SELECTION OF POLYMERASE CHAIN-REACTION PRIMERS FROM AN RNA INTERGENIC SPACER REGION FOR SPECIFIC DETECTION OF CLAVIBACTER-MICHIGANENSIS SUBSP SEPEDONICUS

Specific polymerase chain reaction (PCR) primers targeting genomic DNA were selected for sensitive detection of Clavibacter michiganensis subsp. sepedonicus, causal agent of bacterial ring rot disease of potato. The intergenic spacer region, approximately 500 bp, between the 16S and 23S rRNA genes of Clavibacter michiganensis subsp. sepedonicus, michiganensis, insidiosus, nebraskensis, and tessellarius were initially amplified and sequenced. Subsequently, a pair of PCR primers (Sp1f and Sp5r) was selected on the basis of the sequence data. The primers specifically amplified a 215-bp fragment when Clavibacter michiganensis subsp. sepedonicus genomic DNA was used as template but did not amplify DNA from phenotypically related bacteria, including species of Rathayibacter (formerly Clavibacter) and other subspecies of C. michiganensis; serologically related bacteria isolated from potato stems; or other unknown saprophytic bacteria isolated from potato tubers. Detection of Clavibacter michiganensis subsp, sepedonicus by PCR using these primers was more sensitive than enzyme-linked immunosorbent assay (ELISA) and immunofluorescence tests based on monoclonal antibodies. Amplification products were obtained by PCR for all potato tuber samples that were positive for Clavibacter michiganensis subsp. sepedonicus by ELISA and immunofluorescence. In addition, tubers from ring rot infected plants, which tested negative in ELISA and immunofluorescence, were positive in PCR. However, 12 tubers from healthy plants were all negative in both the serological tests and PCR.