Development of a colorimetric loop‐mediated isothermal amplification (LAMP) assay to facilitate monitoring of carbendazim‐resistant isolates of Colletotrichum truncatum based on the β‐tubulin E198A mutation

Abstract

AbstractAnthracnose, a disease mainly caused by Colletotrichum truncatum, is a serious threat to vegetable soybean production worldwide. Carbendazim, a methyl‐benzimidazole carbamate fungicide, has been frequently applied to control the disease. Unfortunately, carbendazim‐resistant isolates of C. truncatum have emerged, resulting in inefficient disease control. Therefore, a rapid and simple method for monitoring the fungicide‐resistant isolates in fungal populations is essential to select the most appropriate strategy to control the disease and to handle fungicide resistance in a timely manner. In this study, we developed a colorimetric loop‐mediated isothermal amplification (LAMP) assay to detect carbendazim‐resistant isolates by using the E198A mutation in the TUB2 gene as the marker. With the optimal primers, the developed LAMP assay could detect resistant isolates after incubation for 40 min at 67°C. The results could be simply observed with the naked eye, and the assay provided 10‐fold higher sensitivity than conventional PCR. Moreover, the specificity, accuracy, and reproducibility of the generated LAMP were shown in testing with 85 C. truncatum isolates from five locations in northern Thailand. Resistant isolates carrying the F200Y mutation were not detected with the LAMP assay. Thus, the developed colorimetric LAMP assay could be used as a rapid and simple detector of certain carbendazim‐resistant isolates of C. truncatum.