The vineyards of Palatinate (Germany) and Alsace (France) in the Upper Rhine valley are still free from Flavescence dorée (FD) disease, but the main vector in grapevine, the leafhopper Scaphoideus titanus, has recently been detected. Knowledge of reservoir plants and alternative vectors of FD phytoplasmas (FDp) is crucial for management strategies. An interregional survey showed that among 12 main wild woody plants and liana species sampled in the environments of vineyards, only alders were infected by FDp, with high prevalence of MAP-FD2 genotypes. Yellow sticky traps were used to record the abundance and analyze the natural infection rate of potential leafhopper vectors on alders, clematis and cultivated grapevine. Live specimens were collected for inoculation trials. High infection rates and transmission capacities of resident Allygus spp. and the invasive Orientus ishidae were confirmed. In addition, five other Deltocephalinae species were identified as potential vectors of FDp. Lamprotettix nitidulus showed a high abundance and high infection rates with FDp in both regions. Its transmission competence of FDp to alder and broad bean could be demonstrated through repeated trials and high FDp titers were measured in infective individuals. Similar titers were measured in Synophropsis lauri, suggesting it could also transmit FDp. Fieberiella florii, Euscelidius variegatus and Japananus hyalinus may also play a role for the spread of 16SrV group phytoplasmas. Six new MAP-FD1 and FD2 genotypes were detected in leafhoppers. The prevalent and only transmitted genotype was M38: a vectotype II compatible with S. titanus transmission. The results show that the natural spread of FDp among alders is more complex than previously thought, and thus, the risk of transmissions to grapevine is more difficult to evaluate.

Within the 16SrII phytoplasma group, subgroups A-X have been classified based on restriction fragment length polymorphism of their 16S rRNA gene, and two species have been described, namely 'Candidatus Phytoplasma aurantifolia' and 'Ca. Phytoplasma australasia'. Strains of 16SrII phytoplasmas are detected across a broad geographic range within Africa, Asia, Australia, Europe and North and South America. Historically, all members of the 16SrII group share ≥97.5 % nucleotide sequence identity of their 16S rRNA gene. In this study, we used whole genome sequences to identify the species boundaries within the 16SrII group. Whole genome analyses were done using 42 phytoplasma strains classified into seven 16SrII subgroups, five 16SrII taxa without official 16Sr subgroup classifications, and one 16SrXXV-A phytoplasma strain used as an outgroup taxon. Based on phylogenomic analyses as well as whole genome average nucleotide and average amino acid identity (ANI and AAI), eight distinct 16SrII taxa equivalent to species were identified, six of which are novel descriptions. Strains within the same species had ANI and AAI values of >97 %, and shared ≥80 % of their genomic segments based on the ANI analysis. Species also had distinct biological and/or ecological features. A 16SrII subgroup often represented a distinct species, e.g., the 16SrII-B subgroup members. Members classified within the 16SrII-A, 16SrII-D, and 16SrII-V subgroups as well as strains classified as sweet potato little leaf phytoplasmas fulfilled criteria to be included as members of a single species, but with subspecies-level relationships with each other. The 16SrXXV-A taxon was also described as a novel phytoplasma species and, based on criteria used for other bacterial families, provided evidence that it could be classified as a distinct genus from the 16SrII phytoplasmas. As more phytoplasma genome sequences become available, the classification system of these bacteria can be further refined at the genus, species, and subspecies taxonomic ranks.

