1. Grapevine flavescence dorée (FD) follow up Vitisens, GRAFDEPI and Qdetect (GRAFDEPI2) : final reportMarina Dermastia, Helga Reisenzein, Luca Ferretti, 2015, končno poročilo o rezultatih raziskav Povzetek: 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.
Ključne besede: phytoplasmas, grapevine yellows diseases, LAMP, real-time PCR
Objavljeno v DiRROS: 16.09.2024; Ogledov: 30; Prenosov: 17
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2. 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 : project title (FLADO-VIGILANT)Nataša Mehle, Zala Kogej Zwitter, Marianne Loiseau, Xavier Foissac, Sylvie Malembic-Maher, Michael Maixner, Kerstin Zikeli, Stefano Costanzo, Luca Ferretti, Esmeraldina Sousa, 2023, končno poročilo o rezultatih raziskav Povzetek: 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
Ključne besede: Euphresco, Grapevine flavescence dorée, phytoplasmas, hazelnut, grapevine
Objavljeno v DiRROS: 02.09.2024; Ogledov: 103; Prenosov: 72
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3. Interlaboratory comparison study on ribodepleted total RNA high-throughput sequencing for plant virus diagnostics and bioinformatic competenceYahya Gaafar, Marcel Westenberg, Marleen Botermans, László Krizbai, Kris De Jonghe, Yoika Foucart, Luca Ferretti, Denis Kutnjak, Anja Pecman, Nataša Mehle, Jan F. Kreuze, Giovanna Müller, Nikolaos Vakirlis, Despoina Beris, Christina Varveri, Heiko Ziebell, 2021, izvirni znanstveni članek Povzetek: High-throughput sequencing (HTS) technologies and bioinformatic analyses are of growing interest to be used as a routine diagnostic tool in the field of plant viruses. The reliability of HTS workflows from sample preparation to data analysis and results interpretation for plant virus detection and identification must be evaluated (verified and validated) to approve this tool for diagnostics. Many different extraction methods, library preparation protocols, and sequence and bioinformatic pipelines are available for virus sequence detection. To assess the performance of plant virology diagnostic laboratories in using the HTS of ribosomal RNA depleted total RNA (ribodepleted totRNA) as a diagnostic tool, we carried out an interlaboratory comparison study in which eight participants were required to use the same samples, (RNA) extraction kit, ribosomal RNA depletion kit, and commercial sequencing provider, but also their own bioinformatics pipeline, for analysis. The accuracy of virus detection ranged from 65% to 100%. The false-positive detection rate was very low and was related to the misinterpretation of results as well as to possible cross-contaminations in the lab or sequencing provider. The bioinformatic pipeline used by each laboratory influenced the correct detection of the viruses of this study. The main difficulty was the detection of a novel virus as its sequence was not available in a publicly accessible database at the time. The raw data were reanalysed using Virtool to assess its ability for virus detection. All virus sequences were detected using Virtool in the different pools. This study revealed that the ribodepletion target enrichment for sample preparation is a reliable approach for the detection of plant viruses with different genomes. A significant level of virology expertise is needed to correctly interpret the results. It is also important to improve and complete the reference data.
Ključne besede: high-throughput sequencing, ribodepletion, interlaboratory comparison, test performance study, proficiency test, Virtool
Objavljeno v DiRROS: 19.07.2024; Ogledov: 210; Prenosov: 134
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4. Development and validation of a one-step reverse transcription real-time PCR assay for simultaneous detection and identification of tomato mottle mosaic virus and tomato brown rugose fruit virusAntonio Tiberini, Ariana Manglli, Anna Taglienti, Ana Vučurović, Jakob Brodarič, Luca Ferretti, Marta Luigi, Andrea Gentili, Nataša Mehle, 2022, izvirni znanstveni članek Povzetek: Tobamovirus species represent a threat to solanaceous crops worldwide, due to their extreme stability and because they are seed borne. In particular, recent outbreaks of tomato brown rugose fruit virus in tomato and pepper crops led to the establishment of prompt control measures, and the need for reliable diagnosis was urged. Another member of the genus, tomato mottle mosaic virus, has recently gained attention due to reports in different continents and its common features with tomato brown rugose fruit virus. In this study, a new real-time RT-PCR detection system was developed for tomato brown rugose fruit virus and tomato mottle mosaic virus on tomato leaves and seeds using TaqMan chemistry. This test was designed to detect tomato mottle mosaic virus by amplifying the movement protein gene in a duplex assay with the tomato brown rugose fruit virus target on the CP-3’NTR region, which was previously validated as a single assay. The performance of this test was evaluated, displaying analytical sensitivity 10−5–10−6-fold dilution for seeds and leaves, respectively, and good analytical specificity, repeatability, and reproducibility. Using the newly developed and validated test, tomato brown rugose fruit virus detection was 100% concordant with previously performed analyses on 106 official samples collected in 2021 from different continents.
Ključne besede: real-time PCR, tomato mottle mosaic virus, tomato brown rugose fruit virus, leaves detection, seeds detections, performance criteria
Objavljeno v DiRROS: 16.07.2024; Ogledov: 204; Prenosov: 182
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