Novel diamide resistance-linked mutation in Korean Spodoptera exigua and a LAMP assay based on a mutation-associated intronic InDel

Abstract

Recently, resistance to diamide insecticides has been reported in various lepidopteran pests, including Spodoptera exigua. Six field populations and a local population were tested, and results revealed high levels of diamide resistance. We selected a diamide-resistant strain, showing LC50 values 28,950- and 135,286-fold higher than those of a susceptible strain against chlorantraniliprole and flubendiamide, respectively. However, G4946E mutation in the ryanodine receptor, one of the well-known diamide resistance mechanisms, was not found in the tested resistant field populations. Instead, through genome sequencing, we found an I4790M mutation and some InDels, particularly a 29-bp insertion in the neighboring intron that was associated with this resistant allele. By using this distinct region, resistant allele diagnostic primers were designed and applied in a lamp loop-mediated isothermal amplification (LAMP) assay and general PCR. The applicable temperature of the LAMP assay was from 63 to 65 °C for 2 h with four primers. Addition of a loop primer further increased the amplification efficiency. LAMP was feasible with a broad range of DNA concentrations, with a minimum detectable concentration of 100 pg. A DNA releasing method comprising 5 min of incubation at 95 °C allowed DNA detection from some larva and adult samples. The combination of this DNA releasing technique and LAMP resulted in a rapid (up to 100 min) and accurate diagnostic strategy that could be applied to monitor and manage diamide resistance in S. exigua.