoshioka, for being open with their input and providing me with the necessary tools to facilitate this research. Lastly, I would like to thank my labmates, particularly the three Patricias, for helping and entertaining me throughout the Masters. iii Table of Contents List of Tables ............................................................................................................................. vi List of Figures .......................................................................................................................... viii List of Appendices ...................................................................................................................... xi List of Abbreviations .................................................................................................................. xii Chapter 1: Introduction ................................................................................................................ 1 1.1 The Plant Microbiome ........................................................................................................... 1 1.2 The Endosphere and Endophytes ......................................................................................... 2 1.3 Beneficial Functions of Plant Growth-Promoting Microbes .................................................... 4 1.3.1 Production of Phytochemicals and Phytohormones ............................................................ 4 1.3.2 Nutrient Acquisition ............................................................................................................ 5 1.3.3 Abiotic Stress Tolerance .................................................................................................... 5 1.3.4 Protection from Pathogens ................................................................................................. 6 1.4 Practical Application of Plant Growth-Promoting Microbes .................................................... 7 1.4.1 Biofertilizers and Biopesticides ........................................................................................... 7 1.4.2 Other Uses ......................................................................................................................... 8 1.5 Methods of Studying Plant Growth-Promoting Microbes........................................................ 8 1.5.1 In Vitro vs. In Vivo Methods ................................................................................................ 8 1.5.2 Arabidopsis thaliana as a Model Laboratory Plant .............................................................. 9 1.6 Research Objectives and Hypotheses ................................................................................. 10 Chapter 2: Identification of Direct Plant Growth-Promoting Bacterial Endophytes Using In Planta Screening Tests ........................................................................................................................ 12 2.1 Introduction ......................................................................................................................... 12 2.2 Materials and Methods ........................................................................................................ 13 2.2.1 Endophyte Collection and Bacterial Culture Growth ......................................................... 13 2.2.2 Surface Sterilization of Arabidopsis Seeds ....................................................................... 13 2.2.3 In Planta Screening Tests ................................................................................................ 14 2.2.4 Statistical Analysis ........................................................................................................... 15 2.3 Results ................................................................................................................................ 15 2.4 Discussion........................................................................................................................... 19 Chapter 3: Quantification and Explanation of Direct Plant Growth Promotion by Select Bacterial Endophytes ............................................................................................................................... 21 3.1 Introduction ......................................................................................................................... 21 3.2 Materials and Methods ........................................................................................................ 21 iv 3.2.1 16S rRNA Gene Sequencing of Select Endophytes ......................................................... 21 3.2.2 In planta Tests Using Select Endophytes ......................................................................... 22 3.2.3 Statistical Analysis ........................................................................................................... 23 3.2.4 Plantibacter flavus strain 251 Genome Annotation and Search ........................................ 23 3.3 Results ................................................................................................................................ 24 3.3.1 Identification of Select Isolates ......................................................................................... 24 3.3.2 Select in planta tests ........................................................................................................ 25 3.3.3 Plant Beneficial Genes in Plantibacter flavus Strain 251 .................................................. 34 3.4 Discussion........................................................................................................................... 35 Chapter 4: Effects of Bacterial Endophytes on Plant Nitrogen Acquisition ................................. 41 4.1 Introduction ......................................................................................................................... 41 4.2 Materials and Methods ........................................................................................................ 43 4.2.1 nifH PCR .......................................................................................................................... 43 4.2.2 Growth in Nitrogen-Free Medium ..................................................................................... 43 4.2.3 Nitrogen-Limiting In Planta Tests ..................................................................................... 44 4.3 Results ................................................................................................................................ 44 4.3.1 nifH PCR .......................................................................................................................... 44 4.3.2 Growth in Nitrogen-Free Medium ..................................................................................... 45 4.3.3 Nitrogen-Limiting In Planta Tests ..................................................................................... 46 4.4 Discussion........................................................................................................................... 47 Chapter 5: Effects of Bacterial Endophytes on Plant Phosphorus Uptake ................................. 51 5.1 Introduction ......................................................................................................................... 51 5.2 Materials and Methods .......................................................................................................