Flavescence Doree Phytoplasma Research Articles

Flavescence Dorée Strain-Specific Impact on Phenolic Metabolism Dynamics in Grapevine (Vitis vinifera) throughout the Development of Phytoplasma Infection.

Flavescence dorée phytoplasma (FDp) is a phytopathogenic bacterium associated with Grapevine yellowS disease, which causes heavy damage to viticultural production. Epidemiological data revealed that some FDp strains appear to be more widespread and aggressive. However, there is no data on mechanisms underlying the variable pathogenicity among strains. In this research, we employed chromatographic and spectrophotometric techniques to assess how two strains of FDp influence the levels of grapevine phenolic compounds, which are frequently utilized as indicative markers of stress conditions. The results pointed to the upregulation of all branches of phenolic metabolism through the development of infection, correlating with the increase in antioxidative capacity. The more aggressive strain M54 induced stronger downregulation of phenolics' accumulation at the beginning and higher upregulation by the end of the season than the less aggressive M38 strain. These findings reveal potential targets of FDp effectors and provide the first functional demonstration of variable pathogenicity between FDp strains, suggesting the need for future comparative genomic analyses of FDp strains as an important factor in exploring the management possibilities of FDp.

Investigation on Flavescence Dorée in North-Western Italy Identifies Map-M54 (16SrV-D/Map-FD2) as the Only Phytoplasma Genotype in Vitis vinifera L. and Reveals the Presence of New Putative Reservoir Plants.

Flavescence dorée (FD) is the most important phytoplasma-associated disease of the grapevine yellows complex in Europe. Recent studies highlighted a great genetic diversity within FD phytoplasma (FDp) strains and demonstrated that their diffusion is not related exclusively to the pathosystem including Vitis vinifera L. and Scaphoideus titanus but involves additional vectors and reservoir plants. This study aimed to investigate FD epidemiology in north-western Italy, with a particular focus on FDp hosts. During field surveys, leaf samples were collected from symptomatic grapevines and other symptomless plant species, and insects were collected within and around vineyards. Phytoplasmas belonging to the ribosomal group 16SrV were detected and typed using nested-PCR-based amplification and nucleotide sequence analyses of the map gene. All symptomatic grapevines were found to be infected by the FDp genotype M54, prevalent in S. titanus and also identified in other known and newly reported hosts. Interestingly, other FDp strains (M38, M50, M51, M121) and FDp-related strains (M39, M43, M48), never detected in grapevines, were largely identified in several known and newly reported host plants and insects including S. titanus. Such evidence confirmed the complexity of FD ecology, expanding the knowledge on the range of FDp host plants putatively involved in the disease spread.

A successful defence strategy in grapevine cultivar ‘Tocai friulano’ provides compartmentation of grapevine Flavescence dorée phytoplasma

BackgroundFlavescence dorée (FD) is a grapevine disease caused by phytoplasma and it is one of the most destructive pathologies in Europe. Nowadays, the only strategies used to control the epidemics are insecticides against vector, but more sustainable techniques are required. Completely resistant Vitis vinifera varieties have not been uncovered yet, but differences in susceptibility among cultivars and spontaneous recovery from FD symptoms have been observed. The grapevine cultivar ‘Tocai friulano’ shows very low susceptibility to FD but its defence strategy to counteract the phytoplasma spread has not been deciphered yet. In this work, the mechanisms occurring within ‘Tocai friulano’ FD-infected plants were examined in depth to identify the phytoplasma distribution and the defence pathways involved.ResultsIn ‘Tocai friulano’ symptoms of FD-infection remained confined near the area where they appeared during all the vegetative season. Analyses of secondary phloem showed a total absence of FD phytoplasma (FDp) in the trunk and its disappearance in 2-year-old arms from July to November, which was different from ‘Pinot gris’, a highly susceptible variety.Diverse modulations of defence genes and accumulation of metabolites were revealed in 1-year-old canes of ‘Tocai friulano’ FD-infected plants, depending on the sanitary status. Symptomatic portions showed high activation of both jasmonate- and salicylate-mediated responses, together with a great accumulation of resveratrol. Whereas activation of jasmonate-mediated response and high content of ε-viniferin were identified in asymptomatic 1-year-old cane portions close to the symptomatic ones.ConclusionSuccessful defence mechanisms activated near the symptomatic areas allowed the compartmentation of FD symptoms and phytoplasmas within the infected ‘Tocai friulano’ plants. These results could suggest specific agronomical practices to be adopted during FD management of this variety, and drive research of resistance genes against FD.

