Yashiro Toshihisa, Sanada-Morimura Sachiyo
Division of Agro-Environment Research, Kyushu Okinawa Agricultural Research Center, National Agriculture and Food Research Organization, Koshi, Kumamoto, Japan.
PLoS One. 2021 Apr 23;16(4):e0250471. doi: 10.1371/journal.pone.0250471. eCollection 2021.
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.
水稻（Oryza sativa L.）是许多亚洲国家的主要谷类作物。亚洲稻飞虱，褐飞虱（Nilaparvata lugens (Stål)）、白背飞虱（Sogatella furcifera (Horváth)）和灰飞虱（Laodelphax striatellus (Fallén)）（半翅目：飞虱科），是水稻最具经济重要性的害虫。这三种稻飞虱物种经常在同一稻田中共存。传统上，这三种稻飞虱物种个体的种类鉴定依赖于形态特征，但即使是专业研究人员，准确区分低龄若虫也非常困难。在本研究中，我们开发了一种快速一步多重PCR检测方法，使用保守和物种特异性的5.8S-ITS2 rDNA基因引物，用于同时鉴定这三种稻飞虱物种的个体。多重PCR结果表明，无论个体发育阶段如何，都可以根据所得扩增子的长度准确鉴定这三种稻飞虱物种。此外，我们首次将该检测方法应用于稻田中每种稻飞虱低龄若虫的精确定量。值得注意的是，我们发现低龄若虫的物种组成不能从成虫的物种组成推断出来。因此，这里开发的多重PCR检测方法有助于在稻田害虫爆发初期检测每种稻飞虱物种。
