Molecular monitoring and multiplex qPCR-based detection of Cydia pomonella and Grapholita molesta in apple orchards of Kazakhstan.

INTRODUCTION: Apple production in Kazakhstan is highly vulnerable to damage caused by fruit-infesting tortricid pests, particularly codling moths (Lepidoptera: Tortricidae). Accurate and rapid species identification is essential for effective pest management and phytosanitary monitoring in apple orchards. METHODS: Field surveys were conducted in apple orchards of southern and southeastern Kazakhstan to investigate the codling moth complex. Collected specimens were identified morphologically and confirmed by sequencing a mitochondrial cytochrome c oxidase subunit I (COI) region. Comparative COI sequence analysis was used to develop a multiplex quantitative polymerase chain reaction assay employing Minor Groove Binder hydrolysis probes for simultaneous detection and differentiation of Cydia pomonella and Grapholita molesta. RESULTS: Field surveys identified C. pomonella and G. molesta, with C. pomonella accounting for 83.3% of collected specimens. COI sequencing enabled reliable discrimination between the two species. The multiplex assay demonstrated high specificity, produced no false-positive amplification in non-target species, and achieved 100% detection down to 5 pg DNA per reaction. DISCUSSION: The developed COI-based multiplex quantitative polymerase chain reaction assay provides a rapid, sensitive, and reliable tool for simultaneous identification of C. pomonella and G. molesta. It is suitable for phytosanitary monitoring, early detection, and management of quarantine-relevant tortricid pests in Kazakhstan.