Evaluation of PCR assays for quantifying seed-borne infection by Fusarium and Microdochium seedling blight pathogens.

ABSTRACT

AIMS: To evaluate competitive PCR assays for quantifying seed-borne Microdochium and Fusarium seedling blight pathogen DNA and to determine test and year repeatability and sources of variability. METHODS AND RESULTS: Relationships between DNA and plate counts were significant for Fusarium and Microdochium seedling blight pathogens in 152 seed batches from 3 years. Coefficient of determinations, however, differed greatly (Fusarium; R(2) = 0.25, P = 0.029, Microdochium; R(2) = 0.73, P < 0.001). Significant differences between years were observed in the regression slopes for Microdochium. Pathogen DNA quantified in 16 extractions after sampling was highly correlated to results following storage for 1-2 years (R(2) > 0.90). Residual maximum likelihood analysis showed that the least and greatest variance components of the testing procedure were DNA extraction subsampling and PCR assay respectively. CONCLUSIONS: Amount of pathogen DNA is a useful estimator of seed batch contamination for Microdochium but not Fusarium seedling blight pathogens. Although reproducible over time, improvements to the testing procedure should focus on repeated PCR amplifications to reduce assay variability. SIGNIFICANCE AND IMPACT OF THE STUDY Replacing plate counts with competitive PCR for determining the severity of seed batch contamination is feasible in areas where Microdochium seedling blight pathogens predominate.