Bois noir, a disease of the grapevine yellows complex, is associated with ‘Candidatus Phytoplasma solani’ and transmitted to grapevines in open fields by the cixiids Hyalesthes obsoletus and Reptalus panzeri. In vine-growing areas where the population density of these vectors is low within the vineyard, the occurrence of bois noir implies the existence of alternative vectors. The aim of this study was to identify alternative vectors through screening of the Auchenorrhyncha community, phytoplasma typing by stamp gene sequence analyses, and transmission trials. During field activities, conducted in Northern Italy in a vineyard where the bois noir incidence was extremely high, nine potential alternative insect vectors were identified according to high abundance in the vineyard agro-ecosystem, high infection rate, and harbouring phytoplasma strains characterized by stamp gene sequence variants found also in symptomatic grapevines. Transmission trials coupled with molecular analyses showed that at least eight species (Aphrodes makarovi, Dicranotropis hamata, Dictyophara europaea, Euscelis incisus, Euscelidius variegatus, Laodelphax striatella, Philaenus spumarius, and Psammotettix alienus/confinis) are alternative vectors of ‘Candidatus Phytoplasma solani’ to grapevines. These novel findings highlight that bois noir epidemiology in vineyard agro-ecosystems is more complex than previously known, opening up new perspectives in the disease management.

The vintage effect overcomes the terroir effect: a three year survey on the wine yeast biodiversity in Franciacorta and Oltrepò Pavese, two northern Italian vine-growing areas analyses, the year of isolation (vintage) proved to be a factor that significantly affected the biodiversity of the yeast species, whereas the geographical site (terroir) was not. Seventy-five per cent of S. cerevisiae isolates gathered in a unique cluster at a similarity level of 82 %, while the remaining 25 % were separated into minor groups without any evident relationship between d-PCR profile and territory, year or source of isolation. However, in six cases a similar strain appeared at the harvesting time both in Franciacorta and Oltrepò Pavese areas, whereas surprisingly no strain was reisolated in the same vineyard or cellar for consecutive years. INTRODUCTIONIn winemaking, yeasts are essential for the transformation of grape sugars into ethanol and carbon dioxide through alcoholic fermentation; nonetheless, due to their specific enzymic activities and cell autolysis, they can also generate typical sensorial characteristics in wine, like secondary flavours and smoothness (Romano et al., 2003a). Although selected Saccharomyces strains are usually added by oenologists as starter cultures to control the fermentative process, several micro-organisms enter the must from the vineyard environment, winery facilities and cellar equipment, and these can affect the quality of the end product. Nowadays, for a certain style of wines, the use of the 'so called' autochthonous yeasts is considered essential in providing for the valorization and preservation of the environmental microbial biodiversity (Pretorius, 2000). In 3These authors contributed equally to this paper.Abbreviations: ADY, active dry yeast; CE, capillary electrophoresis; ITS, internal transcribed spacer; LSD, least significant difference; PCA, principal component analysis; UPGMA, unweighted pair group method with arithmetic means.

