Pseudomonas entomophila 23S, a PGPR with potential for control of bacterial canker disease in tomato (Solanum lycopersicum L.) against Clavibacter michiganensis subsp. michiganensis Yoko Takishita Department of Plant Science Faculty of Agricultural and Environmental Sciences Macdonald Campus of McGill University 21111 Lakeshore Road, Stainte-Anne-de-Bellevue, Quebec H9X 3V9 May 2018 A thesis submitted to McGill University in partial fulfillment of the requiremnets of the degree of DOCTOR of PHLOSOPHY © Yoko Takishita, Canada, 2018 Table of Contents Acknowledgements ..................................................................................................................... v Abstract ....................................................................................................................................... x Résumé ..................................................................................................................................... xiii List of Tables ............................................................................................................................ xvi List of Figures ......................................................................................................................... xvii List of Appendices ................................................................................................................... xix Preface ....................................................................................................................................... xx Contributions of Authors ....................................................................................................... xx Contributions to knowledge .................................................................................................. xxi Chapter 1 Introduction .................................................................................................................... 1 1.1. Introduction ...................................................................................................................... 1 1.2. Objectives of this thesis .................................................................................................... 4 Chapter 2 Literature review ............................................................................................................ 6 2.1. Tomato .............................................................................................................................. 6 2.2. Tomato bacterial canker ................................................................................................... 8 2.3. Clavibacter michiganensis subsp. michiganensis .......................................................... 11 2.4. Pathogenicity of Cmm .................................................................................................... 12 2.5. Plant defense responses to the pathogen......................................................................... 14 2.6. Induced systemic resistance (ISR) elicited by plant-growth-promoting-rhizobacteria (PGPR)................................................................................................................................... 17 2.7. Molecular response of tomato plants to Cmm ................................................................ 19 2.8. Control of bacterial canker ............................................................................................. 22 2.9. Work in our laboratory ................................................................................................... 25 Connecting text ......................................................................................................................... 29 Chapter 3 Characterization of rhizobacterium Pseudomonas entomophila 23S having antagonistic activity against Clavibacter michiganensis subsp. michiganensis - a bacterial canker causing pathogen in tomato .......................................................................................................... 30 3.1. Abstract ........................................................................................................................... 31 3.2. Introduction .................................................................................................................... 32 3.3. Materials and Methods ................................................................................................... 34 3.4. Results ............................................................................................................................ 42 iii 3.5 Discussion ........................................................................................................................ 47 3.6 Conclusions ..................................................................................................................... 58 3.7 Acknowledgements ......................................................................................................... 59 Connecting text ......................................................................................................................... 77 Chapter 4 Isolation of antimicrobial compounds produced by Pseudomonas entomophila 23S against Clavibacter michiganensis subsp. michiganensis, a pathogen of tomato causing bacterial canker ............................................................................................................................................ 78 4.1. Abstract ........................................................................................................................... 79 4.2. Introduction .................................................................................................................... 80 4.3. Materials and Methods ................................................................................................... 82 4.4. Results ............................................................................................................................ 89 4.5. Discussion ....................................................................................................................... 93 4.6. Conclusions .................................................................................................................... 98 4.7. Acknowledgements ........................................................................................................ 99 Connecting text ....................................................................................................................... 113 Chapter 5 The biocontrol rhizobacterium Pseudomonas entomophila 23S induces systemic resistance in tomato (Solanum lycopersicum L.), against bacterial canker Clavibacter michiganensis subsps. michiganensis ......................................................................................... 114 5.1. Abstract ......................................................................................................................... 115 5.2. Introduction .................................................................................................................. 116 5.3. Materials and Methods ................................................................................................. 119 5.4. Results .......................................................................................................................... 123 5.5. Discussion ..................................................................................................................... 126 5.6. Conclusions .................................................................................................................. 131 5.7. Acknowledgements ...................................................................................................... 132 Chapter 6 Summary, discussion, conclusions and future research ............................................. 140 6.1. Summary of Results...................................................................................................... 140 6.2. General Discussion ....................................................................................................... 142 6.3. General Conclusions ..................................................................................................... 144 6.4. Future research directions ............................................................................................. 144 Chapter 7 References .................................................................................................................. 146 Appendix: Supplemental information for Chapter 3 proteomics ............................................ 173 iv Acknowledgements Although my undergraduate major was in agricultural sciences, my master’s research was on ecology, rather close to geography. When I started the PhD in McGill in 2012, my knowledge of plant science and my relevant laboratory skills were only at the undergraduate level, and most of them I had somewhat forgotten. A number of people have supported me and given me help during my PhD. Starting as such an immature scientist, I am completing this PhD project today. This accomplishment would not be possible without the support of these generous, kind and wonderful people. I cannot thank enough my supervisor Professor Donald Smith. Whenever I encountered hardships during my research, his constant positive attitude made me calm and relaxed, and often made me laugh, and I was able to carry on. His broad knowledge and experience taught me many