PCR DETECTION OF ERWINIA-CAROTOVORA SUBSP ATROSEPTICA ASSOCIATED WITH POTATO TISSUE

The nucleotide sequence for a DNA hybridization probe specific for Erwinia carotovora subsp. atroseptica was determined, and primers were selected for detection of the blackleg pathogen using the polymerase chain reaction (PCR). Primers ECA1f (5'-CGGCATCATAAAAACACG-3') and ECA2r (5'-GCACACTTCATCCAGCGA-3') specifically amplified a 690-bp DNA fragment of all E. carotovora subsp. atroseptica strains tested but not strains of other E. carotovora subspecies isolated from various hosts and geographic regions or other plant- and soil-associated bacteria. Visualization of the E. carotovora subsp. atroseptica-specific PCR product on ethidium bromide-stained agarose gels required a minimum of 250 to 500 CFU per mi. A total of 170 samples of potato stem and tuber tissue was tested by PCR and compared with reactiors in enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody specific for the lipopolysaccharide of E. carotovora subsp. atroseptica. Although 50.6% of these samples were positive in PCR compared to 46.5% in ELISA, some of the ELISA-positive samples were negative in PCR. When E. carotovora subsp. atroseptica DNA was added to these samples, amplified products were obtained in all but two samples after repeating the PCR, indicating that in most cases the failure to obtain PCR amplification for some of the ELISA-positive samples was not due to the presence of inhibitors.