Validation of a duplex TaqMan real-time PCR for detection of Colletotrichum coccodes and Rhizoctonia solani AG-3 on potato tubers

Summary. The potato pathogens Colletotrichum coccodes, (causing black dot), and Rhizoctonia solani anastomosis group 3 (AG-3) (causing black scurf and stem canker) are economically important, and are common in potato production regions, including Northern Italy. A duplex TaqMan quantitative real-time PCR, based on two previously validated singleplex methods, was tested for identification of these species in potato propagation material. This validation was carried out according to EPPO PM 7/98 standard guidelines. The limit of detection (LOD) for the method, assessed using serial dilutions of targets DNA, was 10 fg. Specificity was assessed by testing ten C. coccodes and four R. solani AG-3 isolates, and 19 non-target species including other Colletotrichum and Rhizoctonia spp. and potato pathogens, respecting inclusivity and exclusivity criteria. Selectivity of the test showed no influence of DNA obtained from potato tubers. Repeatability and reproducibility of the duplex qPCR were also validated. The assay was able to detect and distinguish, within a single run, the two fungi allowing early detection in potato tubers.