Quantitative estimates of vector populations and their infectivity in the wild and in cultivated compartments of agroecosystems have been carried out to elucidate the role of the wild compartment in the epidemiology of Flavescence dorée (FD). Seven sites were selected for the investigations in the Piedmont Region of Italy. They were characterized by a high variety of agricultural and ecological landscape features, and included a vineyard surrounded by wild vegetation. In order to describe abundance and prevalence of FD-infected vectors in the cultivated and wild compartments of the vineyard agroecosystem, adults of Scaphoideus titanus were collected by yellow sticky traps inside and outside the vineyard over the period July 10th-September 9th, 2015. They were counted and singly analyzed for the presence of FD phytoplasmas by PCR. Multifactorial correlations among vector population level, prevalence of infected insects inside and outside the vineyards, disease prevalence in cultivated and wild Vitis plants, and location of wild Vitis plants with respect to the vineyard were analyzed. Abundance of S. titanus adults significantly decreased from the end of July onwards, particularly inside the vineyard (average range 22.7 2.5 insects/trap). Percentage of FD-positive S. titanus was significantly higher outside the vineyard (up to 48% on average) compared to inside the vineyard (up to 34% on average), and increased during the season in both compartments.

The aim of this work was to evaluate the susceptibility to flavescence dorée (FD) of 12 Vitis vinifera cultivars grown in Piedmont, and representative of the wine-making tradition of this area. The experiments were conducted under controlled conditions to ensure constant infection pressure. Test plants were ex vitro potted vines, singly inoculated with four Scaphoideus titanus infected by FD-C phytoplasma (FDp), under greenhouse conditions. Vines were tested for FDp at 5 and 8 weeks postinoculation (wpi) and the phytoplasma load was measured in leaves and roots at 8 wpi. Within the 14 V. vinifera accessions (belonging to 12 cultivars), three susceptibility clusters were identified. Cultivars within the low susceptibility group showed low phytoplasma loads and low percentages of infected plants, suggesting a tolerant behaviour. To confirm these results, four Vitis cultivars, representing extremes of FD susceptibility from low to high, were grafted onto Kober 5BB rootstocks and inoculated with laboratory-infected S. titanus, under semi-field conditions. The transmission experiments onto grafted cuttings confirmed that susceptibility to the disease depends on the scion genotype. The data indicated that none of the tested V. vinifera genotypes are resistant to FD, although some cultivars with low susceptibility are available, and can be explored for identifying genetic traits involved in disease tolerance/resistance. Moreover, ranking Vitis genotypes for their susceptibility to FD is in itself a valuable tool to support vine growers in their decision management, by helping them to choose the most appropriate varieties according to their specific FD epidemiological contexts.

Flavescence dorée (FD) is a quarantine grapevine disease caused by a phytoplasma transmitted by the leafhopper Scaphoideus titanus Ball. FD management relies on compulsory insecticide treatments, roguing of infected plants, and substitution with certified material. Some grapevine cultivars show a spontaneous remission of symptoms (recovery). To determine if recovery is a suitable strategy to co-exist with disease in areas of strong infestation, the qualitative aspects of grapes, musts, and wines obtained from recovered Barbera and Chardonnay grapevines were investigated in two productive vineyards. Following field observations, about 1500 plants in each vineyard were divided into healthy (asymptomatic and negative in phytoplasma molecular diagnosis) and recovered (asymptomatic the year of observation but infected the year before). Maturation curves and microvinification tests followed by oenological and sensory analyses showed that maturation trends of recovered grapes were in line with those from healthy plants and the final qualities of wines were comparable. The spread of FD has strongly increased in Piedmont (Italy) in recent decades. Management strategies to cope with the disease are necessary to preserve traditional wine production. Despite the yield from recovered grapevines is quantitatively lower than that from healthy ones, we showed here that the wine quality is, however, preserved.

