Molecular detection assay of the bud mite Trisetacus juniperinus on Cupressus sempervirens in nurseries of central Italy

Trisetacus juniperinus (Nalepa) sensu Keifer (Acari: Eriophyoidea: Phytoptidae) causes irregular development of buds, shoot deformations and stunted growth of trees, resulting in a serious threat to nurseries and young stands of Cupressus sempervirens L. (Mediterranean cypress). Recently, some cypress clones selected for their resistance to the fungal canker agent Seiridium cardinale (Wag.) have shown high susceptibility to the mite. Considering its tiny body, its hidden lifestyle inside the buds and the probable occurrence of other species (the vagrant Epitrimerus cupressi (Keifer) is common on the Mediterranean cypress in Italy), detection and monitoring of T. juniperinus require taxonomic expertise and are often time-consuming and challenging before serious damage is discernible. In the present study, a rapid, cost-effective PCR-based method was developed and validated to detect T. juniperinus on cypresses. The cytochrome c oxidase subunit I (COI) gene was amplified with degenerate and specific primers, but the latter were the only ones able to discriminate between T. juniperinus and E. cupressi. PCR products distinguished the two species both in a pool of individuals in a mixed population of both species and in single individuals, indicating the sensitivity of the detection method. PCR-RFLP (restriction fragment length polymorphism) by means of XmnI and XbaI endonucleases separated the two species. Furthermore, a washing-sieving protocol was used to make mite collection from the tree sample faster and simpler; this procedure did not interfere with the molecular detection of the species. The possibility of the routine use of this assay to monitor quarantine eriophyoids infesting plant material is discussed.