Platforms to differentiate exotic pathovars of plant bacteria

Many of the EPPs that pose the biggest threat to the biosecurity of Australia’s plant industries are bacterial, but difficulties in identification to the subspecific or ‘ pathovar ’ level can seriously delay incursion management and affect market access. Pathovars are defined by host specificity so bio assays remain the definitive means of identification, but these require high level physical containment and can be slow and subjective , delaying diagnosis . Some pathovar - specific serological and molecular tests are available but better diagnostic methods are often required. This project used proteomics and metabolomics, platforms that identify functional molecules potentially associated with plant - pathogen interactions, to identify biomarkers that differentiate pathovars in species of Xanthomonas . Membrane - associated proteins from a collection of bacterial isolates were compared on 2Dimensional gels. Proteins that were found to be differentially expressed between distinct pathovars may be important modulators of host specificity so they were identified and the genes that encode them located by reference to genomic sequences . DNA - based assays targeting these genes were designed and validated for their specificity to the pathovar level . We have developed two new assays that provide levels of specificity not reported elsewhere in the literature. These assays specifically target the bacteria causing the different forms of citrus canker, but without cross - reaction to the closely - related organisms causing bacterial blight on cotton and Citrus Bacterial Spot. The molecular assays will be incorporated into the National Diagnostic Protocol for citrus canker through the SPHDS process. The metabolomics component has analysed metabolite expression in selected bacterial pathovars. Results showed separation between the different pathovars based on differential levels of expression of particular metabolites. These metabolites may be important determinants of pathogenicity. Neither proteomics nor metabolomics had been implemented before in the study of phytopathogenic bacteria and whilst both proved to be technically demanding, each delivered new biomarkers that differentiate phytopathogenic bacteria to a subspecific level . This confirmed the viability of these approaches as platforms to discover novel diagnostic targets. The new methods developed will be implemented into the national incursion response capability , improving the specificity of diagnostic testing available and reducing the possibility of false positive diagnosis . The project has fostered new collaborative partnerships both nationally (NSW, Victoria, WA) and internationally (to Thailand and the USA). The next phase of this work will provide a strong start - up project to the Plant Biosecurity Cooperative Research Centre ( PBCRC ) . This project has directly enhanced the plant bacteriology capacity of NSW and Australia trough the recruitment and training of science professionals and an undergraduate student , and supported the specialist training of a Thai scientist through allied project CRC20093

Highthroughput genomic sequencing of cassava bacterial blight strains identifies conserved effectors to target for durable resistance

Published online: 13 June 2012Cassava bacterial blight (CBB), incited by Xanthomonas axonopodis pv. manihotis (Xam), is the most important bacterial disease of cassava, a staple food source for millions of people in developing countries. Here we present a widely applicable strategy for elucidating the virulence components of a pathogen population. We report Illumina-based draft genomes for 65 Xam strains and deduce the phylogenetic relatedness of Xam across the areas where cassava is grown. Using an extensive database of effector proteins from animal and plant pathogens, we identify the effector repertoire for each sequenced strain and use a comparative sequence analysis to deduce the least polymorphic of the conserved effectors. These highly conserved effectors have been maintained over 11 countries, three continents, and 70 y of evolution and as such represent ideal targets for developing resistance strategies