Submitted to: Journal of Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/23/2003
Publication Date: 12/1/2003
Citation: VANDEMARK, G.J., BARKER, B.M. QUANTIFYING PHYTOPHTHORA MEDICAGINIS IN SUSCEPTIBLE AND RESISTANT ALFALFA WITH A REAL-TIME FLUORESCENT PCR ASSAY. JOURNAL OF PHYTOPATHOLOGY, Vol. 151:577-583. 2003. Interpretive Summary: 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 Phytophthora medicaginis, which is responsible for root rot disease of alfalfa 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. Resistant plants had significantly less pathogen DNA than susceptible plants. Varieties that are used as highly resistant, 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 varieties. This assay will be used in conjunction with a similar assay previously developed for Aphanomyces euteiches, another cause of root rot disease in alfalfa, to help breeders more accurately select multiple disease resistance.
Technical Abstract: A real time fluorescent PCR assay using a set of specific primers and a fluorochrome-labeled probe (TaqMan) was developed to quantify the amount of Phytophthora medicaginis DNA in alfalfa plants that were classified as either resistant or susceptible to the pathogen based on visual assessment of disease response. The assay clearly discriminated among three standard check alfalfa populations with different levels of resistance based on the analysis of DNA extracted from the roots of bulked plant samples. In two independent experiments, the Spearman rank correlation between pathogen DNA content and the number of resistant plants in a bulked sample was greater than '0.89 and highly significant (P < 0.0001). Significantly less pathogen DNA was detected in bulked samples of a highly resistant check population than in bulked samples from more susceptible check populations. Analysis of individual plants indicated that significantly less pathogen DNA was detected in resistant plants than in susceptible plants. The results clearly demonstrate that reduced pathogen colonization is characteristic of a resistant response. The assay provides a considerably more precise method of quantifying pathogen content in root systems than the previously employed method of hyphal staining. Applications of the assay are considered for breeding programs and the study of microbial population dynamics in plants simultaneously infected with different pathogens.