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Keywords: enrichment, grapevine, HTS, nanopore, plant pathogen
Published in DiRROS: 27.03.2026; Views: 175; Downloads: 214
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Abstract: 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.
Keywords: Xylophilus ampelinus, grapevine bacterial blight, molecular diagnostics, Vitis vinifera, real-time PCR, genome-informed assay development
Published in DiRROS: 05.09.2025; Views: 794; Downloads: 407
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Abstract: Europe is the world’s main producer and exporter of grapevine planting material and wine. This important economic sector is facing epidemic threats of at least 10 grapevine yellows diseases (GY) caused by phytoplasmas. In Europe the main phytoplasmas associated with GY are ‘Candidatus’ phytoplasma solani’ (BNp), which is a causal agent of bois noir and FDp, which causes flavescence dorée. Phytoplasmas are notoriously difficult to detect and identify and their specific detection relies exclusively on the molecular methods. Recently new methods, which are reliable, sensitive, fast, less expensive and suitable for using onsites, have been introduced. Among them is a loop-mediated isothermal amplification (LAMP) method, with several advantages (e.g. low sensitivity to plant extracts inhibitors, speed, robustness, simplicity of use) over the other methods (e.g. the real-time PCR). In a recently finished FP7 project VITISENS, a new LAMP protocols have been developed for specific detection of FDp, however, they have not been tested in the interlaboratories trials. In addition, there is no validated LAMP protocol available for the specific detection of BNp at the moment. The main objectives of this project were: (1) Development of new loop-mediated isothermal amplification (LAMP) based protocols for accurate, reliable, fast and affordable diagnostics of ‘Candidatus Phytoplasma solani’ (BNp), which will be applicable in-field (2) To study new possible hosts plants and insect vectors of phytoplasma FDp. (3) To organize an interlaboratory test performance study (TPS) to obtain validation parameters for the selected LAMP protocols for BNp, as well as for the LAMP assay for FDp detection developed in the course of the FP7 project VITISENS.
Keywords: phytoplasmas, grapevine yellows diseases, LAMP, real-time PCR
Published in DiRROS: 16.09.2024; Views: 1186; Downloads: 618
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Abstract: Development of efficient methods and identification of barcodes for discriminating Grapevine flavescence dorée sensu-stricto from other related phytoplasmas and investigation of potential correlation between taxonomic identity and grapevine, alders and hazelnut plant hosts
Keywords: Euphresco, Grapevine flavescence dorée, phytoplasmas, hazelnut, grapevine
Published in DiRROS: 02.09.2024; Views: 1257; Downloads: 951
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Abstract: The use of more salt stress-tolerant vine rootstocks can be a sustainable strategy for adapting traditional grapevine cultivars to future conditions. However, how the new M1 and M4 rootstocks perform against salinity compared to conventional ones, such as the 1103-Paulsen, had not been previously assessed under real field conditions. Therefore, a field trial was carried out in a young ‘Tempranillo’ (Vitis vinifera L.) vineyard grafted onto all three rootstocks under a semi-arid and hot-summer Mediterranean climate. The vines were irrigated with two kinds of water: a non-saline Control with EC of 0.8 dS m–1 and a Saline treatment with 3.5 dS m–1. Then, various physiological parameters were assessed in the scion, and, additionally, gene expression was studied by high throughput sequencing in leaf and berry tissues. Plant water relations evidenced the osmotic effect of water quality, but not that of the rootstock. Accordingly, leaf-level gas exchange rates were also reduced in all three rootstocks, with M1 inducing significantly lower net photosynthesis rates than 1103-Paulsen. Nevertheless, the expression of groups of genes involved in photosynthesis and amino acid metabolism pathways were not significantly and differentially expressed. The irrigation with saline water significantly increased leaf chloride contents in the scion onto the M-rootstocks, but not onto the 1103P. The limitation for leaf Cl– and Na+ accumulation on the scion was conferred by rootstock. Few processes were differentially regulated in the scion in response to the saline treatment, mainly, in the groups of genes involved in the flavonoids and phenylpropanoids metabolic pathways. However, these transcriptomic effects were not fully reflected in grape phenolic ripeness, with M4 being the only one that did not cause reductions in these compounds in response to salinity, and 1103-Paulsen having the highest overall concentrations. These results suggest that all three rootstocks confer short-term salinity tolerance to the scion. The lower transcriptomic changes and the lower accumulation of potentially phytotoxic ions in the scion grafted onto 1103-Paulsen compared to M-rootstocks point to the former being able to maintain this physiological response in the longer term. Further agronomic trials should be conducted to confirm these effects on vine physiology and transcriptomics in mature vineyards.
Keywords: osmotic adjustment, gas exchange, gene expression, water relations, Vitis vinifera L. (grapevine), salinity tolerance
Published in DiRROS: 17.07.2024; Views: 1346; Downloads: 898
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