The grapevine metabolite profile of phloem sap is modified by flavescence dorée

Flavescence dorée (FD) has been recorded in French vineyards since the mid-1950s; it has rapidly become a major threat to vineyard survival in different European grape-growing areas. Infection by the phytoplasma ‘Candidatus Phytoplasma vitis’ causes leaf chlorosis and disturbs primary and secondary core metabolic pathways; it can also occlude sieve plates, therefore affecting normal solute transport through the phloem. Thus, in the present study we hypothesised that the metabolomic fingerprint of the phloem sap changes in FDp-infected vines. The metabolome of the phloem sap collected by centrifugation from the bark of the stem of healthy and infected vines (cv. ‘Loureiro’) was profiled by ESI-HRMS at two snapshot time points: E-L 33 (grape berry still hard and green) and E-L 38 (grape berry at maturity). An untargeted analysis of the phloem sap enabled the identification of 476 metabolites, whereas the targeted analysis focused on eight main classes, namely acids, sugar and polyols, amino acids, phenolic acids, hormones, vitamins and N compounds, as well as on several minor classes like fatty acids, phenols or diols. Depending on the developmental stage of the vine, major differences were observed in the composition of cv. ‘Loureiro’ phloem sap in response to FD infection in terms of sugar, organic acid and amino acid content; furthermore, compounds involved in plant defense, such as salicylic acid, ABA, vitamin C and gallic acid also significantly changed. Overall, these results have contributed towards increasing knowledge about the complex interactions between vine and FD phytoplasma.

Flavescence dorée phytoplasma enters insect cells by a clathrin-mediated endocytosis allowing infection of its insect vector

To perform its propagative and circulative cycle into its insect vector, the flavescence dorée phytoplasma invades different cell types. Clathrin-mediated endocytosis is used by a wide range of bacteria to infect eukaryote cells. Among the insect proteins interacting with the phytoplasma adhesin VmpA, we identified the adaptor protein complex AP-1 and AP-2 suggesting that phytoplasmas could enter the insect cells via clathrin-mediated endocytosis. By infection assays of insect cells in culture, we showed that phytoplasmas entry into Drosophila S2 cells was more efficient than infection of the Euva cell line developed from the insect vector Euscelidius variegatus. Chlorpromazine, cytochalasin D and knockdown of clathrin heavy chain (chc) gene expression using RNA interference inhibited entry of phytoplasmas into S2 cells. During invasion of S2 cells, phytoplasmas were observed very closed to recombinant GFP-labelled clathrin light chain. To verify the role of clathrin in the insect colonization by phytoplasmas, RNAi was performed via artificial feeding of chc dsRNA by the vector E. variegatus. This decreased the expression of chc gene in the midgut and heads of E. variegatus. The chc lower expression correlated to a decreased of midgut and salivary gland cells colonization after the insects had ingested phytoplasmas from infected plants. In conclusion, results indicate that clathrin is important for the FD phytoplasma to enter insect cells and colonize its insect vector.

Transcriptional profile of selected flavescence dorée phytoplasma genes during infection of insect vectors and host plants

Flavescence dorée is an economically important vector-borne disease of grapevine in Europe caused by phytoplasmas belonging to the 16SrV ribosomal group. Expression profiles of 11 genes of the Flavescence dorée phytoplasma (FDp) were analysed over time following infection of natural (Vitis vinifera and the leafhopper vector Scaphoideus titanus) and experimental (Vicia faba and Euscelidius variegatus) hosts. Infected and symptomatic grapevine plants (Chardonnay) were sampled under natural field conditions in a productive vineyard in north-western Italy. Broad bean samples were assayed after the experimental inoculation with infectious E. variegatus. Adults of both vector species were analyzed following FDp acquisition from infected broad bean plants. The selected FDp genes were grouped according to their putative functions within different categories, namely ‘Membrane proteins’ (imp), ‘Regulative elements’ (spoVG, rpoD), ‘Protein metabolism, transport and secretion’ (tldD, ysdC, ftsY), ‘Stress response’ (comp83, osmC), ‘ABC transporters’ (CoABC) and ‘Unknown’ function (contig12, comp115). All analyzed genes were expressed in the four different host species suggesting their crucial role during the FDp infection cycle. Moreover, some of them (contig12, CoABC, comp83, and imp) might be considered essential for phytoplasma survival irrespective of the host, while comp115 seems to be required for insect infection. We showed that FDp is metabolically more active in insects than in plants, at least according to the pool of selected genes for this study, indicating that FDp behaves differently in the two hosts compared to other phytoplasma species/strains.

