Establishment and Application of a Rapid Detection Method for Stemphylium vesicarium in Gray Leaf Spot of Welsh Onion

Abstract

For rapid determination of whether gray leaf spot disease in field-grown Welsh onion ( Allium fistulosum L.) is caused by Stemphylium vesicarium , we targeted the glycerol-3-phosphate dehydrogenase ( gapdh ) gene and designed specific primers (sv-gpdF/R) that amplify a 194-bp fragment, showing high specificity. In quantitative polymerase chain reaction (qPCR) assays, its sensitivity was 10 5 -fold higher than that of conventional PCR. The standard curve constructed showed a strong linear relationship between the cycle threshold (Ct) and the logarithm of template concentration, with a coefficient of determination ( R 2 ) of 0.9902. When this qPCR method was applied to detect S. vesicarium on inoculated Welsh onion leaves at various time points, the pathogen was detected as early as 3-h postinoculation, and it exhibited rapid proliferation between 72 and 96 h. Additionally, the alkaline lysis method allowed rapid DNA extraction from lesion sites, and subsequent PCR using the specific primers could determine whether Welsh onion plants were infected with S. vesicarium . In summary, this study established a rapid detection method for S. vesicarium , the causal agent of gray leaf spot disease in Welsh onion. This method facilitates rapid pathogen identification, enables informed selection of fungicides and provides an effective technical tool for disease prevention and control.