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The Potential of Pseudomonas Bacteria As Biocontrol Agents Against Multiple Plant Pathogens

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The Potential of Pseudomonas Bacteria As Biocontrol Agents Against Multiple Plant Pathogens The potential of Pseudomonas bacteria as biocontrol agents against multiple plant pathogens Sophie Elizabeth Clough Doctor of Philosophy University of York Biology September 2020 Abstract Plant pathogenic bacterium Ralstonia solanacearum (the causative agent of bacterial wilt), and plant parasitic nematodes Globodera pallida (white potato cyst nematode) and Meloidogyne incognita (root-knot nematode) have devastating impacts on several economically important crops globally. While these pathogens are traditionally treated with agrochemicals, their use is in decline due to legal restrictions and harmful impacts on the environment. One environmentally- friendly alternative to agrochemicals could be biocontrol, which takes advantage of naturally occurring plant growth-promoting bacteria. While recent studies have shown promising results, there is no clear screening pipeline to identify and validate successful biocontrol strains with broad activity against multiple different pathogen species. This thesis establishes a screening method to identify effective Pseudomonas biocontrol agents to both bacterial and nematode pathogens using a combination of in vitro laboratory assays, comparative genomics, mass spectrometry and greenhouse experiments. It was found that Pseudomonas strains suppressed both pathogens, with strains CHA0, MVP1-4 and Pf-5 showing high activity. Several secondary metabolite clusters were identified and the suppressive role of DAPG, orfamides A and B and pyoluteorin antimicrobials were experimentally verified. Experimental evolution was used to show that R. solanacearum can evolve tolerance to Pseudomonas strains during prolonged exposure in vitro. However, further work is needed to test whether the evolution of tolerance could limit Pseudomonas biocontrol efficiency in the field. High nematode suppression by all Pseudomonas strains was observed in laboratory assays. Moreover, clear behavioural and developmental changes were observed in response to tested individual compounds. Despite clear nematode suppression observed in vitro, no protection by Pseudomonas was observed in greenhouse experiments. However, Pseudomonas strains reduced bacterial wilt disease incidence caused by one of the tested R. solanacearum strains in vivo. Despite promising in vitro results, more research is needed to translate Pseudomonas biocontrol potential into an agricultural context. 2 Table of Contents Abstract .............................................................................................................................................. 2 Table of Contents ............................................................................................................................... 3 List of Tables ....................................................................................................................................... 5 List of Figures ...................................................................................................................................... 5 Acknowledgements ............................................................................................................................ 8 Declaration by Author ...................................................................................................................... 10 Chapter 1. General Introduction ...................................................................................................... 11 1.1 Threats to food security .......................................................................................................... 11 1.2 A brief history of biocontrol .................................................................................................... 20 1.3 Introduction to Ralstonia solanacearum plant pathogenic bacterium .................................. 25 1.4 Introduction to plant parasitic nematodes (PPNs) ................................................................. 32 1.5 Consideration of the potential antagonisms with biocontrol ................................................. 41 1.6 Development of general biocontrol strategies against both R. solanacearum and PPNs ...... 42 1.7 Project Aims and Objectives ................................................................................................... 43 General Methods............................................................................................................ 48 In vitro and in vivo screening of efficient Pseudomonas biocontrol strains against Ralstonia solanacearum pathogen and investigation of potential underlying mechanisms of inhibition .......................................................................................................................................... 55 3.1 Abstract ................................................................................................................................... 55 3.2 Introduction ............................................................................................................................ 56 3.3 Methods .................................................................................................................................. 59 3.4 Results ..................................................................................................................................... 65 3.5 Discussion ................................................................................................................................ 78 Plant pathogenic Ralstonia solanacearum bacterium can rapidly evolve tolerance to antimicrobials produced by Pseudomonas biocontrol bacteria ..................................................... 84 4.1 Abstract ................................................................................................................................... 84 4.2 Introduction ............................................................................................................................ 85 4.3 Materials and methods ........................................................................................................... 88 4.4 Results ..................................................................................................................................... 92 4.5 Discussion .............................................................................................................................. 109 Screening and identification of effective Pseudomonas biocontrol strains against plant parasitic nematodes and testing their efficacy in vitro and in vivo .................................... 116 5.1 Abstract ................................................................................................................................. 116 5.2 Introduction .......................................................................................................................... 117 3 5.3 Materials and methods ......................................................................................................... 120 5.4 Results ................................................................................................................................... 134 5.5 Discussion .............................................................................................................................. 156 General Discussion ....................................................................................................... 163 6.1 Overview ............................................................................................................................... 163 6.2 The importance of Pseudomonas PGPB genotypic variation when screening for biocontrol activity against R. solanacearum ................................................................................................ 164 6.3 The importance of R. solanacearum genotypic variation when screening for Pseudomonas biocontrol activity ....................................................................................................................... 166 6.4 Evolution of tolerance to biocontrol agents in R. solanacearum ......................................... 168 6.5 Considering Pseudomonas biocontrol efficacy against C. elegans nematode model and PPNs .................................................................................................................................................... 170 6.6 Considering the Pseudomonas strains with the strongest biocontrol potential against both bacterial plant pathogen R. solanacearum and plant parasitic nematodes G. pallida and M. incognita ..................................................................................................................................... 172 6.7 Future development and limitations of biocontrol .............................................................. 173 6.8 Concluding remarks .............................................................................................................. 175 Appendices ..................................................................................................................................... 177 Appendix A: Chapter 3 ................................................................................................................ 177 References ...................................................................................................................................... 182 4 List of Tables Table 2.1 Pseudomonas strains used in these experiments, and their geographical origin. ........... 48 Table 2.2 Ralstonia
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