Multilocus sequence typing of phytoplasmas associated with Flavescence dorée disease in Tuscany vineyards identifies a highly homogeneous lineage in the subgroup 16SrV–C

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.

Scaphoideus titanus fitness on grapevine varieties with different susceptibility to Flavescence dorée phytoplasma

AbstractScaphoideus titanus (Ball) is a Nearctic leafhopper (Cicadellidae: Deltocephalinae), monophagous on Vitis spp., naturalized in European vineyards since the 20th century. Following its introduction and establishment in Europe, S. titanus became the main vector of Flavescence dorée phytoplasma (FDp) to grapevine. FDp causes heavy economic losses to viticulture in Europe. The control of the disease mainly relies on insecticide applications against the vector and on the removal of infected plants. The exploitation of plant resistance or tolerance against pathogens and pests can represent a valuable tool for a more sustainable viticulture. The first step in identifying resistance traits in grapevine varieties towards S. titanus is the evaluation of insect fitness parameters on different grapevine genotypes. Therefore, in this work, nymph mortality and developmental time, adult survival and prolificacy of S. titanus have been studied on three grapevine varieties, characterized by different susceptibility to FD. Scaphoideus titanus showed highest fitness when reared on Barbera, whereas the worst performances were recorded on Moscato, with a significant reduction in nymph and adult survivals, a slowed nymph development, and a decreased number of available eggs. On Brachetto, S. titanus showed an intermediate level of fitness parameters. Consistently with previous studies on feeding behaviour, Barbera is the most suitable host for the FD‐vector. The high suitability of Barbera for S. titanus may partly explain the high susceptibility to FD of this variety. On the contrary, the low suitability of Moscato, poorly susceptible to FD, may be due to antibiosis and antixenosis defence mechanisms that act against S. titanus.

In-Field LAMP Detection of Flavescence Dorée Phytoplasma in Crude Extracts of the Scaphoideus titanus Vector

One of the most destructive diseases affecting grapevine in Europe is caused by Flavescence Dorée phytoplasma (FDp), which belongs to the 16Sr-V group and is a European Union quarantine pathogen. Although many molecular techniques such as loop-mediated isothermal amplification (LAMP) are widely used for the rapid detection of FDp in infected grapevine plants, there is no developed isothermal amplification assay for FDp detection in the insect vectors that are fundamental for the spread of the disease. For this reason, a simple in-field real-time LAMP protocol was optimized and developed for the specific detection of FDp in the insect vector Scaphoideus titanus. The LAMP assay was optimized to work with crude insect extracts obtained by manually shaking a single insect in a buffer for 5 min. Such a simple, sensitive, specific, economic, and user-friendly LAMP assay allowed the detection of FDp in S. titanus in less than half an hour, directly in the field. The developed insect tissue preparation procedure, combined with the LAMP protocol, promptly revealed the presence of FDp in infected S. titanus directly in the vineyards, allowing for monitoring of the spread of the pathogen in the field and to apply timely strategies required for the mandatory control of this pathogen.

Impact of the "Flavescence Dorée" Phytoplasma on Xylem Growth and Anatomical Characteristics in Trunks of 'Chardonnay' Grapevines (Vitis vinifera).

Simple SummaryAnnual rings of the susceptible grapevine cultivar ‘Chardonnay’ were measured and used in order to analyse the impact of the Flavescence dorée (FD) infection on the growth in diameter and the anatomical structure of grapevine trunks. Grapevines are susceptible to water shortage and reduce their growth in diameter in the case of summer drought. However, in the case of the expression of FD symptoms, the ring width reductions are extreme and supersede the drought-induced effects. In addition, in coincidence of the FD symptomatic expression, the anatomy of the phloem tissue of infected grapevines appears heavily disarranged. Moreover, sometimes also the formation of the woody ring is incomplete (early wood only). In conclusion, even though the FD phytoplasma does not inhabit and replicate inside the xylem tissue, our results confirm existing indirect inhibiting effects on the ring growth and the xylem tissue formation in FDp-infected grapevines.Flavescence dorée (FD) is a grapevine disease caused by ‘Candidatus Phytoplasma vitis’ (FDp), which is epidemically transmitted by the Nearctic leafhopper Scaphoideus titanus. In this study, we applied dendrochronological techniques to analyse the response to FDp infections in terms of wood ring widths and anatomical structures of the xylem and phloem tissues of the trunk of the susceptible grapevine cultivar ‘Chardonnay.’ As a rule, grapevines are susceptible to water shortage and reduce their growth in diameter in case of summer drought. In the season of the external expression of FD symptoms, however, the ring width reductions are extreme and supersede any drought-induced effects. In addition, the anatomy of the phloem tissue in the year of the FD symptom expression appears heavily disarranged. Moreover, in the most suffering individuals, the xylem formation remains incomplete and mostly limited to the early wood tissue. In conclusion, even though the FD phytoplasma does not inhabit and replicate inside the xylem tissue, our results confirm existing indirect inhibiting effects on the ring growth and the xylem tissue formation in FDp-infected grapevines.

