Title: REAL-TIME PCR ASSAY TO QUANTIFY FUSARIUM GRAMINEARUM WILD-TYPE AND RECOMBINANT MUTANT DNA IN PLANT MATERIAL Authors
Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 16, 2006
Publication Date: December 1, 2006
Citation: Dyer, R.B., Kendra, D.F., Brown, D.W. 2006. Real-time PCR assay to quantify Fusarium graminearum wild-type and recombinant mutant DNA in plant material. Journal of Microbiological Methods. 67(2006):534-542. Interpretive Summary: Fusarium verticillioides is a fungus that causes some of the most important diseases of corn world-wide. These diseases significantly affect the yield of corn and therefore represent an economic burden to farmers. In addition, the fungus produces a toxin called fumonisin that can contaminate corn kernels thereby posing a serious health risk for both livestock and humans. Of particular concern is the ability of the fungus to escape detection by establishing an asymptomatic (ie. non-disease producing) infection in host plants. Lack of knowledge on this host-fungus relationship in this asymptomatic or endophytic infection makes implementing control strategies difficult. We developed a test to detect and measure the amount of fungus within various tissues of the corn plant as the plant matures from the seedling stage to maturity. We showed that our test is reproducible and sensitive and we confirmed the results of others who used more laborious methods for endophytic fungal detection. This test will allow the dynamics of the endophytic relationship to be studied in a less laborious and time consuming fashion.
Technical Abstract: Fusarium verticillioides is one of the most important world-wide pathogens of maize causing yield loss as well as health problems for livestock and humans through the ingestion of fumonisin contaminated grain. Of particular concern is the ability of F. verticillioides to establish an asymptomatic endophytic relationship with the host plant and the potential contamination of visually healthy corn with fumonisins. The lack of understanding of the dynamics of the endophytic relationship is a limiting factor in implementing effective control strategies. Current methods for studying the host-pathogen relationship are tedious, time consuming, and not very sensitive. We describe the first report that applies real-time PCR to the detection and quantification of F. verticillioides in various tissues of corn during plant development from the seedling stage to ear development. We document the reproducibility and sensitivity of our assay. Four independent standard curves had a reproducible titration of F. verticillioides genomic DNA from 40 ng to 2.56 pg. In some cases, the sensitivity could be increased 5-fold to 0.512 pg. Using our method, we confirm the results of others on the ability of F. verticillioides to colonize root, stem and leaf tissue of seedlings. In addition we show that seed infection can be a route to colonization of stalk and ear tissue of more mature plants.