Diversity of Clavibacter michiganensis subsp michiganensis populations in tomato seed:: What is the significance?

Clavibacter michiganensis subsp. michiganensis (Cmm), causal agent of bacterial canker of tomato, coexists with numerous saprophytic bacteria in seed, some of which share similar colony morphologies. Weakly virulent and avirulent Cmm are also frequently isolated from seed and plants. The challenges to seed health lie in accurately differentiating Cmm from yellow seed contaminants and establishing the etiological role of Cmm subpopulations with varying levels of virulence. Eighteen seed lots were assayed using semiselective media followed by ELISA. A monoclonal antibody (MAb Cmm1) was used to confirm identity of 59 strains of Cmm isolated from seed and to differentiate Cmm from other yellow-pigmented bacteria. Subsets of strains were tested using bacteriological tests, the MicroLog (TM) identification system, rep-PCR using the BOXA1R primer, 16S rRNA gene sequence analysis, and pathogenicity to determine which methods most accurately differentiated Cmm from other yellow bacteria. Consistent identifications were obtained using MAb Cmm1, MicroLog (TM), rep-PCR, and 16S rRNA gene sequence analysis. A filtration and colony blot immunoassay was developed to increase sensitivity and efficiency of the seed assay. Eighty-one percent of the Cmm strains isolated from seed and tested for pathogenicity were hypovirulent or avirulent; thus, studies were initiated to determine their significance. Co-inoculation of virulent and avirulent Cmm strains did not alter the capacity of either strain to colonize tomato stems. The etiological significance of weakly virulent and avirulent Cnint strains is unknown, but until it is established that they pose no threat to seed health, they cannot be ignored when developing new detection assays.