Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 12, 2000
Publication Date: September 1, 2000
Citation: VANDEMARK, G.J., KRAFT, J.M., LARSEN, R.C., GRITSENKO, M.A., BOGE, W.L. A PCR-BASED ASSAY USING SEQUENCE CHARACTERIZED DNA MARKERS FOR THE IDENTIFICATION AND DETECTION OF APHANOMYCES EUTEICHES. PHYTOPATHOLOGY, 90:1137-1144. 2000. Interpretive Summary: All plants, both weeds and cultivated varieties, are susceptible to root and stem diseases caused by fungi that live in the soil. Losses in production due to diseases caused by soilborne fungi amount to several billion dollars annually in the United States. Because it is often prohibitively expensive to use chemical fungicides for controlling plant diseases caused by fungi, and because the application of chemical fungicides may have adverse health or environmental effects, an alternative method for controlling losses is to survey production fields for the presence of disease causing fungi prior to planting. If the grower knows that a disease causing fungus is present, the grower can often choose to cultivate a plant variety that is resistant to the fungus. One particularly destructive disease of alfalfa, peas and beans is root rot caused by the soilborne fungus Aphanomyces euteiches. We found that it was possible to rapidly and accurately detect the presence of this fungus in both infected plant roots and in field soil using molecular tools we have developed. Our system can detect the presence of the fungus in less than three hours, as compared to three weeks required for detecting the fungus using previously developed procedures. These results will help growers of alfalfa, peas, and beans by making it easier for them to know prior to planting if the fungus is present in their fields. This will result in reductions in losses due to disease caused by the fungus.
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.