Flavescence dorée (FD) is the most threatening grapevine yellows (GY) disease in Europe. Despite strict control measures, alarming signs of the spread of the disease in viticultural areas continue to ...be detected. FD is attributed to infection by phytoplasma strains of an incidentally cited species, ‘Candidatus Phytoplasma vitis’. In 2017, a GY field survey was carried out in traditional viticulture areas of Tuscany, central Italy. FD phytoplasma (FDp) was detected in 85 GY symptomatic vines, accounting for 17% of a total of 500 symptomatic samples screened. The FDp-positive vines were scattered in 50 vineyards across seven Tuscan provinces, indicating the distribution of FDp has further extended to central and southwestern parts of Tuscany including Florence and Livorno. Multilocus sequence typing of 15 representative FDp strains from six affected vineyards revealed that the Tuscan FDp strains constitute a highly homogeneous lineage within the subgroup 16SrV–C (FD-C). Single nucleotide polymorphisms (SNPs) were identified in the 16S rRNA, rp, and secY genes of the Tuscan FDp lineage. Such SNP markers provide clues to understanding the genetic relationships among different FDp lineages present in Europe and are useful for searching potential vectors and reservoirs involved in the spread of the FDp in the Tuscan region.
•Flavescence dorée (FD) is a destructive grapevine disease in Europe.•FD disease has been detected in Florence and Livorno, Italy for the first time.•The FD phytoplasma strains found in Tuscan vineyards are highly homogenous.•Single nucleotide polymorphism markers of the phytoplasma lineage are identified.
The focus of this research was the development and evaluation of different complex liquid and solid media for the isolation and growth of phytoplasma strains infecting grapevine plants. Previously ...reported media supporting phytoplasma isolation are commercial and not easy to modify in order to improve performance and selectivity towards obtaining pure cultures of ‘Candidatus Phytoplasma’ species. Three media (Piv®, CB and MB) were therefore evaluated for phytoplasma isolation and colony formation under microaerophilic growing conditions, using grapevine canes from plants showing yellows symptoms, and infected by “flavescence dorée”, “bois noir” and aster yellows phytoplasmas as sources. The newly developed methodology was applied for two years at three sample collection times. Broad applicability and a good repeatability in supporting phytoplasma colony formation were obtained in Pivs® and CBs media. While the MB medium did not support phytoplasma isolation and growth, the CB media support a phytoplasma growth comparable to the one obtained in the previously reported media. This medium has the advantage of a formulation that allow its modification to implement specificity towards selective phytoplasma growth. Moreover preliminary trials on serial dilutions and tetracycline addition confirmed some phytoplasma growth behaviours.
•Phytoplasmas were isolated from infected field-collected grapevine samples.•Complex media for phytoplasma isolation and growth were tested.•Unreported microaerophilic conditions are settled for phytoplasma plate colony growth.•“Flavescence dorée”, “bois noir” and aster yellows phytoplasma colonies are obtained from field infected materials.•Phytoplasma presence in colonies was detected by nested-PCR and sequencing on two genes.
Phytoplasmas are associated with important bacterial diseases, causing severe symptoms in agricultural and ornamental crops. ‘Candidatus Phytoplasma rubi’, associated with the Rubus stunt in ...raspberries (Rubus idaeus) and blackberries (Rubus subgenus Rubus), causes shortened internodes, elongated sepals, proliferation, phyllody, and virescence. The recently published genome of ‘Ca. P. rubi’ RS enabled a comprehensive genomic comparison to the complete genomes of 16SrV phytoplasmas, comprising strains of the flavescence dorée-associated phytoplasma CH and two ‘Candidatus Phytoplasma ziziphi’ strains. Besides the typical transporters and metabolic features of phytoplasmas, the phosphorolysis of sucrose and the utilization of the carboxylic acid L-lactate became apparent for the 16SrV-group. With respect to the effector repertoire and the encoded immunodominant membrane proteins involved in host colonization, the group revealed conserved features that comprise the variable membrane proteins A and B. However, SAP11- and SAP54 orthologs were limited to ‘Ca. P. rubi’ RS and ‘Ca. P. ziziphi’. Genome-sequence-based phylogenetic analysis supports the close relationship of these genomes relative to alder yellows phytoplasmas. The analyses supported the impact of the mobilome on phytoplasma evolution but also highlighted that there is the possibility of identifying phytoplasmas with a larger metabolic repertoire in the future.
