A PCR-based assay by sequence-characterized DNA markers for the identification and detection of Aphanomyces euteiches

Polymerase chain reaction (PCR) products were identified and amplified from isolates of Aphanomyces euteiches and A. cochlioides. The products were cl oned and sequenced, and the data were used to design pairs of extended PCR primers to amplify sequence-characterized DNA markers. The primer pair OPC7 -FS-30 and OPC7-RS-25 amplified a single 1,332-bp product from all isolates of A. euteiches that were not amplified from any other isolates tested. A single 718-bp product was selectively amplified only from isolates of A. co chlioides with the primer pair OPE 10-FS-25 and OPB10-RS-25. A. euteiches w as detected in roots of several varieties of field-grown peas collected fro m a root rot trial site. PCR also detected A. euteiches in the organic frac tion of field soil samples. Both pairs of extended primers were used in a m ultiplex reaction to unambiguously discriminate between A. euteiches and A. cochlioides. Both pairs of primers were used in two-step PCR reactions in which annealing and extension was done in a single step at 72 degrees C. Th is reduced the time required for amplification of the diagnostic PCR produc t and its resolution by electrophoresis to less than 3 h.