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Title: Real-time PCR suggests that Aphanomyces euteiches is associated with reduced amounts of Phytophthora medicaginis in alfalfa that is co-inoculated with both pathogens

Author
item Vandemark, George
item Ariss, Jennifer
item HUGHES, TERESA - UNIVERSITY OF WISCONSIN

Submitted to: Journal of Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/27/2009
Publication Date: 2/15/2010
Citation: Vandemark, G.J., Ariss, J., Hughes, T. 2010. Real-time PCR suggests that Aphanomyces euteiches is associated with reduced amounts of Phytophthora medicaginis in alfalfa that is co-inoculated with both pathogens. Journal of Phytopathology. 158:117-124.

Interpretive Summary: As a consequence of being grown for multiple years, alfalfa often is infected at the same time by numerous pathogens. Accordingly, alfalfa varieties that have resistance to multiple pathogens are commercially desired. When a plant is infected by several pathogens at the same time it can be very difficult to determine what pathogen is responsible for disease symptoms. Historically, breeders and plant pathologists have tried to determine what pathogen is causing disease by measuring the amounts of different pathogens present in plants at any given time. Unfortunately, these historical methods, such as staining plant roots for the presence of pathogens, cannot be used with any precision or confidence when the two pathogens are very similar in appearance. We have developed methods for quantifying pathogens in infected plants based on the detection of DNA sequences that are pathogen-specific. These methods (assays) can be used to both detect and quantify the soilborne plant pathogens Aphanomyces euteiches and Phytophthora medicaginis, which are responsible for root rot disease of alfalfa and other legumes. These two pathogens are closely ‘related’ and very difficult to distinguish based on structure, but the assays are very precise and can clearly distinguish between the two pathogens. We took an alfalfa population that was resistant to both pathogens and another population that was susceptible to both pathogens and inoculated both populations at the same time with both A. euteiches and P. medicaginis. Significantly more of both pathogens could be detected in the susceptible alfalfa population than in the resistant population. Most interesting however, was that less P. medigaginis could be detected in both the resistant and susceptible populations when these plants were inoculated at the same time with both A. euteiches and P. medicaginis than when inoculated with only P. medicaginis. On the other hand, the amount of A. euteiches was relatively constant whether or not plants were inoculated with both P. medicaginis and A. euteiches or with A. euteiches alone. These results suggest that the presence of A. euteiches is inhibiting the proliferation of P. mediganinis, but not vice versa. One possible explanation is that the presence of A. euteiches induces the plant to produce a chemical that is inhibitory to P. medicaginis. This would be an excellent adaptive strategy, since both pathogens are often found in the same field. This is the most successful example to date of being able to quantify two closely related pathogens at once in the same plant, and emphasizes the accuracy and utility of these DNA-based assays. These assays can be applied to screen alfalfa for resistance to two diseases at the same time, and can also be used to study mechanisms involved in the interactions between different plant pathogens.

Technical Abstract: Aphanomyces euteiches and Phytophthora medicaginis are two oomycete pathogens of seedling and mature alfalfa (Medicago sativa L.) that are frequently found in the same field sites. In order to investigate possible interactions of these two pathogens, two greenhouse factorial experiments were conducted on seedling alfalfa representing the phenotypic resistance classes of dual susceptibility and dual resistance to both pathogens. Seedlings were challenged with multiple inoculum concentrations of A. euteiches and P. medicaginis. The measured responses were the proportion of plants rated resistant, and quantities of pathogen DNA as detected by real-time polymerase chain reactions (PCR) assays. For both pathogens, there was significantly more pathogen DNA detected as determined by real-time PCR in the susceptible population compared to the resistant population in all treatment combinations. In general, co-inoculation with both A. euteiches and P. medicaginis reduced the amount of P. medicaginis DNA detected when compared to inoculation with P. medicaginis alone. Co-infestation by both pathogens did not reduce the quantity of A. euteiches detected. These results suggest that A. euteiches is inhibiting the ability of P. medicaginis to prolifierate in plants co-inoculated with both pathogens. A possible explanation is that A. euteiches is inducing the production of a phytoalexin in infected plants that is does not affect A. euteiches but has an inhibitory effect against P. medicaginis. This is the first report to accurately quantify microbial population dynamics in plants co-inoculated with two different oomycete pathogens.