Flavescence dorée (FD) phytoplasma from 16SrV-C and -D subgroups cause severe damage to grapevines throughout Europe. This phytoplasma is transmitted from grapevine to grapevine by the sap-sucking leafhopper Scaphoideus titanus. European black alder and clematis serve as perennial plant reservoirs for 16SrV-C phytoplasma strains, and their host range has recently been extended to hazelnuts. In Slovenia, hazelnut orchards are declining due to 16SrV phytoplasma infections, where large populations of the non-autochthonous leafhopper Orientus ishidae have been observed. To better characterise the phytoplasma-induced decline of hazelnut and possible transmission fluxes between these orchards and grapevine, genetic diversity of 16SrV phytoplasmas in grapevine, hazelnut and leafhoppers was monitored from 2017 to 2022. The nucleotide sequence analysis was based on the map gene. The most prevalent map genotype in grapevine in all wine-growing regions of Slovenia was M54, which accounted for 84% of the 176 grapevines tested. Besides M54, other epidemic genotypes with lower frequency were M38 (6%), M51 (3%), M50 (2%) and M122 (1%). M38, M50 and M122 were also detected in infected cultivated hazelnuts and in specimens of O. ishidae leafhopper caught in declining hazelnut orchards. It suggests that this polyphagous vector could be responsible for phytoplasma infection in hazelnut orchards and possibly for some phytoplasma exchanges between hazelnuts and grapevine. We hereby describe new genotypes: M158 in grapevine as well as four never reported genotypes M159 to M162 in hazelnut. Of these four genotypes in hazelnut, one (M160) was also detected in O. ishidae. Analysis of additional genes of the new genotypes allowed us to assign them to the VmpA-III cluster, which corresponds to the 16SrV-C strains previously shown to be compatible with S. titanus transmission.

Flavescence dorée (FD) is a European quarantine grapevine disease transmitted by the Deltocephalinae leafhopper Scaphoideus titanus. Whereas this vector had been introduced from North America, the possible European origin of FD phytoplasma needed to be challenged and correlated with ecological and genetic drivers of FD emergence. For that purpose, a survey of genetic diversity of these phytoplasmas in grapevines, S. titanus, black alders, alder leafhoppers and clematis were conducted in five European countries. Out of 132 map genotypes, only 11 were associated to FD outbreaks, three were detected in clematis, whereas 127 were detected in alder trees, alder leafhoppers or in grapevines out of FD outbreaks. Most of the alder trees were found infected, including 8% with FD genotypes M6, M38 and M50, also present in alders neighboring FD-free vineyards and vineyard-free areas. The Macropsinae Oncopsis alni could transmit genotypes unable to achieve transmission by S. titanus, while the Deltocephalinae Allygus spp. and Orientus ishidae transmitted M38 and M50 that proved to be compatible with S. titanus. Variability of vmpA and vmpB adhesin-like genes clearly discriminated 3 genetic clusters. Cluster Vmp-I grouped genotypes only transmitted by O. alni, while clusters Vmp-II and -III grouped genotypes transmitted by Deltocephalinae leafhoppers. Interestingly, adhesin repeated domains evolved independently in cluster Vmp-I, whereas in clusters Vmp-II and-III showed recent duplications. Latex beads coated with various ratio of VmpA of clusters II and I, showed that cluster II VmpA promoted enhanced adhesion to the Deltocephalinae Euscelidius variegatus epithelial cells and were better retained in both E. variegatus and S. titanus midguts. Our data demonstrate that most FD phytoplasmas are endemic to European alders. Their emergence as grapevine epidemic pathogens appeared restricted to some genetic variants pre-existing in alders, whose compatibility to S. titanus correlates with different vmp gene sequences and VmpA binding properties.

