Development of a LAMP method for detecting the N75S mutant in SDHI-resistant Corynespora cassiicola.

ABSTRACT

The replacement of asparagine with serine at codon 75 of the sdhC gene (N75S) confers succinate dehydrogenase inhibitor resistance in Corynespora cassiicola, which caused by consecutive fungicide application. To rapidly detect the mutation of N75S, a method based on loop-mediated isothermal amplification (LAMP) was developed in this study. The optimal primer set among the six primer sets designed could clearly identify N75S from the wild-type genotype. The detection threshold of the optimized LAMP mixture (10 µL) was 8.8 fg of target DNA at 63 °C within 60 min. This method specifically showed a color change and ladder-like band only when DNA extracted from isolates containing the N75S mutation was added. The results of stability tests suggested a satisfactory repeatability of this method. Additionally, the assay could positively distinguish N75S mutants from crude DNA isolated from conidia and mycelia of C. cassiicola. Given the high efficiency, sensitivity, specificity, repeatability and simplicity of operation, the LAMP method established here could be useful to evaluate the shift in the sensitivity of C. cassiicola to SDHIs and will provide significant data for the management of Corynespora leaf spot.