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Submitted to: International Aphanomyces Symposium
Publication Type: Proceedings Publication Acceptance Date: 6/1/2003 Publication Date: 6/15/2003 Citation: VANDEMARK, G.J. REAL TIME PCR ASSAYS FOR EXAMINING RESISTANCE TO APHANOMYCES EUTEICHES AND PHYTOPHTHORA MEDICAGINIS IN LEGUMES AND FOR STUDYING MICROBIAL POPULATION DYNAMICS IN MIXED PLANT INFECTIONS. Proceedings of the 2nd International Aphanomyces Symposium. . 2003. Interpretive Summary: Generally, it is not economical to apply chemical fungicides to control diseases of legumes such as alfalfa and peas. In legumes these diseases are most often controlled by the cultivation of varieties that have genetic disease resistance. 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. Efforts have been made previously to quantify the amount of pathogen present in infected roots in order to identify highly resistant plants. However, these quantification methods have tended to have very limited precision. Additionally, a perennial plant such as alfalfa may survive for over five years in the field and become infected at one time by several different plant pathogens. At the USDA-ARS, Prosser, WA, new methods for quantifying the amount of pathogen present in infected plants have been developed based on molecular assays that are pathogen-specific. These assays are used to quantify several different soilborne plant pathogens, including Aphanomyces euteiches and Phytophthora medicaginis, which are responsible for root rot disease of alfalfa and other legumes. The assays are very precise, and very significant positive correlations have been observed between the amount of pathogen DNA detected with these assay and the severity of disease in infected alfalfa and pea plants. Resistant plants consistently have significantly less pathogen DNA than susceptible plants. In the future, we will use these molecular tools to simultaneously screen alfalfa and peas for resistance to several different diseases. Technical Abstract: Real-time PCR assays using sets of specific primers and dual-labeled probes (TaqMan) were developed to quantify the amount of Aphanomyces euteiches DNA and Phytophthora medicaginis DNA in alfalfa plants. The amount of A. euteiches present in infected peas was also quantified. Spearman rank correlations between A. euteiches DNA content and disease severity index (DSI) ratings were positive and significant for both alfalfa and pea. In all experiments, significantly more pathogen was detected in susceptible populations than in resistant populations. For experiments conducted with P. medicaginis, the Spearman rank correlation between pathogen DNA content and the number of resistant alfalfa plants in a bulked sample was negative and significant. Analysis of individual alfalfa plants indicated that significantly less P. medicaginis DNA was detected in resistant plants than in susceptible plants. These results clearly demonstrate that reduced pathogen colonization is characteristic of the resistance response in alfalfa and peas to two different Oomycete plant pathogens. The two real-time assays may be useful for simultaneously selecting for resistance to both pathogens and for studying microbial population dynamics in mixed plant infections. |
