USE OF REAL-TIME PCR TO EXAMINE THE RELATIONSHIP BETWEEN DISEASE SEVERITY IN PEA AND APHANOMYCES EUTEICHES DNA CONTENT IN ROOTS

Authors: Vandemark, George; Grunwald, Niklaus - Nik

Submitted to: European Journal of Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/21/2004
Publication Date: 5/1/2005
Citation: Vandemark, G.J., Grunwald, N.J. 2005. Use of real-time PCR to examine the relationship between disease severity in pea and Aphanomyces euteiches DNA content in roots. European Journal of Plant Pathology. 111:309-316.

Interpretive Summary: The soilborne plant pathogen Aphanomyces euteiches is a fungus-like organism that causes root rot disease of peas. A. euteiches is probably the most globally destructive root rot pathogen of pea, causing severe losses throughout the United States, Europe, Japan, Australia and New Zealand. Entire pea crops can be destroyed in severely infested fields if cool and wet conditions are prevalent in spring and early summer. The only economically viable method for disease control is to grow peas that are resistant to A. euteiches, but unfortunately, very little progress has been made in developing disease resistant pea varieties. 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. At the USDA-ARS, Prosser, WA, we have developed a new method for quantifying the amount of A. euteiches present in the roots of infected peas. The method is based on our ability to use the 'polymerase chain reaction' (PCR) to detect and quantify strands of DNA that are unique to the pathogen, which are not present in healthy peas. We used this method to quantify the amount of five different isolates of A. euteiches in five different pea varieties. What we typically observed was that the most resistant plants had the least amount of pathogen present in their roots. This assay will help breeders to more accurately select resistant plants, which ultimately will result in the faster development of resistant, high yielding pea varieties.

Technical Abstract: Aphanomyces euteiches causes severe root rot of peas. Resistance is limited in commercial pea cultivars. In this study, we used of a real-time fluorescent PCR assay specific for A. euteiches to study the relationship between disease severity and pathogen DNA content in infected peas. Five pea genotypes ranging in levels of resistance were inoculated with five isolates of A. euteiches. Plants were visually rated with a disease disease index of 0 (healthy) ' 5 (extreme root necrosis) and the amount of pathogen DNA in roots was determined using the PCR assay. The susceptible genotypes Genie, DSP and Bolero tended to have significantly higher DSI and pathogen DNA content than the resistant genotypes 90-2079 and PI 180693. PI 180693 consistently had the lowest DSI ratings, while 90-2079 had the lowest amount of pathogen DNA detected in its root systems. The Spearman correlation between pathogen DNA quantity and DSI was positive and significant (P < 0.05) for three isolates, but was not significant for two other isolates. This suggests that the real-time PCR assay may have limited application as a selection tool for resistance in pea to A. euteiches, which would be dependent on the correlation between DSI and pathogen DNA content for a given pathogen isolate. The accuracy and specificity of the real-time PCR assay suggests considerable application for the assay in the study of mechanisms of disease resistance and the study of microbial population dynamics in plants.