Differentiation of Clavibacter michiganensis subsp michiganensis from Other Clavibacter Species Found in Seed and Plant Tissues

The goal of successful diagnostic test development is to identify specific markers that give positive reactions with all target strains and negative reactions with non-target contaminants. However, this goal is rarely achieved with complete satisfaction. Therefore, a rational compromise between sensitivity and specificity, based on a better understanding of population diversity, is needed. Immunodiagnostic tests based on poly-or monoclonal antibodies are known to produce false positive reactions to two major groups of plant-and seed-borne bacteria. The first group consists of non-pathogenic Clavibacter sp., which have been isolated from many geographical locations and are culturally identical to Clavibacter michiganensis subsp. michiganensis (Cmm). Standard biochemical testing has shown that these Cmm-like strains are in fact Clavibacter, however, they show unique profiles when compared at the subspecies level. The role and/or importance of these strains is unknown. The second group consists of Ochrobactrum and Microbacterium spp., which also are associated with tomato plants and seeds and react positively with a commercial Immunostrip assay, but are culturally distinct from Cmm. Genetic analyses of 351 Cmm and 58 non-Cmm strains revealed varying combinations of six genes, four in the Cmm pathogenicity island and one on each pathogenicity plasmid, explaining the failure of current molecular diagnostic tools to correctly identify Cmm. A Loop-mediated Amplification (LAMP) assay was developed that can discriminate Cmm from these Cmm-like strains, as well as other Immunostrip-positive contaminants. Additionally, three new PCR primer sets have been designed to amplify three distinct genes, each of which can specifically detect Cmm without producing false positives. The LAMP and new PCR assays are based upon conserved markers, distinguishing them from assays that detect the presence of mobile/transmissible genetic elements and have varying results. This emphasizes a need to shift current assay development towards detection of stable/conserved genes that will result in development of more reliable assays for seed health and environmental testing. In this study, the LAMP assay was applied to seed testing, using an enrichment step on selective media. The LAMP assay detected Cmm in a 13-year-old naturally infected seed lot, as well as in an artificially infested seed lot, thus proving to be a useful tool for specific detection of Cmm from seed samples. Its utility for other types of plant and environmental sampling will be investigated.