Asian planthoppers (Hemiptera: Delphacidae) that include brown planthoppers (BPH, Nilaparvata lugens, Stål), white-backed planthoppers (WBPH, Sogatella furcifera, Horváth), and small brown planthoppers (SBPH, Laodelphax striatellus, Fallén) are the primary sucking-type pests of rice. These three insects share morphological and sequence similarities. As insecticide resistance patterns and control strategies vary according to species, the accurate discrimination of these species is important. Here, we developed six species-specific primers based on partial mitochondrial genome sequences. The primers were successfully used in multiplex PCR, loop-mediated isothermal amplification (LAMP) assays, and conventional PCR. Here, we used genomic DNA obtained using the DNA-releasing technique (tissue samples were incubated at 95 °C for 5 min with 30 μL nuclease-free water, and the supernatant was used). We showed that multiplex PCR could analyze the density of each species following a mass collection in the field; the LAMP assay can diagnose the species within 40 min; conventional PCR can be widely applied to a large number of field samples, as well as individuals or mass collections. In conclusion, these results demonstrate the potential of the species-specific primers and DNA-releasing technique for accurate multiplex PCR and LAMP assays, which may assist the intensive field monitoring of integrated management of these species.

The diamondback moth, Plutella xylostella L., is a lepidopteran pest that damages various vegetable plants belonging to the genus Brassica worldwide. Various biological controls, such as parasitoid wasps, have been used to control this pest. Among these, Diadegma semiclausum and Diadegmafenestrale are widely used globally. In field-based biological control research, the investigation of the population dynamics of parasitoids and the rate of parasitism within the pest population is very important. However, achieving profundity in research is difficult when morphologically similar species coexist in the field. The morphological characteristics of D. semiclausum and D. fenestrale are very similar, and they both parasitize P. xylostella larvae. Therefore, to accurately identify these species, in this study, we developed a molecular diagnostic method by using loop-mediated isothermal amplification (LAMP). The mitochondrial genome of D. fenestrale and partial nucleotide sequences, including the ITS region of D. semiclausum, were analyzed for use as species diagnosis markers. The results showed that the homology of D. fenestrale to D. semiclausum was 94%, due to the excessively low homology of the D loop, but the actual homology was higher than 94%, particularly in the coding region. D. fenestrale species-specific primers for LAMP were designed based on the region encoding COX3, and the optimal diagnostic reaction condition for the four primers (F3, B3, FIP, and BIP) was 63 °C for 35 min. A species-specific primer capable of classifying D. semiclausum was developed based on the ITS2 region, and the optimal reaction condition for diagnosis was 63 °C for 40 min. Under optimal conditions for both species, upon addition of the loop primer LB, the reaction efficiency increased, and the reaction time was shortened by more than 5 min. The diagnostic limit concentration was up to 10 pg under both optimal conditions; therefore, it was possible to detect even very low concentrations. For both species, diagnosis was possible by using LAMP assay with a DNA-releasing technique, without a DNA extraction process, and by incubating a tissue sample or the homogenized whole body at 95 °C for 5 min. In the case of D. fenestrale, it was possible to diagnose the parasitoid in P. xylostella larvae. Therefore, the developed LAMP diagnostic method can be used in a variety of ways to determine whether P. xylostella has been parasitized in the process of field research and mass breeding, and to accurately distinguish the species that are parasitic to P. xylostella larvae. This LAMP-based diagnostic method can be applied to identify various parasitoids that are used for the biological control of P. xylostella.