Simultaneous identification of Grapholita molesta Busck and Grapholita dimorpha Komai by PCR-RFLP

Although several molecular diagnostic techniques are available for the identification of the apple-feeding pests Grapholita molesta Busck and Grapholita dimorpha Komai, these pests are severely affecting apple orchards in Korea. These two pests may be misidentified or the available molecular diagnostic techniques may not facilitate the simultaneous identification of the morphological features of both species. In this study, we developed a multiplex assay for these two species using the polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) method. Sixty-two specimens were collected from apples presumed infested with moth larvae and from pheromone traps from 2013 to 2014. Both species were identified morphologically, and a partial region of the cytochromeb gene was sequenced to design primers for PCR-RFLP. Digestion profiles of G.molesta and G.dimorpha, using the Sau3A1 restriction enzyme, were characterized using three DNA fragments each for G.molesta (363bp, 91bp and 31bp) and G.dimorpha (220bp, 234bp and 31bp). The RFLP assay developed for both species in this study was more efficient and accurate than other currently used diagnostic assays and would be helpful to identify field-collected specimens for pest control research.