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Title: QUANTIFYING APHANOMYCES EUTEICHES IN ALFALFA WITH A FLUORESCENT POLYMERASE CHAIN REACTION ASSAY

Author
item Vandemark, George
item Barker, Bridget
item GRITSENKO, M - WSU-IAREC, PROSSER, WA

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/9/2001
Publication Date: 3/1/2002
Citation: VANDEMARK, G.J., BARKER, B.M., GRITSENKO, M.A. QUANTIFYING APHANOMYCES EUTEICHES IN ALFALFA WITH A FLUORESCENT POLYMERASE CHAIN REACTION ASSAY. PHYTOPATHOLOGY, 92:265-272. 2002.

Interpretive Summary: Often the factor that most limits progress for disease resistance breeding programs is the inability to discriminate between plants that appear to have approximately equal levels of disease based on visual examination of symptoms. The majority of plant diseases are caused by fungi, and previous methods for quantifying fungi in plant tissue, such as staining of fungal biomass with dyes or detection of fungal-specific proteins, are very limited in precision. At the USDA-ARS, Prosser, WA, a new method for quantifying the amount of pathogen present in infected plants has been developed based on a "real-time fluorescent PCR" assay. This assay can be used to both detect and quantify the soilborne plant pathogen Aphanomyces euteiches, which is responsible for root rot disease of alfalfa, beans, peas and other legumes. The assay is very precise and a very significant positive correlation has been observed between the amount of pathogen DNA detected with this assay and the severity of disease in infected alfalfa plants. Varieties that are used as resistant and susceptible checks were also examined by randomly bulking together plants, isolating DNA from the roots of the bulked sample, and performing the PCR assay. In all experiments, significantly more pathogen was detected in susceptible check variety than in the resistant check variety. Ranking of commercial alfalfa varieties based on results with this assay very closely approximated rankings based on visual examination of disease severity. This assay will assist breeders to more accurately select the most resistant plants for use in breeding programs, and will accelerate the development of extremely resistant alfalfa varieties.

Technical Abstract: A PCR assay using a set of specific primers and a dual-labeled probe (TaqMan) was developed to quantify the amount of Aphanomyces euteiches DNA in alfalfa plants exhibiting varying levels of disease severity. The study included isolates of race 1 and race 2 of A. euteiches. The assay also discriminated between alfalfa populations for resistance based on analysis of DNA extracted from bulked plant samples. Analysis of individual plants and bulked plant samples of standard check populations with both pathogen isolates resulted in Spearman rank correlations between pathogen DNA content and disease severity index (DSI) ratings that were greater than 0.75 and highly significant (P < 0.0005). In experiments with a race 1 isolate, the amount of pathogen DNA present in the resistant check WAPH-1 was significantly less than in the susceptible check Saranac. In experiments with a race 2 isolate, the amount of pathogen DNA in the resistant check WAPH-5 was significantly less than in either of the susceptible checks, Saranac and WAPH-1. Discrimination between commercial varieties based on quantitative PCR analysis of bulked plant samples was similar to classification based on visual assessment of disease severity.