Bioprospecting and pathovar profiling of plant pathogenic bacteria using novel high throughput technology platforms

Abstract

Pseudomonas syringae is one of the most common and abundant plant pathogens, it invades damaged tissues and produces toxins that kill the surrounding cells, allowing for the bacteria for multiply further. P. syringae has many pathovars, which are bacterial subspecies strains with similar characteristics but different pathogenicity and host specificity. Plants affected by P. syringae can develop blight, cankers, discolouration, and dead dormant buds. All of these symptoms make plants more susceptible to harsh abiotic and biotic stress, such as extreme weather, moisture extremes, insects and additional pathogens. Plant pathogens are a worldwide issue as they affect crop health and therefore yield of economically important crops. This project aims to establish high-throughput methodology for the identification of bacterial cell wall polysaccharides, exopolysaccharides and exoenzymes, which can be utilised as a biomarker for identification of Pseudomonas syringae pathovars. Initial experiments indicated that thin layer chromatography (TLC) is not an appropriate method for a large-scale study, due to complex sample preparation and limited success with obtaining viable fractions for polyand monosaccharide identification. Comprehensive microarray polymer profiling (CoMPP) is a high-throughput method most commonly used for polymer profiling in plants, therefore was a candidate of interest for exploring viability of this methodology when applied to bacterial samples. Due to lack of P. syringae monoclonal antibodies findings were limited when bacterial material was analysed using CoMPP. CoMPP was used at a platform for developing epitope depletion methodology for bacterial supernatant, where enzymatic activity was identified. This methodology was applied further on plant material and immunofluorescent microscopy, where enzyme containing bacterial supernatants visibly depleted relevant epitopes in plant structures, such as root hair cells. P. syringae forms biofilms which is aids in surface adhesion and therefore host infection. Alginate, which is a main component of bacterial biofilms was identified using CoMPP at different monoclonal antibody binding patterns, indicating potential for alginate to be utilised as biomarker for pathovar identification. Together, these approaches have the potential to uncover novel enzymes and be developed further into a tool for pathovar identification.