Artificial diets represent an essential tool for investigations on the intimate relationship between plant pathogens and their vectors. Previous research failed in devising an artificial diet delivery system for the meadow spittlebug Philaenus spumarius, to date considered the most important vector of the bacterium Xylella fastidiosa in Europe. Here, we describe a new delivery "tube system" by which we succeeded in artificial feeding of P. spumarius with holidic diets (one sucrose diet and two amino acids diets). Spittlebug probing and feeding behaviour on either the tube system or a traditional "flat system" realized out of a small Petri dish filled with diet and covered with stretched Parafilm (R) were observed in real time by video-EPG (Electrical Penetration Graph), in order to assess the occurrence of ingestion and excretion. Moreover, we evaluated P. spumarius survival on either the tube system filled with the two holidic diets that gave the best EPG results or an empty tube system serving as control. Contrary to the flat system, where just brief stylet insertions through the Parafilm (R) were recorded, the spittlebug ingested the artificial diets when delivered with the tube system. Survival on the diets provided with the tube system was significantly greater than the control, with no differences between the diets tested. Furthermore, the tube system was suitable also for another spittlebug species shown to be a competent vector of X. fastidiosa, that is Neophilaenus campestris. The tool we devised opens new perspectives for investigations on X. fastidiosa/spittlebugs interactions, as well as for the functional analysis of mutant X. fastidiosa strains in respect to insect colonization and transmission.

The leafhopper Euscelidius variegatus is a known phytoplasma vector. Previous studies indicated a crucial role for insect ATP synthase during phytoplasma infection process. Gene silencing of ATP synthase b was obtained by injection of specific dsRNAs in E. variegatus. Here we present the significant reduction of corresponding protein expression and preliminary results on significant differences found in phytoplasma quantity in silenced insects compared to the controls, following injection with ATP synthase b and successive phytoplasma acquisition on infected plants. An in vivo role for insect ATP synthase b could be hypothesized during phytoplasma infection process.

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.

Phytoplasmas are phloem-limited plant pathogenic bacteria in the class Mollicutes, family Acheloplasmataceae. Phytoplasma classification is based on 16S rRNA and ribosomal protein (rp) gene sequences and the 'Candidatus' concept is applied for well characterized phytoplasmas. Once acquired by an insect, phytoplasma must cross its midgut membrane, spread in the hemolymph, migrate to the salivary glands and enter the saliva in order to be transmitted to another plant. Previous studies suggested the involvement of different types of membrane proteins of phytoplasmas in promoting phytoplasma internalization in the host cell. In the present study, the involvement of immunodominant membrane protein Imp of Flavescence dorèe type D phytoplasma (FD-Dp) in the interaction with insect proteins was investigated. Six Auchenorrhyncha species known as vectors, potential, and non- vectors of FD-Dp were selected from distantly related species groups. Four species of the infraorder Cicadomorpha, all in the family Cicadellidae. The dnaD-imp-pyrG genomic fragment of FD-D strain was amplified and sequenced from FD-D infected vine leaves. A total of 162 amino acids were predicted for full-length FD-D Imp protein, with residues 44-162 exposed outside the membrane. A His-tagged partial Imp protein and a rabbit polyclonal antibody were synthesized and used for Western and Far-Western dot blots (FWdB) experiments. Total native proteins were extracted from batches of each insect species. FWdB showed interaction of Imp fusion protein with total proteins of St, Ev (known efficient vectors) and Mq (potential vector), while no interaction signal was detected with the other three species, Rs, Zp, and Mp (non-vectors).

