A new PCR based molecular method for early and precise quantification of parasitization in the emerging olive pest Dasineura oleae

BACKGROUND: Dasineura oleae (Angelini 1831) (Diptera: Cecidomyiidae) was considered a minor pest in olive orchards, but in recent years severe outbreaks have been registered in several Mediterranean countries. Damage is caused by the feeding activity of larvae that induce gall formations and alters the physiological activity of the leaves. In Italy, this pest may be controlled by four Hymenoptera parasitoid species belonging to Platygaster and Mesopolobus genera such as Platygaster demades Walker 1835, Platygaster oleae Szelenyi 1940 (Hymenoptera: Platygastridae), Mesopolobus aspilus (Walker 1835) and Mesopolobus mediterraneus (Mayr 1903) (Hymenoptera: Pteromalidae), but parasitization becomes evident only after gall dissection. RESULTS: In this study, we aim to: (i) design a primer for the detection of specimens belonging to Platygaster and Mesopolobus genera; (ii) develop a multiplex quantitative polymerase chain reaction (qPCR) protocol combined to a fast samples DNA extraction method; (iii) apply the developed protocol to field-collected specimens and compare this method with traditional techniques based on visual estimation of parasitism rate on larvae. Primers were designed to anneal with cytochrome oxidase subunit I (COI) sequences of Platygaster and Mesopolobus genera while protocols were developed to be fast and capable to process several samples at the same time. Molecular analyses demonstrated to provide almost double of the parasitism rate assessed by visual inspection. Furthermore, on second instar larvae the PCR-based method was able to detect ten-fold times the parasitization rate estimated by visual inspection. CONCLUSION: The application on a greater scale of this newly developed method could be fundamental in the determination of the biological control potential in olive orchards.