Development of a Loop-mediated isothermal amplification assay for the detection of mulberry fruit sclerotiniosis pathogen Scleromitrula shiraiana and Ciboria carunculoides

Mulberry fruit sclerotiniosis is a destructive fungal disease that severely impacts mulberry yield and quality. Due to the long latency period of the disease, developing a rapid, sensitive, and reliable diagnostic method for detecting the disease during its asymptomatic stage is critical. In this study, we designed a set of LAMP primers targeting a species-specific region of the β-tubulin gene sequence for the detection of Scleromitrula shiraiana , and another set targeting a species-specific region of the calmodulin gene sequence for the detection of Ciboria carunculoides . The addition of hydroxynaphthol blue allowed for the visualization of positive results through a color change. Optimization of the LAMP assay conditions determined that the optimal reaction occurred at 65℃ for 45 min. The specificity of the assay was confirmed by testing genomic DNA from S . shiraiana , C . carunculoides , and nine closely related plant pathogens. The sensitivity of the LAMP assay was found to be 100 fg/μL for S . shiraiana and 100 pg/μL for C . carunculoides . The practical application of the LAMP assay was demonstrated by successfully detecting S . shiraiana and C . carunculoides in soil samples from diseased mulberry orchards, illustrating its potential for field use. This diagnostic method offers new insights and technical support for the rapid detection of mulberry fruit sclerotiniosis, providing an essential tool for early detection, timely prevention, and effective control of the disease, ultimately benefiting the mulberry industry.