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A PCR-Based Assay by Sequence-Characterized DNA Markers for the Identification and Detection of Aphanomyces euteiches

October 2000 , Volume 90 , Number  10
Pages  1,137 - 1,144

G. J. Vandemark , J. M. Kraft , R. C. Larsen , M. A. Gritsenko , and W. L. Boge

First, second, third, and fifth authors: Vegetable and Forage Crops Production, Agricultural Research Service, U.S. Department of Agriculture, Prosser, WA 99350; and fourth author: Washington State University-IAREC, Prosser 99350

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Accepted for publication 12 July 2000.

Polymerase chain reaction (PCR) products were identified and amplified from isolates of Aphanomyces euteiches and A. cochlioides. The products were cloned 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. cochlioides with the primer pair OPB10-FS-25 and OPB10-RS-25. A. euteiches was detected in roots of several varieties of field-grown peas collected from a root rot trial site. 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. Both pairs of primers were used in two-step PCR reactions in which annealing and extension was done in a single step at 72°C. This reduced the time required for amplification of the diagnostic PCR product and its resolution by electrophoresis to less than 3 h.

Additional keywords: Aphanomyces root rot, sequence-characterized amplified region.

The American Phytopathological Society, 2000