Development of multiplex PCR-based protocols for simultaneous diagnosis of three Spodoptera and one Mamestra species

Since many Noctuidae species are highly destructive crop pests, it is essential to establish proper management strategies, which primarily require an accurate and rapid species identification. However, accurate diagnosis of Noctuidae species in the field, particularly at the larval stage, is very difficult due to their morphological similarity and individual color variation. In particular, caterpillars of Spodoptera exigua, Spodoptera litura, Spodoptera frugiperda, and Mamestra brassicae are morphologically indistinguishable each other and frequently found on the same host crops such as corn or partially green onion etc. in the same season, thus requiring a reliable species diagnosis method. To efficiently diagnose these species, firstly the species-specific internal transcribed spacer (ITS1) sequences identified then, two molecular species diagnosis protocols developed using the ITS1 markers. The first protocol was the multiplex conventional PCR in conjunction with subsequent gel electrophoresis for species identification based on amplicon size. The second protocol was based on the multiplex real-time PCR using fluorescent dye-labelled primers for a single-step diagnosis. Template genomic DNA (gDNA) prepared by DNA release method was also suitable for both protocols as the template prepared by DNA extraction. The two protocols enabled rapid and robust species diagnosis using a single multiplex PCR step. Depending on laboratory instrumentation, one of the two protocols can be easily adapted for the species diagnosis of the four Noctuidae caterpillars in the field. The multiplex real-time PCR protocol will facilitate accurate diagnosis of the four species in a single step regardless of template gDNA quality.