A Quantitative Assay of Puccinia coronata f. sp. avenae DNA in Avena sativa

Crown rust, caused by Puccinia coronata f. sp. avenae, is the most damaging disease of oat. Quantification of the disease can be done by visual or digital assessments of diseased leaf area, lesion number, lesion size, and latent period. Laborious measurements of sporulation can also be made. As an alternative to these methods, a new quantitative assay was developed. The method employs simple inoculum application, quantitative sampling from inoculated areas, a closed tube DNA extraction method restricting loss of tissue, and real-time polymerase chain reaction (PCR) using a pathogen-specific TaqMan primers/probe set. Image analyses of genotypes with varying levels of crown rust resistance were compared to fungal DNA (FDNA) estimations by the new assay. The moderately resistant genotype IA98822-2 was distinguished from susceptible genotypes at the seedling stage, and the moderately resistant genotype O × T 107 was distinguished from the resistant cultivar TAM-O-301 at seedling and adult plant stages using FDNA. These separations were not evident with digital image analysis. The new assay also detected fungal development earlier and more rapidly in genotypes with lower levels of resistance. The assay can consistently estimate disease and should be useful for studying many aspects of the crown rust host-pathogen interaction where precise assessment of pathogen development is needed.