| dc.description.abstractEn | INTRODUCTIONFlavescence dorée (FD) is a severe disease in European vineyards caused by phytoplasma genotypes of the 16SrV-C and D subgroups. FD phytoplasmas (FDp) also infect wild European alders (Alnus glutinosa) and clematis (Clematis vitalba), considered as the original reservoir plants. The main vector of FDp on grapevine is the North-American leafhopper Scaphoideus titanus (ST), introduced in Europe in the early 1900s. However, other auchenorrhynchan species, such as Allygus spp., Dictyophara europaea and Orientus ishidae were characterized as alternative vectors able to propagate FDp in reservoir plants from the environments and occasionally transmit them to grapevine (Filippin et al., 2009; Lessio et al., 2016; Malembic-Maher et al., 2020). Besides the known hosts, the recent detection of FDp in refuge plants and leafhoppers species could complexify FD epidemiological cycle (Casati et al., 2017). The aim of this study was to investigate the potential origin of FD isolated cases detected in vineyards of North-East France, an area that was still free of FD outbreaks, by characterizing FDp reservoir plants and potential leafhopper vector species in the vineyard landscapes.MATERIALS AND METHODSIsolated FD cases were detected in some vineyards of NE France between 2019 and 2021: 1 site in Alsace in Bergholtzzell (site B), and in 6 sites in Champagne in Arrentières (A), Chouilly-South (Cs), Chouilly-North (Cn), Mancy (M), Reuil (R) and Saudoy (S). In each site, ligneous trees and vines surrounding the vineyards were inventoried and wood canes were sampled from autumn 2020 to 2022. Auchenorrhynchan insects (except Thyphlocybinae subfamily) were captured on 23, 29 and 25 yellow sticky traps installed inside the vineyards and in the wild compartment in 2020, 2021 and 2022 respectively. They were renewed every two weeks, from June to September. Insects were also collected by beating the vegetation with sweeping nets and identified with a taxonomic key (Biedermann & Niedringhaus 2009). Acquisition trials of FDp on infected alders by ST were performed in confined greenhouse. Alive O. ishidae were captured on FDp-infected alders in Bommes (South-West France) and transferred by groups of 10 to 15 individuals on healthy alder seedlings for FDp transmission during 1 week. After 3 months incubation, 2 FDp infected alders were selected by PCR. Groups of 80 ST nymphs (L4-L5 stages) were placed twice on the infected alders during 3 to 4 days for FDp acquisition. ST individuals were transferred on 2 grapevines (Fercal) for a 4 weeks latency period. ST were then transferred by groups of 10 individuals on 12 healthy broad beans (Vicia faba) for FDp transmission during one week. Total DNA from plants and insects was extracted following the CTAB and TNES protocols respectively. Extracts were screened for 16SrV phytoplasmas by nested PCR on the map gene followed by sequencing for genotyping (Arnaud et al., 2007). RESULTS AND DISCUSSIONFDp infecting isolated grapevines were M50 genotype in sites Cn, Cs and M, M50 variant (1 SNP) in sites A and S, and M38 in sites B and R. The age of the stocks, greater than 10 years, precluded a possible introduction of the phytoplasma by infected plant material and favored the hypothesis of a transfer from wild plant reservoirs. A total of 394 plant samples of 42 genera collected in the vineyard environment were tested. Alders were only present in sites B, A and R, and were highly infected by populations of 16SrV-C map genotypes (17/20) as already described in other European regions. The genotypes were identified as FDp and Alder Yellow phytoplasmas (AldYp) compatible and not compatible with ST transmission respectively (Malembic-Maher et al., 2020). Since none of the other plant species were infected by 16SrV-C or -D phytoplasmas, alders were the only reservoir plants identified. A total of 33 921 insects belonging to 57 genera were collected and 1 665 insect samples were tested for phytoplasma infection. Nearly 25% of these samples were infected by 16SrV phytoplasmas: either by FDp M38 genotype, by AldYp genotypes, by Candidatus ‘Phytoplasma ulmi’ (16SrV-A) or by unresolved mixed infections. In Alsace, in site B, where ST is absent, the alternative FDp Deltocephalinae vectors Allygus spp, O. ishidae and Lamprotettix nitidulus (B. Jarausch & M. Maixner, personal communication) captured on alder and grapevine, had a high 16SrV-C infection rate ranging from 60 % to 77 % with FDp M38 genotype being predominant. They could be responsible for the single M38 isolated case detected in grapevine, suggesting a low frequency transfer from alders. Other leafhoppers individuals from the species Euscelidius spp., Fieberiella spp., Japananus hyalinus and Graphocephala fennahi were also infected by the M38 genotype. Their vectoring ability needs to be evaluated by transmission trials. In site R, where several FDp M38 scattered cases were detected in grapevine, only 3 over 48 Allygus spp. captured on alders were infected by M38 genotype and 25 were infected by AldYp or unresolved mixed infections. This species could be responsible for the transfer of FDp M38 from alders to the vineyards. No O. ishidae were captured in this site, and none of the 17 L. nitidulus tested were infected by M38 genotype. High ST populations were monitored in this site. One ST over 347 captured on grapevine was infected by M38 suggesting that the M38 cases in neighboring vineyards could also be the result of a low frequency transmission between grapevines by ST. As infected ST were also collected on alders (10/143 samples, 1 M38), the possible FDp acquisition by ST on alder was assessed. Acquisition trials were performed on two experimentally FDp-infected alders. At the end of the trials, 6 symptomatic broad beans and 2 asymptomatic latency grapevines were detected infected by FDp. These results indicate that ST is experimentally able to acquire FDp on alders and transmit it to grapevine and broad bean. In sites where single M50 and M50 variant genotypes were detected in grapevine, none of the tested insects were infected by these genotypes, regardless of the presence (site A) or the absence (sites Cn, Cs, S and M) of infected alders in the vicinity of the vineyards. In site A, alders and Allygus spp. were only infected by a mixture of AldYp genotypes. The reservoir plants and alternative insect vectors of M50 genotypes are still unknown and the origin of the isolated cases need further investigations. ACKNOWLEDGEMENTSFundings: PNDV CoAct2, ANR Beyond projects. Thanks to: C. Abidon (IFV), M. Delame, B. Doublet I. Riou, D. Roger, L. Henriet, D. Petermann, I. Maurice (SRAL GE), P. Pienne, A. Bonomelli, F. Hainez (CIVC), C. Gisbert, J. Beuzelin (FREDON-GE), B. Jarausch, M. Maixner (JKI).REFERENCESArnaud, G. et al. 2007. Multilocus sequence typing confirms the close genetic interrelatedness of three distinct “flavescence dorée” phytoplasma strain clusters and group 16SrV phytoplasmas infecting grapevine and alder in Europe. Applied and Environmental Microbiology, 73(12): 4001-4010.Biedermann, R. & Niedringhaus, R. 2009. The plant- and leafhopper of Germany - identification key to all species. In: Wissenschaftlich Akademischer buchvertrieb-Fründ, 409 pp. Scheessel, Germany.Casati, P. et al. 2017. New insights on “Flavescence dorée” phytoplasma ecology in the vineyard agro ecosystem in southern Switzerland. Annals of Applied Biology, 171(1): 37-51.Christensen, N.M. et al.2004. Distribution of phytoplasmas in infected plants as revealed by real-time PCR and bioimaging. Molecular Plant-Microbe Interactions, 17(11): 1175-1184.Filippin, L., et al. 2009. Molecular characteristics of phytoplasmas associated with “flavescence dorée” in clematis and grapevine and preliminary results on the role of Dictyophara europaea as a vector. Plant Pathology, 58: 826–837.Lessio, F. et al. 2016. The mosaic leafhopper Orientus ishidae: host plants, spatial distribution, infectivity, and transmission of 16SrV phytoplasmas to vines. Bulletin of Insectology, 69(2): 277-289.Malembic-Maher, S. & Desqué, D. et al. 2020. When a Palearctic bacterium meets a Nearctic insect vector: genetic and ecological insights into the emergence of the grapevine “flavescence dorée” epidemics in Europe. Plos Pathogens, 16: e1007967. | |
