Graduate Thesis Or Dissertation

Evaluation of the colonization and biofilm production by Burkholderia pyroccinia FP62 on geranium leaves

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  • The leaf surface is a stark environment for bacterial colonists with limited availability of nutrients, water and protected niches. Despite these limitations, a plethora of bacteria appear to have adapted ways to survive and flourish on leaf surfaces. One mechanism of survival is the formation of biofilms. Biofilms are a consortium of bacteria and/or other microorganisms encased in an exopolymeric matrix and attached to a surface. Biofilm formation could assist in biological control agents surviving on leaf surfaces. Burholderia pyroccinia FP62 is a superior biological control agent of Botrytis cinerea (Gray mold) on geranium. Evidence suggests that biofilm production is crucial to the biological control activity of FP62. Thus, understanding how FP62 biofilms develop on leaf surfaces would enhance our understanding of the role of biofilm formation in the biological control of foliar pathogens. The spatial and temporal dynamics of FP62 on geranium leaves were examined by quantifying the culturable populations and microscopically examining the changes in bacterial cell distribution and morphology. Inoculated leaf samples were observed with a scanning electron, environmental scanning electron, confocal laser scanning, or a fluorescence stereomicroscope. Both chemically fixed and unfixed samples were observed. In addition, the subsurface structure of biofilms was examined by removing tissue with a focused ion beam then viewed with scanning electron and scanning ion microscopy. Microscopy investigations established that biofilms, produced by FP62, developed in a similar manner as those found in saturated conditions. FP62 cells attach to the surface, form aggregations and these developed into biofilms. Large aggregations were associated with the exopolymeric matrix by 3 days after inoculation; structured biofilms were observed by 7 days. The large biofilms were mainly associated with glandular trichomes and stomata; these spanned numerous plant cells ranging in size from 50μm to 1mm. The surface of mature biofilms had a convoluted surface that masked the underlying leaf cell topography. There was a copious amount of sheet-like or fibril-like matrix materials covering and connecting biofilms to each other and glandular trichomes. Focused ion beam milling removed cross sections with precision; subsurface investigations established that biofilms consist of varying densities of cells which are interconnected by fibril-like matrix materials. There were many empty spaces below the biofilm surface; through sequential milling it was determined that many of these spaces were interconnected and potentially formed microchannels. FP62 biofilms share many traits with saturated biofilms and the extensive biofilm formation of the leaf surface would enhance the potential that the biological control agent could come in contact with foliar pathogens and reduce disease development.
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