Genetic Diversity of Flavescence Dorée Phytoplasmas in Vineyards of Serbia: From the Widespread Occurrence of Autochthonous Map-M51 to the Emergence of Endemic Map-FD2 (Vectotype II) and New Map-FD3 (Vectotype III) Epidemic Genotypes

Flavescence dorée (FD) is a European quarantine disease of grapevine caused by FD phytoplasma (FDp) transmitted by the leafhopper of North American origin Scaphoideus titanus. The disease affects the most important viticultural regions of Europe and all wine-growing regions of Serbia. Unlike the insect vector, the pathogen is native to Europe and associated with several wild host plants among which alder trees as the main source of two out of three map genetic clusters of pathogen variants (Map-FD1 and FD2). Heretofore, the FDp epidemic in Serbian vineyards was thought to be monotypic, i.e., caused by the single genotype of the Map-FD3 cluster, M51, and correlated with clematis as the natural source plant. This study aimed to provide data on genetic diversity, through map and vmpA gene typing, and insights into ecological properties of epidemiological cycles driving the epidemic outbreaks of FD in Serbia today. Map genotyping of 270 grapevine isolates collected from 2017 to 2019 confirmed M51 as autochthonous genotype widespread in all wine producing regions of Serbia and the dominant FDp epidemic genotype in most of the districts (75%, 202/270 isolates), except in north Serbia where multiple outbreaks of M12 Map-FD3 were recorded (54 isolates). Tree of heaven is reported as a new FDp plant reservoir for the Serbian vineyards, hosting the M51 genotype, along with clematis. An outbreak of a new endemic Map-FD3 genotype M144 was documented in grapevine samples from east Serbia (5 isolates), correlating with previous findings of the same genotype in clematis. In addition, single grapevine infections with five new Map-FD3 genotypes (M150-M154) were recorded in central Serbia, thus indicating high endemic potential for new outbreaks. The vmpA typing placed all Map-FD3 isolates into the VmpA-III cluster, i.e., Vectotype III. Finally, we found direct evidence that at least two FDp endemic genotypes, M89 and M148, of the Map-FD2/VmpA-II have escaped from alders and propagated in the grapevine-S. titanus pathosystem in Serbia (Vectotype II). Our findings confirm the high complexity of the FDp ecological cycle and provide evidence of a unique, autochthonous Balkan epidemiology sourced endemically.

New insights on Scaphoideus titanus biology and their implications for integrated pest management

The Nearctic leafhopper Scaphoideus titanus is known to be the main vector of Flavescence doree phytoplasma (FDP), a serious disease of the grapevine in temperate European areas. Adult lifespan and oviposition rate were investigated under semi-natural conditions to assess potential fecundity of this species. Egg load was detected under field conditions in North Italy. Females lived longer than males, with a sex ratio in their favour during the late season when they are still able to lay eggs. Both sexes have a high average lifespan, females can live over 60 days whilst males over 40 days. Furthermore, 25% of the females lived over 70 days, and laid an average of 60–65 eggs. A number of approximately 70 to 130 eggs was laid by 25% females. The comparison between the egg load trend and the oviposition rate shows that both indices remain constant until September and then decrease in the following months. The presence of the vector was detected until late season in the vineyards. In fact, some females with mature eggs were collected until October 30 th , whilst in seminatural condition few individuals were able to lay eggs until the end October to early November. This research shows as S. titanus biological parameters have been largely underestimated so far. A serious implication of the high longevity is the prolongation of the adult inoculation period, and a temporal expansion of the risk of infecting the vineyards no longer protected by insecticide treatments. This suggests to reconsider the phytosanitary management of the vineyards after harvesting.

