Spatial and Seasonal Structure of Bacterial Communities Within Alpine Vineyards: Trentino as a Case Study.

Bacteria have a fundamental role in determining the fitness of grapevine, the composition of grapes and the features of wines but at present, little information is available. In this work, the bacteria colonizing the different portions of grapevine (bark, leaves and grapes) were explored in the vineyards of the Alpine region of Trentino, considering the impact of different environmental and agronomical variables. The vineyards included in the work were selected based on their different geographical positions (altitude) and grapevine training systems in order to explore the whole variability of the grapevine ecosystem. Moreover, the surface amount of copper was measured on grapes and leaves during the vegetative growth. Bacterial analysis, performed using plate counts and Illumina MiSeq, revealed an increase in the concentration of grape bacteria proportional to the progress of the ripening stage. Conversely, the peak of bacterial concentration onto leaf and bark samples occurred in August, probably due to the more favourable environmental conditions. In bark samples, the bacterial microbiota reached the 7 log CFU/cm2, while 6 log UFC/g were measured in grape samples. A remarkable biodiversity was observed, with 13 phyla, 35 classes, 55 orders, 78 families and 95 genera of bacteria present. The presence of some taxa (Alphaproteobacteria, Desulfovibrionaceae, Clostriadiales, Oscillospira, Lachnospiraceae and Bacteroidales) was ubiquitous in all vineyards, but differences in terms of relative abundance were observed according to the vegetative stage, altitude of the vineyard and training system. Bacteria having oenological implication (Lactobacillus, Pediococcus and Oenococcus) were detected in grape samples collected in August, in low abundance. The data revealed a complex bacterial ecosystem inside the vineyard that, while maintaining common traits, evolves according to environmental and agronomical inputs. This study contributes to define the role of bacteria in the complex balance established in each vineyard between human actions and agricultural environment, known as terroir.

Selection of Lactic Acid Bacteria Species and Strains for Efficient Trapping of Drosophila suzukii.

(1) Monitoring of Drosophila suzukii is based on the use of effective traps and baits. The current baits are insufficient to provide efficient monitoring. The use of bacteria as bio-catalyzers to produce bioactive volatiles may improve flies' attraction. Thus, we conducted this work to improve Droskidrink® bait's attractiveness using lactic acid bacteria. (2) Different baits that were based on the use of Droskidrink® were assessed for flies' attraction in a Droso-Trap® in a vineyard. Oenococcus oeni, Pediococcus spp., and Lactobacillus spp. were used. The performance of the most attractive species, O. oeni, inoculated into Droskidrink® was assessed in laboratory tests. The responses of female flies to volatiles produced by Droskidrink® with O. oeni strains were recorded by electroantennography. (3) Preliminary field assessment of baits recorded O. oeni as the most attractive species. Three strain groups showed adaptation to test conditions. Volatiles extracted by the headspace of baits inoculated with O. oeni, elicited electroantennographic responses from fly antennae. (4) Droskidrink® inoculated with O. oeni is a highly attractive bait for monitoring. These findings will be useful for improving the attractiveness of D. suzukii commercial baits based on the utilization of LAB volatiles in a strain-dependent manner.

Biodiversity and oenological attitude of Saccharomyces cerevisiae strains isolated in the Montalcino district: biodiversity of S. cerevisiae strains of Montalcino wines.

