Development of a LAMP-Based Molecular Species Diagnosis Method for Four Major Agricultural Pests in the Genus Spodoptera (Lepidoptera: Noctuidae)

SIMPLE SUMMARY: Four major Spodoptera pests, S. exigua, S. frugiperda, S. litura, and S. littoralis, are widely distributed polyphagous pests affecting various crops. Despite different distribution areas, these four species cause serious damage to agriculture worldwide. As these species are morphologically similar at the larval stage, diagnostic methods have been developed and utilized for their identification. Here, we developed a loop-mediated isothermal amplification (LAMP) assay for rapid and effective species diagnosis, along with PCR, to identify Korean field-collected or overseas samples. The optimal conditions for the LAMP assay were 61 °C for 60 min with four LAMP primers. Additional loop primers increased the amplification efficiency in S. exigua, whereas increased non-specific amplification was found in other species. A broad range of DNA concentrations was observed in the LAMP assay, and the minimum detectable DNA concentration was 1 pg. The DNA release method for LAMP involved incubation of larval or adult tissue samples for 5 min at 95 °C, without a DNA extraction step. Considering the gradual diversification invasive pest incidence, this simple and accurate LAMP assay can be used for intensive field monitoring of invasive pests and integrated management of these species. ABSTRACT: Molecular-based species identification tools are helpful to identify tiny insect and lepidopteran pests that show morphological similarities in the larval stage and are essential for quarantine as well as agricultural research. Here, we focused on four major Spodoptera pests: S. exigua, S. frugiperda, S. litura, and S. littoralis. S. exigua and S. litura mitochondrial genome sequences were newly identified and species-specific sequence regions were identified in the cytochrome c oxidase subunit II and III regions. Species primers were designed and applied in loop-mediated isothermal amplification (LAMP) and PCR to identify Korean field-collected or overseas samples. The optimal incubation conditions for LAMP were 61 °C for 60 min with four LAMP primers. Additional loop primers increased the amplification efficiency for S. exigua, and the nonspecific amplification for other species. The LAMP assay could detect a wide range of DNA concentrations, with the range 1 ng–1 pg in dependence of four LAMP primers. The DNA-releasing technique, without DNA extraction, in the LAMP assay involved larval or adult tissue sample incubation at 95 °C for 5 min. The entire process takes approximately 70 min. This new molecular diagnostic method is simple and accurate, with application in the field and laboratory and for monitoring and ecological studies.