Simultaneous detection of three crown rot pathogens in field-grown strawberry plants using a multiplex PCR assay

Fusarium wilt, Phytophthora root rot, and Anthracnose crown rot of strawberry are three infectious diseases that cause growth retardation and even death in strawberry plants. Because these diseases seriously harm the commercial strawberry industry, it is critically important to quickly detect the three causal pathogens. In this study, we established a multiplex PCR (mPCR) system to detect the three strawberry pathogens Fusarium oxysporum f. sp. fragariae (Fof), Phytophthora nicotianae (Pn), and Colletotrichum gloeosporioides (Cg). We identified three primer pairs for specifically detecting these pathogens. This was followed by determining the ideal concentration of each primer pair for mPCR. The optimum annealing temperature was found to be 59.2 °C and the optimal number of cycles was 35. The minimum detection limit of mPCR was 1 ng/μL, which was lower than the single detection sensitivity for each pathogen. Strawberry field samples were randomly selected and 34 diseased plants were assayed using the mPCR system. In the actual application test, our established mPCR can detect pathogens more quickly than conventional culture method. In conclusion, the mPCR system established in this study provides a rapid and effective method for the simultaneous detection of three important strawberry pathogens.