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Clavibacter michiganensis subsp. michiganensis, the cause of bacterial canker disease, is one of the most destructive pathogens in greenhouse and field tomato. The pathogen is now present in all main production areas of tomato and is widely distributed in the European and Mediterranean Plant Protection Organization region. The inspection and quarantine of the plant pathogens relies heavily on accurate detection tools. Primers and probes reported in previous studies do not distinguish the C. michiganensis subsp. michiganensis pathogen from other closely related subspecies of C. michiganensis, especially the nonpathogenic subspecies that were identified from tomato seeds recently. Here, we have developed a droplet digital PCR (ddPCR) method for the identification of this specific bacterium with primers/TaqMan probe set designed based on the pat-1 gene of C. michiganensis subsp. michiganensis. This new primers/probe set has been evaluated by real-time PCR (qPCR) and ddPCR. The detection results suggest that the ddPCR method established in this study was highly specific for the target strains. The result showed the positive amplification for all five C. michiganensis subsp. michiganensis strains, and no amplification was observed for the other 43 tested bacteria, including the closely related C. michiganensis strains. The detection threshold of ddPCR was 10.8 CFU/ml for both pure C. michiganensis subsp. michiganensis cell suspensions and infected tomato seed, which was 100-fold more sensitive than qPCR performed using the same primers and probe. The data obtained suggest that our established ddPCR could detect C. michiganensis subsp. michiganensis even with low bacterial load, which could facilitate both C. michiganensis subsp. michiganensis inspection for pathogen quarantine and the routine pathogen detection for disease control of black canker in tomato.
A 2021 study studied this question.