Molecular diagnosis of three major Asian planthoppers (Hemiptera: Delphacidae) through species-specific primers and optimized reaction conditions

Asian planthoppers, including brown planthoppers (BPH), white-backed planthoppers (WBPH), and small brown planthoppers (SBPH), are major pests of rice crops. Despite morphological and sequence similarities, accurate discrimination of these species is crucial for effective pest control. To address this issue, we developed six species-specific primers based on partial mitochondrial genome sequences. These primers were successfully utilized in multiplex PCR, loop-mediated isothermal amplification (LAMP) assays, and conventional PCR using DNA obtained through a DNA-releasing technique (incubating tissue samples at 95 °C for 5 min with 30 μL nuclease-free water). Our study demonstrated that multiplex PCR can analyze the density of each species after a mass collection in the field, the LAMP assay can diagnose the species within 40 minutes, and conventional PCR can be widely applied to a large number of field samples as well as individuals or mass collections. These findings highlight the potential of species-specific primers and the DNA-releasing technique for accurate multiplex PCR and LAMP assays, which may help to improve the intensive field monitoring of these pests and the integrated management of these species