Development of multiplex PCR assays for rapid identification of four morphologically similar lepidopteran pests of temperate fruit orchards

Abstract Temperate fruit orchards are heavily impacted by several lepidopteran pests, including Cydia pomonella (codling moth), Grapholita funebrana (plum fruit moth), Euzophera bigella (quince moth), and Ectomyelois ceratoniae (locust bean moth). These pests cause significant damage to crops, reducing yield and market quality. Accurate and efficient pest identification, particularly during early larval stages, remains a challenge due to their morphological similarities. Molecular diagnostic techniques, including DNA barcoding, offer reliable alternatives, but sequencing methods are costly and impractical for routine monitoring. To address this, we developed and validated two multiplex polymerase chain reaction assays targeting the mitochondrial COI and COII genes for species-specific identification of these four orchard pests. The assays produced distinct, non-overlapping amplicons, allowing for rapid and accurate identification of the pests at all life stages, especially early larvae. The method is cost-effective, simplifies pest detection, and provides a valuable tool for integrated pest management (IPM). The assays may support biological control programs and quarantine inspections by facilitating accurate species identification. Our results demonstrate the utility of these assays for pest monitoring and management, providing a practical molecular tool for routine diagnostics, pest surveillance, quarantine inspection, and IPM in temperate fruit orchards.