A nested-polymerase chain reaction protocol for the detection of Mycosphaerella nawae in persimmon

Mycosphaerella nawae is the causal agent of circular leaf spot of persimmon. A polymerase chain reaction (PCR) based protocol was developed for M. nawae-specific identification from pure culture, or infected symptomatic and asymptomatic persimmon tissues. Variation among the internal transcribed spacer regions (ITS) of the ribosomal DNA (rDNA) sequences of potentially related fungal species in persimmon orchards was analyzed for a primer pair design. Specificity was confirmed using multiple isolates of these species, other fungal pathogens that cause foliar diseases in persimmon and contaminants commonly obtained in the isolation process. The detection threshold for M. nawae DNA was lowered from 50 pg to 500 fg when nested-PCR was evaluated instead of single PCR. The nested-PCR protocol developed in this study showed its suitability to be applied for the specific detection of M. nawae from three types of naturally infected persimmon tissues: from lesions in fresh leaves, from pseudothecia present in lesions in leaf litter, and from infected asymptomatic leaves. The protocol can be useful for routine diagnosis, disease monitoring programs and for epidemiological research.