PCR DETECTION OF ERWINIA-CAROTOVORA SUBSP ATROSEPTICA ASSOCIATED WITHPOTATO TISSUE

The nucleotide sequence for a DNA hybridization probe specific for Erw inia carotovora subsp. atroseptica was determined, and primers were se lected for detection of the blackleg pathogen using the polymerase cha in reaction (PCR). Primers ECA1f (5'-CGGCATCATAAAAACACG-3') and ECA2r (5'-GCACACTTCATCCAGCGA-3') specifically amplified a 690-bp DNA fragmen t of all E. carotovora subsp. atroseptica strains tested but not strai ns of other E. carotovora subspecies isolated from various hosts and g eographic regions or other plant- and soil-associated bacteria. Visual ization of the E. carotovora subsp. atroseptica-specific PCR product o n ethidium bromide-stained agarose gels required a minimum of 250 to 5 00 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 immunos orbent assay (ELISA) with a monoclonal antibody specific for the lipop olysaccharide of E. carotovora subsp. atroseptica. Although 50.6% of t hese samples were positive in PCR compared to 46.5% in ELISA, some of the ELISA-positive samples were negative in PCR. When E. carotovora su bsp. atroseptica DNA was added to these samples, amplified products we re obtained in all but two samples after repeating the PCR, indicating that in most cases the failure to obtain PCR amplification for some o f the ELISA-positive samples was not due to the presence of inhibitors .