Nested PCR-based assay for diagnosis of circular leaf spot disease-causing mycosphaerella nawae and its application

In this study, we developed a nested PCR detection method based on draft genomic sequences of Mycosphaerella nawae and then applied the method to survey primary symptoms of circular leaf spot (CLS) and the seasonal ascospore scattering period. To develop the assay for detecting the fungus, 11 primers for the initial PCR and 19 primers for the nested PCR were designed (based on the glycoside hydrolase family 3 gene) using draft genomic sequences of M. nawae. Consequently, the primer pairs Mn-Fl/ Mn-Rl and Mn-nes-168f/Mn-nes-483r were selected and their specificities examined using regional M. nawae isolates, various healthy persimmon leaves, other fungal pathogens of persimmon, and allied species of M. nawae. Furthermore, the developed nested PCR method was applied for the diagnosis of the initial stage of CLS using both diseased and asymptomatic leaves. Additionally, scattered M. naivae ascospores collected by a spore sampler were surveyed from May to November 2015. Therefore, it is assumed that the nested PCR method can suggest the proper fungicide spraying period for CLS through anticipation of the ascospore quantity, and the early diagnosis of CLS will enable control of its development on persimmon.