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Title: QUANTIFYING THE RELATIONSHIP BETWEEN DISEASE SEVERITY AND THE AMOUNT OF APHANOMYCES EUTEICHES DETECTED IN ROOTS OF ALFALFA AND PEA WITH A REAL-TIME PCR ASSAY

Author
item Vandemark, George
item BARKER, B

Submitted to: Archives of Phytopathology and Plant Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/23/2003
Publication Date: 5/1/2003
Citation: VANDEMARK, G.J., BARKER, B.M. QUANTIFYING THE RELATIONSHIP BETWEEN DISEASE SEVERITY AND THE AMOUNT OF APHANOMYCES EUTEICHES DETECTED IN ROOTS OF ALFALFA AND PEA WITH A REAL-TIME PCR ASSAY. ARCHIVES OF PHYTOPATHOLOGY AND PLANT PROTECTION, Vol. 36, pp. 81-93. 2003.

Interpretive Summary: The soilborne plant pathogen Aphanomyces euteiches is responsible for root rot disease of alfalfa, beans, peas and other legumes. A. euteiches is probably the most destructive root rot pathogen of pea on a global scale, and very little progress has been made in developing commercial varieties of pea with adequate levels of resistance to this pathogen. 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. Previous methods for quantifying pathogens in plant tissue, such as staining of biomass with dyes or detection of pathogen-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. 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 and pea plants. For both alfalfa and pea, significantly more pathogen was detected in susceptible varieties than in resistant varieties. For both peas and alfalfa, the least amount of pathogen DNA was detected in the healthiest plants, while the greatest amount of pathogen DNA was detected in the sickest plants. 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 and pea varieties.

Technical Abstract: A real-time PCR assay was used to quantify the relationship in alfalfa and pea between disease severity and amount of Aphanomyces euteiches detected in roots. The study included isolates of race 1 and race 2 of the alfalfa pathovar of A. euteiches and an isolate obtained from diseased pea. Spearman rank correlations between pathogen DNA content and disease severity index (DSI) ratings were positive (¿ 0.57) and significant (P ¿ 0.0007) for individual alfalfa plants, bulked alfalfa plant samples, and individual pea plants. In all experiments, significantly more pathogen was detected in susceptible populations than in resistant populations. The results clearly demonstrate that resistance to A. euteiches in both alfalfa and pea is characterized by a reduction in pathogen colonization relative to levels observed for susceptible reactions. The assay was very specific for A. euteiches, producing very linear assays with DNA extracted from pathogen isolates obtained from alfalfa, pea, and bean. Possible applications of the assay in conjunction with other real-time PCR assays specific to other legume pathogens are discussed in relation to simultaneous disease screening for multiple plant pathogens and the study of microbial population dynamics in mixed plant infections.