The in-bottle fermentation of sparkling wines is currently triggered by few commercialized Saccharomyces cerevisiae strains. This lack of diversity in tirage yeast cultures leads to a prevalent uniformity in sensory profiles of the end products. The aim of this study has been to exploit the natural multiplicity of yeast populations in order to introduce variability in sparkling wines throughout the re-fermentation step. A collection of 133 S. cerevisiae strains were screened on the basis of technological criteria (fermenting power and vigor, SO2 tolerance, alcohol tolerance, flocculence) and qualitative features (acetic acid, glycerol and H2S productions). These activities allowed the selection of yeasts capable of dominating the in-bottle fermentation in actual cellar conditions: in particular, the performances of FX and FY strains (isolated in Franciacorta area), and OX and OY strains (isolated in Oltrepò Pavese area), were compared to those of habitually used starter cultures (IOC18-2007, EC1118, Lalvin DV10), by involving nine wineries belonging to the two Consortia of Appellation of Origin. The microbiological analyses of samples have revealed that the indigenous strains showed an increased latency period and a higher cultivability along the aging time than the commercial starter cultures do. Results of chemical analyses and sensory evaluation of the samples after 18 months sur lies have shown that significant differences (p < 0.05) were present among the strains for alcoholic strength, carbon dioxide overpressure and pleasantness, whereas they were not observed for residual sugars content, titratable acidity or volatile acidity. Indigenous S. cerevisiae exhibited comparable values respect to the commercial starter cultures. The ANOVA has also proven that the base wine formulation is a key factor, by significantly affecting (p < 0.01) some oenological parameters of wine, like alcoholic strength, volatile acidity, carbon dioxide overpressure, titratable acidity and dry extract. The use of native yeast strains for the re-fermentation step can be considered a convenient way for introducing differentiation to the final product without modifying the traditional technology. In a perspective of “precision enology,” where the wine is designed on specific vine cultivars and microorganisms, this work underlines that exploring yeast biodiversity is a strategic activity to improve the production.

Bois noir (BN) grapevine disease is associated with ‘Candidatus Phytoplasma solani’ (CaPsol), a pathogen with a complex ecology including multiple insect vectors and plant hosts. A key point to improve the effectiveness of BN control strategies consists in determining the epidemiological role of ground‐cover weeds. The present study employed a multidisciplinary approach, based on the application of spatial (spatial analysis by distance indices) and molecular (stamp gene typing) analyses, to identify weeds with a potential role in BN epidemiology in northern Italy. Generated data showed that in addition to Convolvulus arvensis, one of the main CaPsol inoculum sources, Chenopodium album, Polygonum aviculare and Trifolium repens were found associated with BN epidemiology. CaPsol molecular typing highlighted that the strains prevalent in symptomatic grapevines were characterised by stamp sequence variants St19, St11 (nettle‐related) and St5 (bindweed‐related). The latter was prevalent also in Hyalesthes obsoletus and weeds, suggesting their main association with bindweed‐related epidemiology. On the other hand, nettle‐related CaPsol strains were occasionally found in H. obsoletus and weeds. Considering that H. obsoletus‐mediated transmission of CaPsol occurs mainly with young instars, further investigations will confirm if, in addition to bindweed and nettle, weeds associated with BN epidemiology in Franciacorta can represent larval developmental hosts and, consequently, act as CaPsol reservoirs for transmission to grapevine. Moreover, other studies are needed to clarify the relationship between such weeds and CaPsol alternative vectors.

The evolution of the yeast populations was investigated during controlled and spontaneous fermentations of Chardonnay must in two Franciacorta wineries (A and B) that used the same starter culture. Two hundred and three isolates were collected and identified as Saccharomyces cerevisiae (97.5 %), Pichia membranifaciens (2.0 %) and Hanseniaspora vinae (0.5 %) through the analyses of ITS rDNA region by RFLP, D1/D2 of 26S rDNA partial sequence and scHO gene. A high intraspecific diversity of S. cerevisiae isolates was detected by means of the inter-delta sequence PCR analysis: 117 profiles corresponding to different strains were distinguished (at level of similarity <90.5 %) and monitored to follow the dynamics of cell populations. In winery A, the commercial strain maintained the predominance since its δ-PCR profile constituted most of the colonies recovered at different times of sampling (from 44 to 100 % of plate counts), in this case only 18 different genotypes out of 74 isolates were recognized. In winery B, where spontaneous fermentations were performed in the same environment, the starter culture never took control and a succession of indigenous populations overcame without one prevailed on the others; actually, 40 genotypes out of 53 isolates can be identified. The highest level of biodiversity was observed in spontaneous fermentation (winery B) where 59 genotypes out of 71 S. cerevisiae isolates were discriminated; a continuous change in cell populations was noticed with the simultaneous presence from 6 to 10 different genotypes. The management of the starter culture and the environmental hygiene was shown to be fundamental to control the inoculated fermentations.