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Validation of methods for Flavescence dorée phytoplasma sensu stricto identification through Test Performance Studies.

hal.structure.identifierAgence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail [ANSES]
dc.contributor.authorLOISEAU, Marianne
hal.structure.identifierLaboratoire de santé des végétaux [LSV Angers]
dc.contributor.authorCOUSSEAU-SUHARD, Pascaline
hal.structure.identifierLaboratoire de santé des végétaux [LSV Angers]
dc.contributor.authorRENAUDIN, Isabelle
hal.structure.identifierNational Institute of Biology [Ljubljana] [NIB]
hal.structure.identifierJozef Stefan International Postgraduate School [Ljubljana, Slovenia]
dc.contributor.authorKOGEJ ZWITTER, Zala
hal.structure.identifierNational Institute of Biology [Ljubljana] [NIB]
dc.contributor.authorMEHLE, Natasa
hal.structure.identifierJulius Kühn-Institut - Federal Research Centre for Cultivated Plants [JKI]
dc.contributor.authorZIKELI, Kerstin
hal.structure.identifierJulius Kühn-Institut [JKI]
dc.contributor.authorMAIXNER, Michael
hal.structure.identifierUSDA-APHIS PPQ
dc.contributor.authorCOSTANZO, Stefano
hal.structure.identifierCREA Research centre for plant Protection and Certification [CREA]
dc.contributor.authorFERETTI, Luca
hal.structure.identifierInstituto Nacional de Investigação Agrária e Veterinária = National Institute for Agrarian and Veterinary Research [Oeiras, Portugal] [INIAV]
dc.contributor.authorSA-PEREIRA, Paula
hal.structure.identifierInstituto Nacional de Investigação Agrária e Veterinária = National Institute for Agrarian and Veterinary Research [Oeiras, Portugal] [INIAV]
dc.contributor.authorSOUSA, Esmeraldina
hal.structure.identifierDienstleistungszentrum Ländlicher Raum DLR Rheinpfalz [DLR Rheinpfalz]
dc.contributor.authorWETZEL, Thierry
hal.structure.identifierAgroscope Changins
dc.contributor.authorDEBONNEVILLE, Christophe
hal.structure.identifierDepartment of Agriculture, Food and the Marine, Plant Health Laboratories
dc.contributor.authorDELLA BARTOLA, Michele
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorBEY, Jean-Said
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorSALAR, Pascal
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorMALEMBIC-MAHER, Sylvie
hal.structure.identifierBiologie du fruit et pathologie [BFP]
dc.contributor.authorFOISSAC, Xavier
dc.date.accessioned2024-09-18T02:03:01Z
dc.date.available2024-09-18T02:03:01Z
dc.date.conference2024-05-14
dc.identifier.urihttps://oskar-bordeaux.fr/handle/20.500.12278/201635
dc.description.abstractEnINTRODUCTIONFlavescence dorée (FD), the most impacting grapevine yellows in Europe, is associated to Scaphoideus titanus-transmitted phytoplasmas also called Flavescence dorée phytoplasmas sensu stricto (FDp). FDp variants belong to taxonomic subgroups 16SrV-C and 16SrV-D. Up to current knowledge, the single 16SrV-D map-genotype variant M54 as well as the more than 150 variants of subgroup 16SrV-C are all listed on the EU quarantine list, regardless of their epidemic properties in the vineyard. FD phytoplasma epidemic nature is particularly tied to specific forms of the adhesin-like variable membrane proteins (VMP), determining Vectotypes II and III that are epidemic in European vineyards and Vectotype I (16SrV-C subgroup) that are non-epidemic in the vineyard. Vectotype I of European alders, occasionally transmitted from alder trees to grapevine by Oncopsis alni, poses no outbreak risks (Malembic-Maher et al., 2020). Distinguishing between epidemic and non-epidemic isolates is important for an effective and sustainable management. Current diagnostic tests (EPPO, 2016) target 16SrV group without differentiation, while vectotypes determination offer the advantage to directly identify vectotypes. This study aims to validate identification methods capable to distinguish FDp from other 16SrV phytoplasmas.MATERIALS AND METHODSDesign of the study. Eleven participant laboratories from 9 different countries tested an identical series of double-blind samples following the provided working protocols and using their own reagents and machines.Samples. The tested samples consisted of total nucleic acid (TNA) extracts, instead of fresh plant leaf samples, to avoid problems of homogeneity between laboratory extraction methods and stability over shipping. To evaluate analytical specificity, the samples consisted of 15 target isolates of FDp from different plant hosts, different geographical origins and different genotypes and 15 plants contaminated by other phytoplasmas of 16SrV group or phytoplasmas responsible of grapevine yellows or healthy grapevines. Each sample was tested in duplicate. To evaluate the analytical sensitivity, the repeatability and the reproducibility, a 5-level dilution series of grapevine infected by FDp diluted in healthy grapevine was analyzed 6 time.Tested protocols. After intra-laboratory adaptation and selection, three molecular protocols were submitted to the interlaboratory trial: 1) a nested-PCR 16SrV map adapted from Arnaud et al. (2007), followed by sequence analysis (Method1); 2) a nested-PCR VmpA-R1 adapted from Rossi et al. (2019) and Malembic-Maher et al. (2020), followed by sequence analysis (Method2); 3) a real-time PCR Vmp-RK-A23B1 (Foissac et al., this congress) (Method3).Processing of the results. The following parameters were calculated: analytical specificity, representing