Development of LAMP and droplet digital PCR methods for differentiation between Cydia pomonella (L.) and Grapholita molesta (Busck)

Abstract

The codling moth, Cydia pomonella (L.), is an economically important key fruit pest worldwide. In China, C. pomonella was first discovered in 1953 and has since been introduced into at least eight provinces. The monitoring of C. pomonella using sex pheromones is essential for controlling this destructive pest and preventing its spread from infested areas. However, the sex pheromone of C. pomonella also has strong attractive effects on Grapholita molesta (Busck), which results in the mixture of the two pest insects. Furthermore, capturing individuals, especially during the early phase of spread, is challenging due to the limited number of introductions. Thus, it is crucial to provide an accurate and rapid diagnostic method to differentiate them. To develop such a method for distinguishing between C. pomonella and G. molesta, we initially selected a set of C. pomonella specific-LAMP primers from seven designed sets of candidate primers and its sensitivity was evaluated using DNA. Finally, the effectiveness of the method was proven using insect tissue and a temperature-controlled, insulated cup. Additionally, the optimal reaction temperature, specificity, and sensitivity of the C. pomonella ddPCR-primer were determined. The development of the C. pomonella LAMP and ddPCR methods provide tools for the monitoring of C. pomonella in China.