PCR-based species identification applied in Japanese pear orchards to survey seasonal proportion changes of phytoseiid mite species

Abstract

For this study, we developed a PCR-based method to identify the ribosomal internal transcribed spacer (ITS) sequences of six phytoseiid mite species found in Japanese pear orchards: Neoseiulus californicus (McGregor), Neoseiulus womersleyi (Schicha), Neoseiulus makuwa (Ehara), Amblyseius eharai Amitai and Swirski, Gynaeseius liturivorus (Ehara), and Amblyseius swirskii Athias-Henriot. Using the method, we can show seasonal proportion changes of phytoseiid mite species composition in Japanese pear orchards. Genomic DNA extracted individually from phytoseiid mites collected in the orchards are subjected to PCR of the ITS sequences using a universal primer set of which nucleotide sequences are conserved among the six phytoseiid mite species. Then, DNA samples showing PCR products with the universal primer set are used for another PCR of the ITS sequences using species-specific primer sets designed for respective phytoseiid mite species. Species-specific PCR is conducted in order of decreasing dominance of phytoseiid mite species. The method, which specifically identifies the six phytoseiid mite species irrespective of their sexes and developmental stages, might be useful for researchers who are lacking adequate morphological identification skills and nucleotide sequencing systems at their institutions.