20.500.12556/DiRROS-23506 Development of a multi-targeted real-time PCR assay for the detection of the grapevine pathogen Xylophilus ampelinus Background Xylophilus ampelinus is a plant pathogenic bacterium that causes bacterial blight in grapevines, which can lead to severe yield losses and economic damage. Owing to its fastidious growth on culture media, detection is primarily based on molecular methods. However, existing tests have produced inconsistent results, particularly when used to detect latent infections and non-validated matrices. There is a risk of false-positive results, with economic consequences such as restrictions on international trade. To enhance the diagnostics of X. ampelinus, a genome-informed approach was utilised to identify new potential targets for specific detection. On the basis of these sequences, multiple real-time PCR assays were designed, and their specificity and sensitivity were assessed, as well as their performance validated across three different grapevine tissues, including leaves, roots, and xylem. Results The newly designed real-time PCR assays were evaluated via high throughput testing for specificity and sensitivity and compared with a reference assay. The most promising assays were selected and validated in different grapevine tissues and included in a test performance study to validate their reproducibility and robustness. Three new assays (Xamp_BA_2, TXmp22.4, and Xamp_BA_7) demonstrated high specificity and sensitivity for X. ampelinus detection. The Xamp_BA_2 assay exhibited the best overall performance, offering high diagnostic sensitivity and robustness across diverse plant matrices. Importantly, the assays exhibited no cross-reactivity with non-target bacterial species and maintained high detection accuracy across diverse grapevine tissue types. Conclusions The newly developed real-time PCR assays provide an enhanced diagnostic framework for the detection of X. ampelinus in various plant matrices, significantly improving the applicability of molecular testing. The Xamp_BA_2 assay demonstrates superior performance and is recommended for routine diagnostics, with other validated assays being employed for confirmation of identification. The development of these new assays represents a significant expansion of our toolkit for the precise detection of X. ampelinus in grapevines, with the potential to contribute to the mitigation of grapevine bacterial blight, the prevention of yield losses, and the protection of international trade in grapevine material. Further implementation of these assays will support regulatory and phytosanitary efforts to mitigate the spread of X. ampelinus. Xylophilus ampelinus grapevine bacterial blight molecular diagnostics Vitis vinifera real-time PCR genome-informed assay development Xylophilus ampelinus bakterijski ožig vinske trte molekularna diagnostika Vitis vinifera PCR v realnem času načrt testov na podlagi genomskih podatkov true false true Angleški jezik Slovenski jezik Neznano 2025-09-05 13:59:15 2025-09-05 13:59:16 2025-10-24 03:54:28 0000-00-00 00:00:00 2025 0 0 Nasl. z nasl. zaslona; Soavtorji: Alexandra Bogožalec Košir, Janja Matičič, Manca Pirc, Neža Turnšek & Tanja Dreo; Opis vira z dne 29. 7. 2025; str. 1-15 Vol. 21, [article no.] 99 2025 0000-00-00 Zaloznikova Objavljeno apc 0000-00-00 0000-00-00 2025-07-18 577.2 1746-4811 10.1186/s13007-025-01422-4 244205571 https://plantmethods.biomedcentral.com/articles/10.1186/s13007-025-01422-4 1 c2cc0aae-8a4f-11f0-9bb7-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=34774 RAZ_Bencic_Aleksander_2025.pdf RAZ_Bencic_Aleksander_2025.pdf 1 8BDF47F212339EBB6F697A77057723E5 f3414eac224fe3b7217e8f66741c7a6c0689a534c06489fc1d0b74d01e4ce4fa 13533c43-8a50-11f0-9bb7-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=34775 Nacionalni inštitut za biologijo 0 0 0