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

Simple Summary The three planthoppers, brown planthoppers (Nilaparvata lugens), white-backed planthoppers (Sogatella furcifera), and small brown planthoppers (Laodelphax striatellus), are major economically important pests of rice. However, conventional morphology-based methods are inefficient in differentiating these pests at a species level. In this study, we report the successful design of species-specific primers and their use in general and multiplex PCR and loop-mediated isothermal amplification (LAMP) assays, the widely used tools for molecular species diagnosis. Each primer comprised a species-specific sequence of at least 2-mer or more in both forward and reverse primers. The primers displayed high diagnostic efficiency in conventional and multiplex PCR. Furthermore, the LAMP performed at 61 degrees C for 30 min using the inner, loop, and species-specific primers confirmed the reactions and successfully identified the species. Here, we used the DNA-releasing technique (reported elsewhere) to obtain the DNA from the tissue samples of the insects. The DNA yield was sufficient to perform the conventional and multiplex PCRs and LAMP assays. Additionally, we demonstrate the high sensitivity of LAMP to achieve positive amplification using from 100 pg to 10 pg genomic DNA. Overall, the techniques can effectively diagnose the planthoppers in a large number of field-collected or individual samples. Asian planthoppers (Hemiptera: Delphacidae) that include brown planthoppers (BPH, Nilaparvata lugens, Stal), white-backed planthoppers (WBPH, Sogatella furcifera, Horvath), and small brown planthoppers (SBPH, Laodelphax striatellus, Fallen) 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 degrees C for 5 min with 30 mu L 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.