Flavescence dorée phytoplasmas (FDp, 16SrV) are plant pathogenic non-cultivable bacteria associated with a severe and epidemic disease of grapevine and are transmitted by the Cicadellidae Deltocephalinae leafhoppers Scaphoideus titanus Ball (natural vector) and Euscelidius variegatus Kirschbaum (vector under laboratory conditions only). By living in two worlds, the plant and insect life style of phytoplasmas may drive differential gene expression, as known from literature, but also select different populations inside the plant and insect hosts. Genetic variation of phytoplasmas associated with FD (based on dnaK, vmpA and malG genes) in insects and plants revealed the presence of a wide range of genotypes infecting/co-infecting plants and vectors. FDp genetic variation is not homogeneous between insects and plants, as some genotypes are more represented in insects and others in plants. This skewed genotypic distribution indicates that insect vector drives phytoplasma populations that infect plants and, on the other way, host-plant drives the phytoplasma populations that can be acquired by and infect insects. The competition of the different genotypes inside plant and insect hosts and the presence of FD-infected plants inside and outside the vineyards, all of them representing source of inoculum for the vector, increase the complexity of this phytoplasma/plant/vector epidemiological system. Moreover, in the field, different plant species can harbour different FD phytoplasma genotypes and are visited by different vector/potential vector species. Some of these complex interrelationships have been studied at the field level as well as under controlled laboratory conditions. Although there is some convergence in the phytoplasma genotype composition infecting insects and plants at each site, a higher genetic variation of FD was consistently found in the insects compared to plants in all sites. Moreover, some genotypes were only found in vector or plant hosts. The ecological and evolutionary significance of this finding is discussed. 

The spittlebug Philaenus spumarius is by far the most abundant xylem-sap feeder insect in the Italian olive agroecosystems and is the proven vector of Xylella fastidiosa CoDIRO strain to olive in Apulia. This highly polyphagous species has one generation per year, nymphs develop in late winter-early spring, and adults are present over an extended period from spring onwards. Four olive orchards of one ha in size were chosen in the Liguria (North-Western Italy) and in Apulia (South-Eastern Italy) regions. Host-plant association of nymphal stages on the herbaceous cover was investigated while, for the adults, the presence on herbaceous and woody hosts was recorded all through the season. Within 30 sample units of 0.25 m2 randomly distributed in each olive orchard, all nymphs were counted and their host-plants identified. Adults were sampled with sweep net on 10-30 random sample units of different vegetation compartments: grass cover, olive trees, and spontaneous shrubs. Nymphs were mainly associated to species of the families Asteraceae, Fabaceae, Umbelliferae. Host-plant shifting and a different location on the plant was recorded between early and late nymphal stages, suggesting that nymphs are rather mobile. Adult population moved from grass cover to woody plants in late spring and summer and moved back to grass cover in late summer-early autumn, thus showing a clear host-shifting during the season. This information is relevant for the development of control strategies of the vector in the X. fastidiosa infected area, based on both insecticide application and agronomic measures, as well as for risk assessment in non-infected areas.

Background. Flavescence dorée of grapevine (FD) is a phytoplasma-associated disease transmitted by the leafhopper Scaphoideus titanus. The disease spreads through primary infections (from gone-wild vines surrounding the vineyard to cultivated grapevines) and secondary infections (from vine to vine within the vineyard). To start looking for disease resistance in grapevine, FDp transmission experiments to the main Piedmontese cultivars were carried out with the vector S. titanus. Susceptibility was estimated by measuring the proportion of infected plants and the phytoplasma load, as estimated by qPCR. PhD research project. The research will address the possibility that susceptibility/tolerance would possibly be related to the vector fitness/feeding behaviour on the different grapevine genotypes and/or to the plant genetic characteristics. For this, Electric Penetration Graph (EPG) studies will be conducted on the most susceptible and tolerant cultivars, and vector fitness will be described by measuring S. titanus adult longevity and female prolificacy on the same cultivars. Moreover, the possibility of manipulating vector-FDp interactions using RNA interference will be investigated. For this purpose, dsRNA complementary to insect genes presumably involved in phytoplasma transmission will be produced and delivered to the leafhoppers by injection and feeding. Gene silencing and its effect on vector transmission ability will be evaluated. The expected results, together with the ones obtained by plant physiologists on the identification of grapevine genetic traits possibly involved in resistance, will greatly enhance the comprehension of the epidemiology of FD and open new control perspectives.