A real-time BIO-PCR assay for detection of Ralstonia solanacearum race 3, biovar 2, in asymptomatic potato tubers

Ralstonia solanacearum, the causal agent of brown rot of potato, can often be carried latently in seed potato tubers. We designed real-time PCR (polymerase chain reaction) primers and probe and a highly sensitive BIO-PCR assay for specific detection of the strains of race 3, biovar (bv.) 2 of R. solanacearum in asymptomatic potato tubers. The biovar-specific primers and probe reacted with all 17 strains of bv. 2 of R, solanacearum tested, including 12 from potato and 5 from geranium. None of the other 35 strains of R. solanacearum reacted, including 4 strains of bv. I from potato or the closely related blood disease bacterium from banana. None of 13 other bacteria reacted, including 5 Erwinia and 2 Clavibacter species from potato. Using undiluted potato tuber extract, inoculated with R. solanacearum bv. 2, as few as 30 cells/mL of extract could be detected. Two of 14 naturally infected potato tubers with no disease symptoms were positive by the newly described real-time BIO-PCR assay, whereas none were positive with a standard real-time PCR assay. This is the first report of the detection of R. solanacearum bv. 2 in asymptomatic, latently infected potato tubers by PCR. The real-time assay is very simple and much less time consuming than classical PCR.