Establishment and Application of a Multiplex PCR Assay for the Rapid Detection of Anastomosis Group (AG)-3PT, the Pathogen Causing Potato Black Scurf and Stem Canker.

anastomosis group 3 (AG-3) is the main causative agent of the soil-borne disease known as potato black scurf, which poses a huge threat to potato production. Rapid and accurate identification of AG-3 isolates in soil and potato seed tubers prior to planting is essential for good production. In this study, a multiplex PCR assay was established for the detection of AG-3. Two pairs of target-specific primers were designed from sequences for endopolygalacturonase and pyridoxine biosynthesis genes downloaded from GenBank. The main factors influencing PCR amplification, such as annealing temperature and primer concentration, were optimized. Results show that the proposed multiplex PCR assay is highly sensitive and specific for the target genes in the pathogen even when the DNA concentration is reduced to 20 fg/μL. The resulting calibration plot shows a linear relationship between electrophoretic band peaks and genomic DNA concentration (R = 0.98). The primer specificity was confirmed by applying them to other AG groups and plant pathogen species on which no amplicons were produced. Using the primers, we successfully detected small amounts of AG-3 present in soil and potato tuber samples. Taken together, the detection assay developed in this study has high sensitivity, strong specificity, and accuracy and can be used to detect and identify soil and potato seed tubers infected with AG-3.