A rapid multiplex PCR assay for species identification of Asian rice planthoppers (Hemiptera: Delphacidae) and its application to early-instar nymphs in paddy fields

Rice (Oryza sativa L.) is the main cereal crop in many Asian countries. The Asian rice planthoppers, Nilaparvata lugens (Stål) (brown planthopper), Sogatella furcifera (Horváth) (white-backed planthopper), and Laodelphax striatellus (Fallén) (small brown planthopper) (Hemiptera: Delphacidae), are the most economically important pests of rice. These three rice planthopper species often co-occur in the same paddy field. Traditionally, species identification of individuals of the three rice planthopper species has relied on morphological characters, but accurate discrimination of early-instar nymphs is very difficult, even for expert researchers. In this study, we developed a rapid one-step multiplex PCR assay using conserved and species-specific 5.8S-ITS2 rDNA gene primers for simultaneous identification of individuals of the three rice planthopper species. The multiplex PCR results showed that the three rice planthopper species could be identified accurately based on the length of the resultant amplicon, regardless of the individual developmental stage. Furthermore, we applied this assay for the first accurate quantification of early-instar nymphs of each rice planthopper species in paddy fields. Notably, we found that the species composition of early-instar nymphs cannot be extrapolated from that of adults. Thus, the multiplex PCR assay developed here facilitates detection of each rice planthopper species at the beginning of outbreaks in paddy fields.