Detection of Clavibacter michiganensis subsp sepedonicus in potato tubers by BIO-PCR and an automated real-time fluorescence detection system

Ring rot of potato, caused by Clavibacter michiganensis subsp. sepedonicus, is one of the most regulated diseases of potatoes world wide. The organism is often difficult to detect in symptomless tubers because of low populati ons and slow competitive growth on available media. Polymerase chain reacti on (PCR) primers and a fluorescent probe for use in the Perkin Elmer 7700 a utomated real time PCR detection system (TaqMan) were designed from a C. mi chiganensis subsp. sepedonicus-specific genomic DNA fragment for developmen t of a BIO-PCR assay for C. michiganensis subsp, sepedonicus in potato tube rs. Results of screening the primers with strains of C. michiganensis subsp . sepedonicus and other bacteria showed the primers to be specific. A total of 30 naturally infected ring rot suspect tubers were sampled by the core extract, shaker incubation procedure and assayed by (i) plating aliquots on to agar media, (ii) classical PCR, and (iii) BIO-PCR. In all, 4 tubers were positive by agar plating and pathogenicity tests, 8 by classical TaqMan PC R, and 26 by TaqMan BIO-PCR. We conclude that BIO-PCR combined with the Taq Man automated closed detection system is a rapid, reliable method of assayi ng large numbers of potato tuber extracts for C, michiganensis subsp. seped onicus. Furthermore, for a large central laboratory running large numbers o f PCR assays, the high-throughput TaqMan system can reduce costs per sample over the more labor-intensive classical PCR.