Identification of the Melon Fruit Fly Zeugodacus cucurbitae (Coquillett) (Diptera, Tephritidae) by Real-Time PCR with SYBR Green I Dye

The melon fly Zeugodacus cucurbitae (Coquillett) is a fruit pest mostly damaging plants of the family Cucurbitaceae and capable of causing significant economic losses. This species of Asian origin has spread to Africa, Australia, and Oceania. Preventing further dispersal of the melon fly in other regions requires a quick and reliable method for its identification, especially at the preimaginal stages that are most often found in fruits and are also more difficult to identify by morphological characters. During our study, species-specific primers for Z. cucurbitae mtDNA COI gene region were designed for the first time and used in real-time PCR with SYBR Green I dye to differentiate Z. cucurbitae from some other Tephritidae species occurring on the same host plants: Zeugodacus tau (Walker), Bactrocera dorsalis (Hendel), B. latifrons (Hendel), Anastrepha grandis (Macquart), Ceratitis capitata (Wiedemann), and two Dacus species. The results of sensitivity analysis of real-time PCR showed that the detection limit for DNA template was 1 ng/μL. The proposed method was successfully tested on both adults and third-instar larvae, so it can be used for research and in the future, for practical tasks of plant quarantine.