Grapevine yellows (GY) affecting Lebanese vineyards are reported to be associated with ‘Candidatus Phytoplasma solani’, the bois noir (BN) etiological agent. However, during a field survey in June 2014 for BN in Mansoura municipality of West Bekaa, Lebanon, ‘Candidatus Phytoplasma omanense’ was detected in a grapevine sample, cultivar Syrah, exhibiting leaf scorch and discoloration, using a phytoplasma universal nested-PCR and sequencing of the 1.2 kbp 16SrDNA amplicon. The same 1.2 kbp 16SrDNA sequence could be amplified from Hyalesthes obsoletus and Reptalus sp. Cixiidae planthoppers collected on Convolvulus arvensis (bindweed) plants in June 2014 in Aammiq municipality of West Bekaa. Yellowing and stunted bindweed plants collected in 2015 in Kefraya and Aamiq municipalities of West Bekaa were also found infected with the same phytoplasma strain. A 16S rDNA RFLP assay was designed to differentiate this phytoplasma from ‘Ca. P. solani’ and ‘Ca. P. phoenicium’, which are endemic to the Bekaa region. ‘Ca. P. omanense’ is reported for the first time in grapevine and in Cixiidae planthoppers already known to respectively host and vector ‘Ca. P. solani’ strains associated with BN disease in grapevine. This result highlights the need for a precise survey of phytoplasmas associated to grapevine yellows in the Eastern Mediterranean basin, and for investigating the possible role of ‘Ca. P. omanense’ as a new threat to Euro-Mediterranean viticulture.

A survey of genetic diversity of "flavescence dorée" (FD)-related phytoplasmas in grapevines, alders and clematis as well as alder-feeding leafhoppers was conducted in France, Hungary, Germany, Italy and Serbia.Genotyping was based on the housekeeping gene map and on the vmp genes encoding surface variable membrane proteins.Transmission assays of the phytoplasmas were performed with alder and/or grapevine-feeding leafhoppers.The study demonstrated that European alders constitute an original reservoir of FD phytoplasma by hosting a high diversity of FD-related phytoplasma genotypes, also present in non viticultural areas.The alder phytoplasmas grouping in Vmp-I cluster were transmitted by the Macropsinae O. alni, but were not compatible with the FD phytoplasma vector on grapevine the Deltocephalinae Scaphoideus titanus.The alder phytoplasmas in Vmp-II and -III clusters were transmitted by the Deltocephalinae Allygus spp.and Orientus ishidae.Such pre-existing phytoplasmas were compatible with S. titanus transmissibility and can be responsible for the emergence of FD phytoplasma epidemics in grapevine.VmpA proteins of cluster II better adhered to Euscelidius variegatus and S. titanus insect cells and midguts than those of cluster I.Such adhesins might play a key role in the adaptation to new vectors.

To study the role of wild areas around the vineyards in the epidemiology of flavescence dorée (FD) and track the origin of new foci, two phytoplasma genetic markers, dnaK and malG, were developed for FD phytoplasma (FDp) characterization. The two genes and the vmpA locus were used to genetically characterize FDp populations at seven agroecosystems of a wine-growing Italian region. Vitis vinifera, "gone-wild" V. vinifera and rootstocks, Clematis spp., and Scaphoideus titanus adults were sampled within and outside the vineyards. A range of genotypes infecting the different hosts of the FDp epidemiological cycle was found. Type FD-C isolates were fairly homogeneous compared to type FD-D ones. Most of the FD-D variability was correlated with the malG sequence, and a duplication of this locus was demonstrated for this strain. Coinfection with FD-C and FD-D strains was rare, suggesting possible competition between the two. Similar levels of FDp genetic variation recorded for grapevines or leafhoppers of cultivated and wild areas and co-occurrence of many FDp genotypes inside and outside the vineyards supported the idea of the importance of wild or abandoned Vitis plants and associated S. titanus insects in the epidemiology of the disease. Genetic profiles of FDp found in Clematis were never found in the other hosts, indicating that this species does not take part in the disease cycle in the area. Due to the robustness of analyses using dnaK for discriminating between FD-C and FD-D strains and the high variability of malG sequences, these are efficient markers to study FDp populations and epidemiology at a small geographical scale.IMPORTANCE Flavescence dorée, a threatening disease of grapevine caused by FD phytoplasma (FDp), is distributed within the most important wine-producing areas of Europe and has severe effects on both vineyard productivity and landscape management. FDp is a quarantine pest in Europe, and despite the efforts to contain the pathogen, the disease is still spreading. In this work, new genetic markers for the fine genetic characterization of FDp at local scale are presented. Our findings improve the knowledge of FDp epidemiological cycle and offer the possibility of tracking the route of the FDp infection. In particular, due to its high genetic variability, one of the newly developed markers could be sufficient to track the origin of new infection foci, either from the wild areas or from nurseries.

