During 2013-2016, 277 tomato fields were surveyed across Iran to monitor the status of bacterial canker of tomato, caused by Clavibacter michiganensis subsp. michiganensis. Altogether, 450 plant samples Ð either symptomatic or symptomless Ð were collected, which allowed recovering 35 bacterial strains. The latter were positive for the PCR test performed using the Clavibacter-specific primer pair CMR16F1/CMR16R1. Based on the phylogeny of the gyrB gene sequences, 31, three and one out of 35 strains were identified as C. michiganensis, Microbacterium sp., and Agrococcus sp., respectively. Among the 31 strains, 23, six, and two strains were identified as C. michiganensis subsp. michiganensis, C. michiganensis subsp. tessellarius and Clavibacter spp., respectively, based on the phylogeny of the gyrB gene, which was further confirmed by multilocus sequence analysis (MLSA) of five housekeeping genes (i.e. atpD, gyrB, ppk, recA, and rpoB). In pathogenicity tests, all 23 strains induced wilting symptoms on tomato plants in greenhouse conditions, while no symptoms were observed on eggplant, bell pepper and chili pepper plants. All evaluated pathogenicity determinant genes (i.e. celA, pat-1, tomA, ppaA, chpC, and chpG)were detected, in 18 out of 31 C. michiganensis strains, using eight specific primer pairs. Estimation of the number of nucleotide differences, sequence similarity matrix, and MLSA clustered two peach-colored strains (Tom495 and Tom532) separately from all nine previously described subspecies, thereby suggesting these two strains as a new subspecies of C. michiganensis. However, a detailed taxonomic study, using multi-phased molecular approaches is needed to delineate a formal taxonomic name for these atypical strains.
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