Development of Molecular-Based Species Identification and Optimization of Reaction Conditions for Molecular Diagnosis of Three Major Asian Planthoppers (Hemiptera: Delphacidae)

Abstract

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.