Development of a Species Diagnostic Molecular Tool for an Invasive Pest, Mythimna loreyi, Using LAMP

SIMPLE SUMMARY: Mythimna loreyi is a serious pest of grain crops and reduces yields in maize plantations. M. loreyi is a native species in East Asia for a long time. However, this species has recently emerged as a migration (or invasive from other Asian countries) pest of some cereal crops in Korea. Little is known about its basic biology, ecology, and it is difficult to identify the morphologically similar species, Mythimna separate, which occur at the cornfield in the larvae stage. Species diagnosis methods for invasive pests have been developed and utilized for this reason. Currently, the molecular biology method for diagnosing M. loreyi species is only using the mtCO1 universal primer (LCO1490, HCO2198) and process PCR and sequencing to compare the degree of homology. However, this method requires a lot of time and effort. In this study, we developed the LAMP (loop-mediated isothermal amplification) assay for rapid, simple, and effective species diagnosis. By analyzing the mitochondrial (mt) genome, the species-specific sequence was found at the coding region of the NADH dehydrogenase subunit 5 gene. A broad range of DNA concentration was workable in LAMP assay, in which the minimum detectable DNA concentration was 100 pg. DNA releasing method was applied, which took five minutes of incubation at 95 °C without the DNA extraction process, and only some pieces of tissue from larvae and adult samples were needed. The incidence of invasive pests is gradually diversifying. Therefore, this simple and accurate LAMP assay is possibly used in the intensive field monitoring for invasive pests and integrated management of Mythimna loreyi. ABSTRACT: The Mythimna loreyi (Duponchel) is one of the well-known invasive noctuid pests in Africa, Australia, and many Asian countries. However, it is difficult to identify the invasive and morphologically similar species, Mythimna separate, which occur at the cornfield in the larvae stage. Currently, the molecular biology method for diagnosing M. loreyi species is only using the mtCO1 universal primer (LCO1490, HCO2198), which requires a lot of time and effort, such as DNA extraction, PCR, electrophoresis, and sequencing. In this study, the LAMP assay was developed for rapid, simple, effective species identification. By analyzing the mitochondrial genome, the species-specific sequence was found at the coding region of the NADH dehydrogenase subunit 5 gene. Based on this unique sequence, four LAMP primers and two loop primers were designed. The F3 and B3 primers were able to diagnose species-specific, in general, and multiplex PCR and specifically reacted within the inner primers in LAMP assay. The optimal incubation condition of the LAMP assay was 61 °C for 60 min with four LAMP primers, though additional loop primers, BF and LF, did not significantly shorten the amplification time. The broad range of DNA concentration was workable in LAMP assay, in which the minimum detectable DNA concentration was 100 pg. DNA releasing method was applied, which took five minutes of incubation at 95 °C without the DNA extraction process. Only some pieces of tissue of larvae and adult samples were needed to extract DNA. The incidence of invasive pests is gradually diversifying. Therefore, this simple and accurate LAMP assay is possibly applied in the intensive field monitoring for invasive pests and integrated management of Mythimna loreyi.