Development of a Multiplex PCR to Simultaneously Detect Four Problematic Pathogens in Cymbidium kanran

Colletotrichum gloeosporioides (Cg), Pestalotiopsis sp. (Ps), Fusarium oxysporum (Fo), and Fusarium proliferatum (Fp) are pathogens that cause various diseases of Cymbidium kanran. Species identification based on the morphological characteristics of pathogen-infected orchids is very complex and difficult; therefore, specific and reliable diagnostic methods are needed. Here, we developed a multiplex polymerase chain reaction (PCR) assay for simultaneous detection of Cg, Ps, Fo, and Fp. Four pairs of pathogen-specific primers were designed based on the internal transcribed spacer sequences of each pathogen, generating fragments of 480, 407, 321, and 279 bp, respectively. The multiplex PCR assay using these four pairs of mixed primers effectively detected the four pathogens simultaneously, with a sensitivity of 0.1 ng genomic DNA/pathogen. Application of the optimized multiplex PCR assay to symptomatic C. kanran leaf samples effectively detected single or multiple pathogens. These results indicate that the multiplex PCR developed in this study is not only a rapid and reliable method for detecting four problematic pathogens in C. kanran, but also serves as a very useful tool for early diagnosis.