A PCR-Based Assay for the Identification and Detection
of Aphanomyces euteiches
George J. Vandemark, John M. Kraft, Richard C. Larsen, Marina A. Gritsenko and William L. Boge. USDA-ARS, Department of Crop and Soil Sciences, Washington State University, Prosser, WA 99350
Aphanomyces euteiches is a causal agent of root rot in alfalfa, peas, and bean. Alfalfa is infected by at least two different races of the pathogen, which exhibit differential virulence towards varieties employed as standard checks. It is often difficult to detect the pathogen in an efficient and timely manner because of the need for oospore production for accurate microscopic detection and the presence of other soil microorganisms that can confound identification based on microbiological techniques. 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 SCARs were developed by first identifying randomly amplified polymorphic DNA (RAPD) markers that were selectively amplified by all isolates tested of A. euietches, but which were not amplified by any of the other tested soil microbes. These PCR products were cloned and sequenced, and the nucleotide sequences were used for designing SCAR primers. The primer pair OPC7-FS30/OPC7-RS25 amplified a single PCR product of 1332 bp in all tested isolates of A. euteiches. This primer could be used in a two-step PCR reaction, in which the annealing of primer to template DNA and subsequent primer extension were done in a single step of 1 min at 72oC. 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. The amplification of a single PCR product that is specific to A. euteiches provides a rapid means of detecting the pathogen in both infected plants and soil samples. 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.