Evaluation of PCR methods for the diagnosis of bacterial ring rot infections in potato 1

Abstract

A 434‐bp fragment of Clavibacter michiganensis subsp. sepedonicus DNA was sequenced and four different primers were designed for PCR amplification. PCR products of 200 bp and 300 bp were consistently amplified with genomic DNA of the bacterium. Preliminary specificity experiments indicated that these primers only amplified DNA from the ring‐rot bacterium. Composite 400‐tuber samples were collected from a lot of 20 000 kg of potatoes or individual tuber samples with suspected ring‐rot symptoms. The samples were divided into two parts and parallel tests with immunofluorescence and PCR were done. Potato‐tissue samples for PCR detection were extracted once with 1:1 phenol‐chloroform. The optimal dilution of the sample for PCR‐based detection was 1:10. Altogether 200 samples were analysed by both immunofluorescence and PCR methods. In most cases, the same positive and the same negative samples were revealed. In some cases, PCR revealed the samples with a lower degree of contamination more easily. Positive and negative samples were very easily detected by the PCR‐based assay. Large‐scale specificity experiments will be needed to clarify whether our primers only detected the ring‐rot bacteria and not closely related saprophytic bacteria.