The invasive sap-feeding pest Toumeyella parvicornis (pine tortoise scale) is rapidly spreading across Europe, threatening pine ecosystems, particularly in forest–urban areas of Italy. In this scenario, early detection and monitoring strategies are critical to prevent new outbreaks and mitigate impacts in infested regions. Current surveillance is challenged by the lack of rapid, sensitive tools for indirect detection of this cryptic, canopy-dwelling pest, despite advancements in molecular diagnostics and environmental DNA (eDNA). Here, we established a highly specific qPCR assay using LNA probe chemistry for detecting T. parvicornis DNA from both adult insects and their excreted honeydew. DNA was successfully isolated/quantified from all tested matrices. We recorded average Cq values of 20.9 for insect specimens and 30.3 for collected honeydew samples. Targeting the COI barcoding region, the assay demonstrated excellent specificity in both in silico and in vitro tests, showing no cross-reactivity to other pine-associated taxa. The limit of detection for DNA isolated from insect was 64 fg/µL. This is the first diagnostic protocol to use honeydew as a matrix for indirect detection of T. parvicornis. Optimized for routine application by Plant Health Services, this eDNA-based tool offers a valuable approach for future monitoring of sap-sucking hemipterans in multiple environments.

This study presents a qPCR-based molecular diagnostic method utilizing a TaqMan probe to ac-curately identify Agrilus auroguttatus Schäffer (Coleoptera: Buprestidae) from adult insects, ex-creta samples, and frass residues. The method demonstrated excellent DNA amplification across all sample types, highlighting strong analytical specificity. The protocol exhibited high repeata-bility and reproducibility, with inter-run and intra-run variability standard deviations consist-ently at or below 0.5. This assay distinguished A. auroguttatus samples from non-target species with 100% sequence correspondence. Analytical sensitivity (LoD) for adult and frass samples was determined at 8 pg/µL and at a 1:5 dilution from an initial concentration of 5 ng/µL, yield-ing mean Cq values of 35.14 ± 0.71 and 37.07 ± 0.59, respectively. The indirect diagnostic capabil-ity of this method is particularly valuable for detecting the presence of wood-boring insects, po-tentially aiding in the early detection of A. auroguttatus in new environments or in managing cur-rent outbreak areas. This diagnostic tool could thus play a critical role in biosecurity efforts to limit the spread of this invasive species.

Aleurocanthus spiniferus (Quaintance) (Hemiptera: Aleyrodidae) has rapidly spread, mainly in the central and eastern Mediterranean coastal area, infesting various new host plants alongside known ones. This invasive species poses a significant threat to agricultural ecosystems, necessitating urgent action to monitor and control outbreaks in previously pest-free areas. While entomological and morphological recognitions are crucial for initial detection, challenges often arise in quickly identifying different developmental stages or genus-level distinctions, particularly in surveys conducted by personnel with limited entomological skills. Due to these challenges, a qPCR probe protocol was developed to enhance the diagnostic capacity of laboratories responsible for the territorial control of pests. This biomolecular tool integrates morphological surveys, enabling prompt and reliable proof of A. spiniferus presence in free areas, delimited territories, or during phytosanitary import inspections. The protocol’s high analytical specificity, inclusivity, and exclusivity ensure accurate identification of A. spiniferus, while its low limit of detection and high repeatability and reproducibility reinforce its utility as a standardized diagnostic method. By facilitating prompt and targeted control efforts, this innovative approach strengthens the resilience of agricultural systems against the widespread threat of A. spiniferus infestations.

Ips typographus is one of the most destructive bark beetles affecting conifer forests in Europe, where climatic disturbances and the movement of infested wood can rapidly shift populations from endemic levels to severe outbreaks. Early detection through border inspections and forest monitoring is essential to prevent new introductions and limit the spread of established populations. Here, we developed and validated a probe-based TaqMan qPCR assay, targeting the mitochondrial COI barcode region, for the rapid and species-specific detection of I. typographus from both insect material and environmental DNA recovered from frass and exit-hole wood chips. Validation followed EPPO PM7/98(5) guidelines, assessing analytical specificity, sensitivity, repeatability, reproducibility, and inter-laboratory transferability. High analytical specificity was demonstrated against a broad panel of non-target species, and reliable amplification was obtained across different tested matrices. The method showed strong analytical sensitivity, with limits of detection of 0.32 pg/µL for adult-derived DNA and 1.6 pg/µL for artificial frass. Repeatability, reproducibility, and inter-laboratory blind testing further confirmed the diagnostic reliability of the method. This validated qPCR assay provides a rapid and sensitive molecular tool for the early detection of I. typographus, supporting border inspection and phytosanitary diagnostic laboratories in forest biosecurity activities.

To date, Philaenus spumarius (Linnaeus, 1758), Philaenus italosignus Drosopoulos & Remane, 2000, and Neophilaenus campestris (Fallén, 1805) are proven vectors of the phytopathogenic bacterium Xylella fastidiosa Wells et al., 1987 in Europe. Currently, the identification of these three species relies on the well-documented status of morphological and taxonomical characters, making the discrimination of vector adult males possible by genitalia comparison. This study updates the biomolecular diagnostic tests with a rapid identification tool for P. italosignus, using locked nucleic acid (LNA) probe technology. The test also overcomes the difficulties associated with the morphological identification of females and juveniles. The morphological α-taxonomic identification of the male, achieved through comparison with the type of the species, retains its primary role in specimen identification for probe building. Later, the proposed assay can contribute to the rapid identification of P. italosignus by the secondary (molecular) identification step. The new LNA qPCR test offers high reliability and reproducibility in the identification of P. italosignus instars, thus improving targeted surveys of X. fastidiosa vector populations and allowing discrimination between species collected in the field. The accurate identification and census of vector individuals, regardless of their gender and instar, enhances the efficacy of Xylella IPM-DSS (Integrated Pest Management Decision Support System) strategies.