the ability of the methods to correctly identify the target phytoplasma; analytical sensitivity, representing the last level at 100% positive results; repeatability and reproducibility (adapted from EPPO, 2021).RESULTS AND DISCUSSIONAnalytical specificity varied across the methods. The real-time PCR Vmp-RK-A23B1 (Method3) exhibited the highest analytical specificity for FDp (98%), while the nested-PCR 16SrV map followed by sequence analysis (Method1) showed the lowest analytical specificity (87%). However, for the Vmp I cluster, Method3 generated 32.7 % of false positive results. The best analytical sensitivity is obtained with Method1 and Method3 with 100% detection of FDp at 1x10-1. The repeatability of the evaluated methods ranges from 78 to 100% depending on the laboratory. The best average repeatability is obtained with Method3. The reproducibility also varied across the methods from 81 to 96%, with Method3 demonstrating the highest reproducibility.The performance characteristics of the real-time PCR are most interesting for the identification of FDp. However, the primers and probe designed for the identification of Vectotype I (phytoplasmas of 16SrV group but not FDp) showed low specificity as they target the poorly variable vmpB gene. The Vmp-RK-A1 simplex qPCR method could hence be carried out separately when confirmation of the presence of a Vectotype I variant is needed.Less accurate results were obtained for the two nested-PCRs followed by sequencing analysis. As it can be expected for such methods, their reproducibility was also lower than that of the real-time PCR. These methods remain useful as they allowed the distinction between different map types and vectotypes (here only based on R1-vmpA repeated domain), and the specific identification of the other non-epidemic phytoplasmas that can be detected in grapevine. However, critical points have to be underlined and taken into consideration by users for their proper implementation: risk of micro-contaminations, choose of reliable sequencing service providers, experience and/or training in sequence analysis.By sharing protocols, conducting test performance studies and aligning diagnostic procedures, laboratories involved in routine diagnosis can ensure the accuracy, reliability and reproducibility of diagnostic methods. And, by enhancing our ability to accurately identify FDp, we can significantly improve disease surveillance and sustainable control measures.ACKNOWLEDGEMENTSThis work was carried out in the frame of EUPHRESCO project FLADO VIGILANT. The authors would like to thank Fiona Constable and Bianca Rodrigues Jardim (Australian Government) for providing of DNA extracts of some non-target phytoplasmas.REFERENCESArnaud, G., Malembic-Maher, S., Salar, P., et. al. (2007). Multilocus sequence typing confirms the close genetic interrelatedness of three distinct flavescence doree phytoplasma strain clusters and group 16SrV phytoplasmas infecting grapevine and alder in Europe. Appl Environ Microbiol, 73(12), 4001-4010. https://doi.org/10.1128/AEM.02323-06 EPPO. (2016). PM 7/079 (2) Grapevine flavescence dorée phytoplasma. EPPO Bulletin, 46(1), 78-93. https://doi.org/10.1111/epp.12280 EPPO. (2021). PM 7/98 (5) Specific requirements for laboratories preparing accreditation for a plant pest diagnostic activity. EPPO Bulletin, 51(3), 468-498. https://doi.org/10.1111/epp.12780 Malembic-Maher, S., Desque, D., Khalil, D., et al.. (2020). When a Palearctic bacterium meets a Nearctic insect vector: Genetic and ecological insights into the emergence of the grapevine Flavescence doree epidemics in Europe. PLoS Pathog, 16(3), e1007967. https://doi.org/10.1371/journal.ppat.1007967 Rossi, M., Pegoraro, M., Ripamonti, et al. (2019). Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale. Appl Environ Microbiol, 85(10), e03123-03118. https://doi.org/10.1128/aem.03123-18
dc.language.isoen
dc.rights.urihttp://creativecommons.org/licenses/by/
dc.source.titleProceedings of the sixth European Bois noir workshop and first international Pro-AECOGY conference
dc.title.enValidation of methods for Flavescence dorée phytoplasma sensu stricto identification through Test Performance Studies.
dc.typeCommunication dans un congrès
dc.subject.halSciences du Vivant [q-bio]/Microbiologie et Parasitologie
bordeaux.hal.laboratoriesBiologie du Fruit & Pathologie (BFP) - UMR 1332*
bordeaux.institutionUniversité de Bordeaux
bordeaux.institutionINRAE
bordeaux.conference.titleSixth European Bois noir workshop and first international Pro-AECOGY conference
bordeaux.countryFR
bordeaux.title.proceedingProceedings of the sixth European Bois noir workshop and first international Pro-AECOGY conference
bordeaux.conference.cityBordeaux
bordeaux.peerReviewedoui
hal.identifierhal-04700053
hal.version1
hal.invitednon
hal.proceedingsoui
hal.conference.organizerINRAE and University of Bordeaux
hal.conference.end2024-05-16
hal.popularnon
hal.audienceInternationale
hal.origin.linkhttps://hal.archives-ouvertes.fr//hal-04700053v1
bordeaux.COinSctx_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.btitle=Proceedings%20of%20the%20sixth%20European%20Bois%20noir%20workshop%20and%20first%20international%20Pro-AECOGY%20conference&rft.au=LOISEAU,%20Marianne&COUSSEAU-SUHARD,%20Pascaline&RENAUDIN,%20Isabelle&KOGEJ%20ZWITTER,%20Zala&MEHLE,%20Natasa&rft.genre=unknown


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