Following a request from the European Commission, the EFSA Panel on Plant Health (PLH) performed a quantitative analysis of the risk posed by the Flavescence dorée phytoplasma (FDp) in the EU ...territory. Three scenarios were analysed, one with current measures in place (scenario A0), one designed to improve grapevine propagation material phytosanitary status (scenario A1) and one with reinforced eradication and containment (scenario A2). The potential for entry is limited, FDp being almost non‐existent outside the EU. FDp and its major vector, Scaphoideus titanus, have already established over large parts of the EU and have the potential to establish in a large fraction of the currently unaffected EU territory. With the current measures in place (A0), spread of FDp is predicted to continue with a progression of between a few and ca 20 newly infested NUTS 2 regions during the next 10 years, illustrating the limitations of the current control measures against spread. FDp spread is predicted to be roughly similar between scenarios A1 and A2, but more restricted than under scenario A0. However, even with reinforced control scenarios, stabilisation or reduction in the number of infested NUTS 2 regions has only relatively low probability. Under scenario A0, FDp has a 0.5–1% impact on the overall EU grapes and wine production, reflecting the effectiveness of the current control measures against impact. Under both scenarios A1 and A2, FDp impact is predicted to be reduced, by approximately one‐third (A1) to two‐thirds (A2) as compared to A0, but the associated uncertainties are large. The generalised use of hot water treatment for planting material produced in infected zones has the most important contribution to FDp impact reduction in scenario A1 and has high feasibility. Both increased eradication and containment measures contribute to impact reduction under scenario A2 but the overall feasibility is lower.
The increasing demand for safe and sustainably produced food is leading to the development of strategies of pest control alternative to chemicals. One innovative method is Vibrational Mating ...Disruption (VMD) to disrupt insect communication in plants. VMD was proven effective in preventing mating of the grapevine pest Scaphoideus titanus, vector of flavescence dorée. However, the stress induced by VMD on the target species has the potential to influence other crucial aspects of the insect biology and ethology. Therefore, the goal of this study was to understand side effects of VMD on the flight activity and oviposition of S. titanus. The results of our experiments conducted in the greenhouse showed that in the presence of a receptive female, males fly more if exposed to vibrations than in the silent control but not differently from singles males in silence. Surprisingly, we found that also females subjected to VMD fly more than in the silence. Regarding oviposition, we found that mated females exposed to vibrations and single females (unmated) laid significantly fewer eggs than mated females in silence. In conclusion, this study shows the potential of VMD to interfere, besides with mating, with other important biological aspects of the pest species.
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•Vibrational Mating Disruption (VMD) increases flight activity of Scaphoideus titanus males and females.•VMD reduces oviposition in S. titanus.•VMD could affect pest population density by interfering with multiple behaviors of target species.
Flavescence dorée is a grapevine disease affecting European vineyards which has severe economic consequences and containing its spread is therefore considered as a major challenge for viticulture. ...Flavescence dorée is subject to mandatory pest control including removal of the infected vines and, in this context, automatic detection of Flavescence dorée symptomatic vines by unmanned aerial vehicle (UAV) remote sensing could constitute a key diagnosis instrument for growers. The objective of this paper is to evaluate the feasibility of discriminating the Flavescence dorée symptoms in red and white cultivars from healthy vine vegetation using UAV multispectral imagery. Exhaustive ground truth data and UAV multispectral imagery (visible and near-infrared domain) have been acquired in September 2015 over four selected vineyards in Southwest France. Spectral signatures of healthy and symptomatic plants were studied with a set of 20 variables computed from the UAV images (spectral bands, vegetation indices and biophysical parameters) using univariate and multivariate classification approaches. Best results were achieved with red cultivars (both using univariate and multivariate approaches). For white cultivars, results were not satisfactory either for the univariate or the multivariate. Nevertheless, external accuracy assessment show that despite problems of Flavescence dorée and healthy pixel misclassification, an operational Flavescence dorée mapping technique using UAV-based imagery can still be proposed.
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•RNAi in the phytoplasma vector Euscelidius variegatus is long-lasting.•Silencing of ATP synthase β transcript reduces expression of the cognate protein.•dsRNA injection generates ...siRNAs mapping only on the fragment used to design dsRNAs.•Silenced insects acquire chrysanthemum yellows and Flavescence dorée phytoplasmas.•Silencing of ATP synthase β reduces the multiplication of both phytoplasmas.
The leafhopper Euscelidius variegatus is a natural vector of the chrysanthemum yellows phytoplasma (CYp) and a laboratory vector of the Flavescence dorée phytoplasma (FDp). Previous studies indicated a crucial role for insect ATP synthase α and β subunits during phytoplasma infection of the vector species. Gene silencing of ATP synthase β was obtained by injection of specific dsRNAs in E. variegatus. Here we present the long-lasting nature of such silencing, its effects on the small RNA profile, the significant reduction of the corresponding protein expression, and the impact on phytoplasma acquisition capability. The specific transcript expression was silenced at least up to 37 days post injection with an average reduction of 100 times in insects injected with dsRNAs targeting ATP synthase β (dsATP) compared with those injected with dsRNAs targeting green fluorescent protein (dsGFP), used as negative controls. Specific silencing of this gene was also confirmed at protein level at 15 days after the injection. Total sRNA reads mapping to dsATP and dsGFP sequences in analysed libraries showed in both cases a peak of 21 nt, a length consistent with the generation of dsRNA-derived siRNAs by RNAi pathway. Reads mapped exclusively to the fragment corresponding to the injected dsATPs, probably indicating the absence of a secondary machinery for siRNA synthesis. Insects injected either with dsATP or dsGFP successfully acquired CYp and FDp during feeding on infected plants. However, the average phytoplasma amount in dsATP insects was significantly lower than that measured in dsGFP specimens, indicating a probable reduction of the pathogen multiplication when ATP synthase β was silenced. The role of the insect ATP synthase β during phytoplasma infection process is discussed.
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