Submitted to: Canadian Phytopathological Society Meeting
Publication Type: Proceedings
Publication Acceptance Date: 3/1/2000
Publication Date: 6/1/2000
Citation: VANDEMARK, G.J., KRAFT, J.M., LARSEN, R.C., GRITSENKO, M.A. A PCR-BASED ASSAY USING SEQUENCE CHARACTERIZED DNA MARKERS FOR THE IDENTIFICATION AND DETECTION OF APHANOMYCES EUTEICHES. PROCEEDINGS OF THE 2000 CANADIAN PHYTOPATHOLOGICAL SOCIETY AND THE PACIFIC DIVISION OF THE AMERICAN PHYTOPATHOLOGICAL SOCIETY, JUNE 2000, P. 35. 2000.
Technical Abstract: PCR products were identified that were only amplified from isolates of Aphanomyces euteiches or A. cochlioides. These products were cloned and sequenced, and the sequences 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 1331 bp product from all isolates of A. euteiches that was not amplified from any other fungal isolates. A single 718 bp product was selectively amplified only from all isolates of A. cochlioides using the primer pair OPB10-FS-25 and OPB10-RS- 25. A. euteiches was detected in pea roots one day after inoculation with a zoospore suspension. PCR also detected A euteiches in the organic fraction of field soil samples. Both pairs of extended primers were used in a multiplex reaction to unambiguously discriminate between A. euteiches and A. cochlioides. The primers could be used in two-step PCR reactions, in which annealing and extension was done in a single step at 72oC. This reduced the time needed for the amplification of the diagnostic PCR product and its resolution by electrophoresis to less than three hours. This method provides a rapid and accurate assay for detecting A. euteiches.