Loop-Mediated Isothermal Amplification for the Rapid Detection of the Mutation of Carbendazim-Resistant Isolates in Didymella bryoniae

Abstract

Gummy stem blight (GSB) caused by Didymella bryoniae (D. bryoniae) is a worldwide fungal soil-borne disease that can cause severe yield reduction of watermelon. To shorten the monitoring time of carbendazim-resistant strains of D. bryoniae in the field, in this study, we developed a loop-mediated isothermal amplification (LAMP) assay for rapid detection of carbendazim-resistant strains of D. bryoniae. The β-tubulin gene of carbendazim-resistant strains was selected as the target for primer design. Based on the color change of hydroxy naphthol blue (HNB) and gel electrophoresis, the optimal reaction conditions for LAMP were determined at 65 °C for 50 min. In specificity tests, the LAMP assay was able to distinguish between carbendazim-resistant and sensitive strains of D. bryoniae. Moreover, in sensitivity tests, the detection limit was 1 ng/μL D. bryoniae DNA of the carbendazim-resistant strain. In addition, the LAMP method was successfully applied to detect carbendazim-resistant strains in D. bryoniae-infested samples. Therefore, the developed LAMP assay provides a new method for the rapid detection of carbendazim-resistant strains of D. bryoniae.