Phloem and xylem modifications of Vitis vinifera stems in response to flavescence dorée phytoplasma infection

AbstractAnatomical modifications of xylem and phloem tissues of grapevine (Vitis vinifera) stems of shoots infected by the flavescence dorée phytoplasma (FDp) were first observed and described in the 1960s, but never quantified in detail. In this paper, we describe and quantify the impact of FDp on grapevine stem tissues, and relate it to the level of expression of symptoms and to cultivar‐specific FDp susceptibility. For this purpose, we measured and quantified the anatomical parameters of xylem and phloem tissues of a tolerant (Merlot) and a susceptible (Chardonnay) cultivar. For each cultivar, thin sections of eight shoots with symptoms from FDp‐infected grapevines, eight symptomless shoots from the same FDp‐infected grapevines, and eight symptomless shoots from symptomless grapevines (control) were compared. Results showed general inhibition of xylem growth and proliferation of phloem tissues (hyperplasia) with lack or irregular arrangement of the fibre‐sclereids in the axial phloem of the stems from shoots with symptoms, irrespective of the cultivar. Xylem vessels of infected Merlot shoots were partly occluded by tyloses and a higher number of smaller vessels were produced than in control plants. Thus, the anatomical responses confirmed the detrimental effect of FDp on stems of infected grapevine shoots, including impaired stem development and lack of periderm formation. Statistically significant differences were found between the two cultivars with different levels of susceptibility to FDp infection.

Silencing of ATP synthase β reduces phytoplasma multiplication in a leafhopper vector

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.

Ecology‐based analysis of a recent association between Spartium junceum and 16SrV phytoplasma

AbstractAn outbreak of Spartium witches’ broom (SpaWB) in Sicily prompted us to identify and characterize associated phytoplasmas. Over 80 samples of Spanish broom (Spartium junceum) and around 270 individuals of the potential vector Livilla spectabilis were collected and analysed. Single and mixed infections of 16SrV and ‘Candidatus Phytoplasma spartii’ were detected in Spanish broom samples and for the first time in L. spectabilis. The 16SrV isolates were further characterized by multilocus sequence typing (MLST) to determine their phylogenetic relationship with flavescence dorée phytoplasma (FDp) and to evaluate the risk of host‐jumping to grapevine. Phylogenetic analysis of most of the analysed genes using the MLST approach grouped S. junceum 16SrV‐C isolates with FDp isolates infecting grapevine and Scaphoideus titanus. Notably, phylogenetic analysis of the vmpA gene clustered the S. junceum isolates with FDp genotypes transmitted by S. titanus. This study extends the knowledge of SpaWB epidemiology, focusing on the possible risk of a 16SrV host jump from Spanish broom to grapevine. Spanish broom was identified as a reservoir and potential inoculum source of phytoplasmas that cause severe disease in cultivated crops. Furthermore, the L. spectabilis psyllid may be involved in the epidemiology of this 16SrV‐C phytoplasma, although in the absence of in vivo transmission trials. The study further confirms the strong ability of phytoplasmas to adapt to new hosts and vectors, thus leading to potential phytosanitary emergencies.

Ability of Euscelidius variegatus to Transmit Flavescence Dorée Phytoplasma with a Short Latency Period.

Simple SummaryPhytoplasmas are a group of phloem-restricted phytopathogens that attack a huge number of wild and cultivated plants, causing heavy economic losses. They are transmitted by phloem-feeding insects of the order Hemiptera; the transmission process requires the vector to orally acquire the phytoplasma by feeding on an infected plant, becoming infective once it reaches the salivary glands after quite a long latency period. Since infection is retained for all of the insect’s life, acquisition at the nymphal stage is considered to be most effective because of the long time needed before pathogen inoculation. This work provides evidence for the reduced latency period needed by adults of the phytoplasma vector Euscelidius variegatus from flavescence dorée phytoplasma acquisition to transmission. Indeed, we demonstrate that adults can become infective as soon as 9 days from the beginning of phytoplasma acquisition. Our results support a reconsideration of the role of adults in phytoplasma epidemiology, by indicating their extended potential ability to complete the full transmission process.Phytoplasma transmission takes place by insect vectors through an Acquisition Access Period (AAP), Latency Period (LP) and Inoculation Access Period (IAP). Generally, phytoplasmas are believed to be transmitted more efficiently by nymphs because they need a long LP to reach the salivary glands before becoming infective. The transmission can start from adults as well, but in this case a long LP may exceed the insect’s lifespan. However, previous evidence has indicated that adults can undergo a shorter LP, even though little knowledge is available regarding the phytoplasma temporal dynamics during this period. Here, we investigate the minimum time required by the phytoplasma to colonize the vector midgut and salivary glands, and finally to be inoculated into a plant. We used the leafhopper Euscelidius variegatus to investigate the life cycle of flavescence dorée phytoplasma (FDP). Phytoplasma-free E. variegatus adults were left on broad beans (BBs) infected with FDP for an AAP of 7 days. Subsequently, they were individually transferred onto a healthy BB for seven different IAPs, each one lasting 24 h from day 8 to 14. Molecular analyses and fluorescence in situ hybridization were performed for FDP detection. FDP was found in the leafhopper midgut from IAP 1 with an infection rate reaching 50%, whereas in the salivary glands it was found from IAP 2 with an infection rate reaching 30%. FDP was also detected in BBs from IAP 4, with infection rates reaching 10%. Our results represent an important step to further deepen the knowledge of phytoplasma transmission and its epidemiology.

