LAUSR

Clavibacter michiganensis (Cm) is a Gram-positive bacterial pathogen that proliferates in the xylem vessels of tomato, causing bacterial wilt and canker symptoms. Accurate detection is a crucial step in confirming outbreaks of bacterial canker and developing management strategies. A major problem with existing detection methods are false positive and negative results. Here, we report the use of comparative genomics of 37 diverse Clavibacter strains, including 21 strains sequenced in this study, to identify specific sequences that are Cm detection targets. Genome-wide phylogenic analyses revealed additional diversity within the genus Clavibacter. Pathogenic Cm strains varied in plasmid composition, highlighting the need for detection methods based on chromosomal targets. We utilized sequences of Cm specific loci two develop a multiplex PCR based diagnostic platform using two Cm chromosomal genes (rhuM and tomA) and an internal control amplifying both bacterial and plant DNA (16s rRNA). The multiplex PCR assay specifically detected Cm strains from a panel of 110 additional bacteria, including other Clavibacter species and bacterial pathogens of tomato. The assay was adapted to detect the presence of Cm in seeds and tomato plant materials with high sensitivity and specificity. In conclusion, the described method represents a robust, specific tool for detection of Cm in tomato seeds and infected plants.