Rapid identification of quarantine bacterial plant pathogens by sequence comparisons within single housekeeping genes

Monitoring and surveillance of bacterial plant pathogens is vital in efforts to control disease and maintain freedom from alien and newly emerging diseases. Rapid and reliable identification of species and pathovars provides the primary means of evaluating the disease threat from isolated pathogens. With the development and reduced cost of DNA sequencing technologies, phylogenetic-based identification based on comparative sequence analysis of protein coding genes has been shown to be rapid, reliable, reproducible and cost-effective. Multilocus sequence typing (MLST), multilocus sequence analysis (MLSA) and DNA barcoding methods are all now commonly used procedures for identification of bacteria to genus, species and subspecific levels. These methods enable discrimination of species, strains and pathovars that could not previously be reproducibly resolved using traditional phenotype profiling methods, representing a significant advance in pathogen identification. Full taxonomic identification using MLST, MLSA or multiple barcoding markers from a series of housekeeping genes can be time consuming and expensive and is not always required for routine identification purposes in the diagnostic laboratory. This work was therefore undertaken to validate a number of protocols, each based on partial sequence analysis of a single gene previously used in more comprehensive phylogenetic analyses, for the routine identification of specific plant pathogenic bacteria of quarantine importance. The aim of the project was to collate, evaluate and format data from published phylogenetic studies and ongoing sequencing studies in European diagnostic laboratories to provide standardised protocols for routine use in diagnostic laboratories for rapid identification of bacterial plant pathogens of statutory importance (including certain Xanthomonas spp., pathovars of the Pseudomonas syringae complex, Erwinia amylovora and Dickeya spp).