DETECTION AND IDENTIFICATION OF CLAVIBACTER-MICHIGANENSIS SUBSP SEPEDONICUS AND CLAVIBACTER-MICHIGANENSIS SUBSP MICHIGANENSIS BY NONRADIOACTIVE HYBRIDIZATION, POLYMERASE CHAIN-REACTION, AND RESTRICTION ENZYME ANALYSIS

To develop a rapid and reliable detection and identification method fo r Clavibacter michiganensis subsp. sepedonicus and C. michiganensis su bsp. michiganensis, two biotinylated probes and derived primer sets we re evaluated for specificity using a large number of bacterial strains . Detection in dot blot analysis using the Diagen probe against C. mic higanensis subsp. sepedonicus was possible with all 32 C. michiganensi s subsp. sepedonicus strains tested. Cross-hybridization occurred with all nine C. michiganensis subsp. insidiosus strains tested. No hybrid ization occurred with any of 54 other related and unrelated bacterial strains including C. michiganensis subsp. michiganensis, C. michiganen sis subsp. nebraskensis, C. michiganensis subsp. tessellarius, C. iran icus, C. rathayi, and C, tritici and potato saprophytes. Hybridization of the MIC 1 probe against C. michiganensis subsp. michiganensis was obtained with 22 out of 24 C. michiganensis subsp. michiganensis strai ns. A weak hybridization signal occurred only with two strains of C. m ichiganensis subsp. insidiosus. No hybridization occurred with any of the 71 other related and unrelated bacterial strains tested including tomato saprophytes. Restriction fragment length polymorphisms detected with the Diagen probe allowed differentiation between C. michiganensi s subsp. sepedonicus and the related C. michiganensis subsp. insidiosu s. Restriction fragment length polymorphism analysis using the MIC 1 p robe and BamHI showed at least two groups of patterns within C. michig anensis subsp. michiganensis. By using a primer set derived from the D iagen probe, a DNA sequence could be amplified with all C. michiganens is subsp. sepedonicus strains tested. Only the nontarget organism C. m ichiganensis subsp, insidiosus yielded a similar polymerase chain reac tion product. Restriction enzyme analysis of the polymerase chain reac tion product enabled rapid distinction between the subspecies. With a CMM primer set derived from the MIC 1 probe a DNA sequence was amplifi ed from the same 22 out of 24 C. michiganensis subsp. michiganensis st rains that showed hybridization with the MIC 1 probe. The polymerase c hain reaction product could be verified by restriction enzyme analysis . The Diagen and MIC 1 probes and derived primer sets were shown to be useful for the detection and identification of C, michiganensis subsp . sepedonicus and C. michiganensis subsp. michiganensis. The MTC 1 pro be, however, failed to detect two strains of the latter subspecies.