Development of a diamide resistance diagnostic method using LAMP based on a resistance-specific indel in ryanodine receptors for Spodoptera exigua (Lepidoptera: Noctuidae)
Recently, resistance to diamide insecticides (IRAC group 28) has been reported in various lepidopteran pests, including Spodoptera exigua. In the present study, susceptibility of six field populations was evaluated to two diamide insecticides: chlorantraniliprole and flubendiamide. The bioassay test for resistance revealed a high level of diamide resistance and helped to select a diamide resistant (Di-R) strain, whose LC50 values against chlorantraniliprole and flubendiamide were 28,950- and 135,286-fold higher, respectively, than those of susceptible strains. In the ryanodine receptor, instead of the G4946E mutation, one of the well-known diamide resistance mechanisms, we found a I4790M mutation and identified the resistance allele-specific indel linked to it. Resistance allele diagnostic primers were designed using this distinct region and applied in loop-mediated isothermal amplification (LAMP) and general PCR. LAMP accurately detected the specific indel when conducted for 2 h at temperature range from 63 {degrees}C to 65 {degrees}C and using four LAMP primers; its efficiency was further amplified by an additional loop primer. A broad range of DNA concentrations was workable in the LAMP assay, with the minimum detectable DNA concentration of 100 pg. The new DNA releasing method used for the LAMP assay consisted of 5 min of incubation of a larva or adult tissue at 95{degrees}C. The entire diagnostic process, which included the DNA releasing technique and LAMP, lasted only 100 min. This simple and accurate LAMP assay can be applied to monitor diamide resistance and for integrated resistance management of S. exigua in the field.