Comparison of Different Methods in Detection of Clavibacter michiganensis subsp michiganensis in Tomato Seeds

Several methods used for the detection of Clavibacter michiganensis subsp. michiganensis (Cmm) in artificially infested tomato seed lots were evaluated using two seed extraction methods (grinding and soaking). The detection threshold was evaluated by adding different numbers of artificially infested seeds (one, five and ten) to noninfected seed samples of two different sizes: 2000 and 5000 seeds. Four detection methods were simultaneously compared for their sensitivity in Cmm detection in seeds: isolation on selective media, direct PCR from seed material, Bio-PCR which first cultured bacteria on NBY agar prior to PCR, and Enrichment PCR. In samples of 2000 seeds containing one, five and ten infested seeds, the pathogen was detected in at least four out of five replicates by three detection methods (selective plating, direct PCR and Bio-PCR), using both the grinding extraction method (with an addition of centrifugation step) and soaking. DNA extraction from seed material was needed before the Enrichment PCR and direct PCR amplifications were performed. In samples of 5000 seeds one infested seed could not be detected by direct PCR or Enrichment PCR in any of the five replicates, regardless of the seed extraction method used. In samples containing five infested seeds per 5000 seeds, Cmm was detected by all four detection methods. A comparison of the efficiency of detection of the pathogen by using extraction methods that included grinding and soaking showed no differences between them.