Intraspecies Competition Among Strains in the - PowerPoint Presentation

1. Intraspecies Competition Among Strains in the Ralstonia solanacearum Species Complex (RSSC) as a Potential BiocontrolRoxanne Reyes1 and Dr. Alejandra Huerta2Cornell University, College of Agriculture and Life Sciences, Department of Global DevelopmentNorth Carolina State University, College of Agriculture and Life Sciences, Department of Entomology & Plant PathologyIntroductionMethods 2- Overlay Inhibition AssaysResultsConclusionsReferencesRalstonia solancearum is a soilborne bacterial plant pathogen that causes bacterial wilt on a variety of host species, over 250 across 54 different plant families, including important agronomic crops such as potato, tomato, and eggplant. Currently it is ranked as the second most destructive plant pathogenic bacterium and annual global crop losses are projected at approximately 1 billion USD on potato alone. The destructive nature of the disease is due to the pathogen’s worldwide distribution, genetic diversity, and ability to withstand a variety of environmental conditions. Unlike other similar pathogens which can be easily managed with chemical soil fumigation or antibiotics, these tactics are generally not effective towards R. solanacearum. Current control methods have been largely limited to cultural tactics or breeding hosts for genetic resistance. The goal of my research is to exploit mechanisms of intraspecies competition within strains of the RSSC to develop novel management strategies for this bacterium. Specifically, I am studying the role of Rhs-toxin proteins, which inhibit the growth of bacteria within a species, secreted by the Type 6 Secretion System (T6SS), a syringe like bacterial apparatus that translocates the toxins from the producing cell to the target cell. I first generated a T6SS mutant strain of K60 (K60ΔTssM) by replacing the TssM gene with an antibiotic resistance cassette, Kanamycin. Next, I tested this and other mutants’ ability to inhibit growth of other strains in the RSSC by conducting overlay inhibition assays. This basic research can lay the foundation for a potential biocontrol strategy to manage bacteria wilt.An effective narrow spectrum biocontrol strategy can be an environmentally safer alternative to chemical soil fumigants which can potentially leave residues in soil and produce. In addition, the targeted nature of this biocontrol strategy can ensure that non-target beneficial soil microbes essential for overall plant health are unaffected.  According to preliminary data, the fact that the mutants can inhibit other strains (ex. Molk2) indicates that the type 6 secretion system may not be solely responsible for the secretion of Rhs-toxins. K60▵TssM partially decreases the degree of inhibition on AW1 in comparison to the WT K60.Mutants SA2 and SA5+8 also partially lose their ability to inhibit other RSSC strains.The lack of complete loss of inhibition could be due to only 1/x proteins being mutated within the T6SS.Future WorkI propose the need to mutate more than 1 gene within the T6SS locus.In addition, other Rhs-toxins should be mutated within those represented in K60. “The Current Bacterial Wilt Situation: A Global Overview.” Bacterial Wilt Disease and the Ralstonia Solanacearum Species Complex, by J.G. Elphinstone, American Phytopathological Society, 2005, pp. 9–28. “Management of Bacterial Wilt Disease.” Bacterial Wilt Disease and the Ralstonia Solanacearum Species Complex, by G S Saddler, American Phytopathological Society, 2005, pp. 121–132. Paudel, Sujan, et al. “Taxonomy and Phylogenetic Research on Ralstonia Solanacearum Species Complex: A Complex Pathogen with Extraordinary Economic Consequences.” Pathogens, vol. 9, no. 11, 2020Saprophytic Life stage- Bacteria survives in the soil by means of an alternate host such as weeds, in dead/decaying plant debris, or in waterOnce a susceptible host is perceived, bacteria moves into the roots of the host and penetrates through natural wounds caused by agronomic practices, pests, or the environmentBacteria invade the xylem vessel and multiply quicklyBacteria produce extracellular polysaccharides and virulence factors which block vertical water transport in the plant from roots to shootsThe plant wilts and dies :(Bacteria move back into the soil where it waits to infect the next susceptible hostRalstonia solanacearum Life Cycle Methods 1- Generating the K60ΔTssM MutantGenerate primers using Geneious software and NEBuilder Assembly ToolAmplify upstream and downstream region of TssM geneAmplify the Kanamycin cassetteConfirmation with PCR and specific primersGibson assembly cloning to aneel all 3 fragments togetherInsert into a destination vector (PRK415), then into Ecoli., then into K60Confirmation with PCR and specific primersTransformed in K60 WT (Wild type)Square petri plates with two 25mL layers of CPG agar. Top layer of the overlay is inoculated with 100 uL of bacterial suspension of the target strain at an OD600 of 0.1.Circular wells are poked into the agar with a sterile cork borerWells are filled with 30 uL of supernatant from producing strains Incubate plates at 28C and photograph the plates next to a ruler after 72 hrs.Measure the diameter of the halos of inhibition in cm using ImageJ software.