Lavender decline compromises French lavender production, and preliminary data have suggested the involvement of "Candidatus Phytoplasma solani" in the etiology of the disease. In order to evaluate the epidemiological role of "Ca. Phytoplasma solani," a 3-year survey was conducted in southeastern France. "Ca. Phytoplasma solani" was detected in 19 to 56% of the declining plants, depending on seasons and cultivars, and its prevalence was correlated with symptom severity. Autumn was more favorable than spring for phytoplasma detection, and "Ca. Phytoplasma solani" incidence was higher in Lavandula angustifolia than in Lavandula intermedia hybrids. Detection of the phytoplasma fluctuated over months, supporting the chronicity of infection. Three "Ca. Phytoplasma solani" secY genotypes, S17, S16, and S14, were the most prevalent in lavender fields and were also detected in nurseries, whereas strains detected in surrounding bindweed and wild carrots were mostly of the S1 and S4 genotypes. This suggests that lavender is the main pathogen reservoir of the epidemic. Adults and nymphs of the planthopper vector Hyalesthes obsoletus were commonly captured in lavender fields and were shown to harbor mainly the prevalent phytoplasma genotypes detected in lavenders. The "Ca. Phytoplasma solani" genotype S17 was transmitted to Catharanthus roseus periwinkle by naturally infected H. obsoletus. Finally, the inventory of the bacterial community of declining lavenders that tested negative for "Ca. Phytoplasma solani" by 16S rRNA deep sequencing ruled out the involvement of other phloem-limited bacterial pathogens. IMPORTANCE The etiology and main pathways for the spread of lavender decline, an infectious disease affecting French lavender production since the 1960s, have remained unclear, hampering the development of efficient control strategies. An extensive survey of lavender fields led to the conclusion that "Candidatus Phytoplasma solani" was chronically infecting declining lavenders and was associated with large infectious populations of Hyalesthes obsoletus planthoppers living on the crop itself. Lavender appeared to be the main reservoir host for lavender-specific phytoplasma strains, an unusual feature for this phytoplasma, which usually propagates from reservoir weeds to various economically important crops. These results point out the necessity to protect young lavender fields from the initial phytoplasma inoculum coming from surrounding lavender fields or from infected nurseries and to promote agricultural practices that reduce the development of H. obsoletus vector populations.

Flavescence dorée (FD) is a quarantine disease of grapevine, involving interactions between the plants, leafhopper vectors, and FD phytoplasma. Characterizing the susceptibility of vine varieties could limit disease propagation. After extensive surveys in vineyards, we showed that Cabernet Sauvignon (CS) is highly susceptible, with a high proportion of symptomatic branches and phytoplasma titers, in contrast to Merlot (M). Localized insect transmissions and grafting showed that phytoplasma circulate in the whole plant in the CS cultivar, but in M they are restricted to the transmission point. Insect-mediated transmission under high confinement mimicking natural conditions confirmed these phenotypes and allowed the classification of 28 Vitis accessions into three distinct categories, according to the percentage of infected plants and their phytoplasma titers. Reduced symptoms, low phytoplasma titers, and low percentages of infected plants were found to be associated in the Vitis vinifera cultivars tested. Interestingly, the low susceptibility of M was observed for one of its parents, i.e., Magdeleine Noire des Charentes. Rootstocks and their Vitis parents, although having high percentages of infected plants and intermediate to high phytoplasma titers, shared a symptomless response. This is troubling, because rootstocks can constitute a silent reservoir of contamination in mother plants or when they grow wild nearby vineyards. Altogether, data suggest distribution of genetic traits within the Vitis genus involved in insect-mediated phytoplasma transmission, multiplication, circulation, and symptom development.