Dynamics of Scaphoideus titanus population in southern South Tyrol (Italy) and detection of Grapevine Flavescence Dorée phytoplasma in the insect with a multiplex real-time PCR

In South Tyrol the population of Scaphoideus titanus, the vector of Grapevine Flavescence doree phytoplasma (FD), has been steadily increasing since 2010. The present work provides an overview of the monitoring activity of Scaphoideus titanus in South Tyrolean vineyards coupled with the description of a sensitive and reliable detection method of FD in the insect vector. We have developed an endogenous control of Scaphoideus titanus to be used in a multiplex real-time PCR, amplifying the map gene of FD phytoplasma. We present evidence on the performance of this new vector reference and its compatibility with FD detection.

When a Palearctic bacterium meets a Nearctic insect vector: Genetic and ecological insights into the emergence of the grapevine Flavescence dorée epidemics in Europe.

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.

New Viral Sequences Identified in the Flavescence Dorée Phytoplasma Vector Scaphoideus titanus.

(1) Background: The leafhopper Scaphoideus titanus is the primary vector of Flavescence dorée phytoplasma (FDp) in European vineyards. Flavescence dorée is one of the most severely damaging diseases of Vitis vinifera and, consequently, a major threat to grape and wine production in several European countries. Control measures are compulsory, but they mainly involve large-scale insecticide treatments, with detrimental impacts on the environment. One possible solution is to exploit the largely unexplored genetic diversity of viruses infecting S. titanus as highly specific and environmentally benign tools for biological control. (2) Methods: A metatranscriptomic approach was adopted to identify viruses that may infect individuals caught in the wild in both its native (United States) and invasive (Europe) areas. Reverse transcription PCR was used to confirm their presence in RNA pools and explore their prevalence. (3) Results: We described nine new RNA viruses, including members of “Picorna-Calici”, “Permutotetra”, “Bunya-Arena”, “Reo”, “Partiti-Picobirna”, “Luteo-Sobemo” and “Toti-Chryso” clades. A marked difference in the diversity and abundance of the viral species was observed between the USA population and the European ones. (4) Conclusions: This work represents the first survey to assess the viral community of a phytoplasma insect vector. The possibility to exploit these naturally occurring viruses as specific and targeted biocontrol agents of S. titanus could be the answer to increasing demand for a more sustainable viticulture.

Flavescence Dorée Phytoplasma Has Multiple ftsH Genes that Are Differentially Expressed in Plants and Insects.

Flavescence dorée (FD) is a severe epidemic disease of grapevines caused by FD phytoplasma (FDP) transmitted by the leafhopper vector Scaphoideus titanus. The recent sequencing of the 647-kbp FDP genome highlighted an unusual number of genes encoding ATP-dependent zinc proteases FtsH, which have been linked to variations in the virulence of “Candidatus Phytoplasma mali” strains. The aims of the present study were to predict the FtsH repertoire of FDP, to predict the functional domains and topologies of the encoded proteins in the phytoplasma membrane and to measure the expression profiles in different hosts. Eight complete ftsH genes have been identified in the FDP genome. In addition to ftsH6, which appeared to be the original bacterial ortholog, the other seven gene copies were clustered on a common distinct phylogenetic branch, suggesting intra-genome duplication of ftsH. The expression of these proteins, quantified in plants and insect vectors in natural and experimental pathosystems, appeared to be modulated in a host-dependent manner. Two of the eight FtsH C-tails were predicted by Phobius software to be extracellular and, therefore, in direct contact with the host cellular content. As phytoplasmas cannot synthesize amino acids, our data raised questions regarding the involvement of FtsH in the adaptation to hosts via potentially enhanced recycling of phytoplasma cellular proteins and host protein degradation.