|Kraft, John - RETIRED, USDA ARS|
|Gritsenko, Marina - WSU, IAREC, PROSSER, WA|
Submitted to: Alfalfa Improvement Conference Proceedings
Publication Type: Proceedings
Publication Acceptance Date: June 1, 2000
Publication Date: July 1, 2000
Citation: VANDEMARK, G.J., KRAFT, J.M., LARSEN, R.C., GRITSENKO, M.A., BOGE, W.L. A PCR-BASED ASSAY FOR THE IDENTIFICATION AND DETECTION OF APHANOMYCES EUTEICHES. PROCEEDINGS OF THE 37TH NORTH AMERICAN ALFALFA IMPROVEMENT CONFERENCE, JULY 2000, P. 282. 2000. Technical Abstract: Aphanomyces euteiches is a causal agent of root rot in alfalfa, peas, and bean. It is often difficult to detect the pathogen in an efficient and timely manner. We report here the development of sequence characterized amplified regions (SCARs) that differentiated all tested isolates of A. euteiches from other Aphanomyces sp. and from eight other genera of soilborne microbes, including closely related chromists such as Achlya sp. Phytopthora sp., and Pythium sp. The primer pair OPC7-FS30/OPC7-RS25 amplified a single PCR product of 1332 bp in all tested isolates of A. euteiches. The presence of the pathogen in plant roots could be confirmed with a protocol requiring less than 2 h for completion of both PCR and resolution by gel electrophoresis. A. euteiches could also be detected both in field grown plants and soil samples by amplification of the diagnostic SCAR. The SCAR sequence was also used to design primers and probes for quantitative PCR based on the detection of fluorescently-labeled amplicons We have designed two sets of primers and probes that have been successfully used with quantitative PCR to detect significant differences in inoculum levels of A. euteiches race 2 between the resistant check WAPH-5 and the susceptible checks WAPH-1 and Saranac. This protocol detects the pathogen in infected roots much earlier than can be done based on microscopic or microbiological techniques, and may provide a new tool for the study of processes of pathogenicity and virulence.