PCR detection of Clavibacter michiganensis subsp. sepedonicus
Studies have been carried out in Canada to select and apply PCR primers for specific amplification and detection of Clavibacter michiganensis subsp. sepedonicus (EPPO A2 quarantine pest) in potato tuber samples. Primers targeting the intergenic spacer region between 16S and 23S rRNA genes have been selected. The spacer region between 16S and 23S rRNA genes has been almost completely sequenced for the 5 subspecies of Clavibacter michiganensis; and although it presents similarities among the subspecies, some base-pair differences could be used. The selected primers allowed specific detection of Clavibacter michiganensis subsp. sepedonicus and no reaction was obtained for other subspecies or closely related bacterial species. Amplification was also obtained with several strains of Clavibacter michiganensis subsp. sepedonicus from different geographical areas. PCR detection with these primers is more sensitive than ELISA and immunofluoresence (IF) tests based on monoclonal antibodies. Amplification was obtained for all potato tuber samples that were positive for Clavibacter michiganensis subsp. sepedonicus by ELISA or IF. No reaction was observed on healthy tubers. In addition, tubers from ring rot-infected plants, which tested negative in ELISA and IF, gave positive results by PCR.
Though this PCR test can be applied rather easily on a large number of samples, it is not expected that it will replace serological tests in routine, as it is more time-consuming. However, the authors felt that it is a useful test as it could confirm doubtful results given by serology, and could also provide a useful tool when studying the potential for persistence of the bacteria in the soil outside potato tissues.
Li, X.; De Boer, H. (1995) Selection of Polymerase Chain Reaction primers from an RNA intergenic spacer region for specific detection of Clavibacter michiganensis subsp. sepedonicus.
Phytopathology, 85(8), 837-842.