The biodiversity of Saccharomyces cerevisiae was studied in the Montalcino area (Italy). Two wineries were involved in the study, which compared the genotypic and oenological characteristics of the S. cerevisiae strains isolated in spontaneous fermentations. After isolation yeasts were identified by 26S rRNA gene sequence analysis, and S. cerevisiae strains were characterized through interdelta sequence analysis (ISA). Oenological tests were performed in synthetic grape must by varying the magnitude of the main wine-imiting factors. The evolution of alcoholic fermentation was monitored by measuring sugar consumption and flow cytometry. The results revealed the prevalence of S. cerevisiae from the third day of fermentation and the presence of a wide range of S. cerevisiae strains having ISA profiles characteristic of each winery. From an oenological point of view, the features of such strains, in terms of resistance to wine-limiting factors, seemed to be linked to the main oenological variables applied in the production process of each winery. Extreme fermentation temperatures and copper residues are the variables that mostly depress the yeast population, in terms of fermentation rate and cell viability. Flow cytometry revealed the different impact of limiting factors on the viability of yeast by the quantification of the ratio between live/dead yeast cells of each strain, suggesting different mechanisms of inhibition, for instance stuck of cell growth or cell killing, in response to the different stress factors.

Liquid Baits with Oenococcus oeni Increase Captures of Drosophila suzukii.

The spotted-wing drosophila (SWD), Drosophila suzukii Matsumura (Diptera: Drosophilidae), native to Eastern Asia, is an invasive alien species in Europe and the Americas, where it is a severe pest of horticultural crops, including soft fruits and wine grapes. The conventional approach to controlling infestations of SWD involves the use of insecticides, but the frequency of application for population management is undesirable. Consequently, alternative strategies are urgently needed. Effective and improved trapping is important as an early risk detection tool. This study aimed to improve Droskidrink® (DD), a commercially available attractant for SWD. We focused on the chemical and behavioral effects of adding the bacterium Oenococcus oeni (Garvie) to DD and used a new trap design to enhance the effects of attractive lures. We demonstrate that microbial volatile compounds produced by O. oeni are responsible for the increase in the attractiveness of the bait and could be later utilized for the development of a better trapping system. Our results showed that the attractiveness of DD was increased up to two-fold by the addition of commercially available O. oeni when combined with an innovative trap design. The new trap-bait combination increased the number of male and especially female catches at low population densities.

Bacterial Complexity of Traditional Mountain Butter Is Affected by the Malga-Farm of Production.

Bacteria can play different roles affecting flavors and food characteristics. Few studies have described the bacterial microbiota of butter. In the present paper, next-generation sequencing was used to determine bacterial diversity, together with aromatic characteristics, in raw cow milk butter processed by traditional fermentation, in fourteen small farms called "Malga", located in the Trentino province (Alpine region, North-East of Italy). The physicochemical and aromatic characterization of traditional mountain butter (TMB) showed a low moisture level depending on the Malga producing the butter. Counts of lactic acid bacteria, Staphylococci, and coliforms, as well as diacetyl/acetoin concentrations exhibited changes according to the geographical origin of Malga and the residual humidity of butter. MiSeq Illumina data analysis revealed that the relative abundance of Lactococcus was higher in TMB samples with the highest values of acetoin (acetoin higher than 10 mg/kg). The traditional mountain butter bacterial community was characterized by a "core dominance" of psychrotrophic genera, mainly Acinetobacter and Pseudomonas, but according to ANCOM analysis, a complex bacterial population emerged and specific bacterial genera were able to characterize the TMB bacteria community, with their high abundance, based on the Malga producing the butter.

Addition of volatile sulfur compounds to yeast at the early stages of fermentation reveals distinct biological and chemical pathways for aroma formation.

Volatile sulfur compounds (VSCs) greatly influence the sensory properties and quality of wine and arise via both biological and chemical mechanisms. VSCs formed can also act as precursors for further downstream VSCs, thus elucidating the pathways leading to their formation is paramount. Short-term additions of exogenous hydrogen sulfide (H2S), ethanethiol (EtSH), S-ethylthio acetate (ETA), methanethiol (MeSH) and S-methylthio acetate (MTA) were made to exponentially growing fermentations of synthetic grape medium. The VSC profiles produced from live yeast cells were compared with those from dead cells and no cells. Interestingly, this experiment allowed the identification of specific biochemical and/or chemical pathways; e.g. most of the conversion of H2S to EtSH, and the further step from EtSH to ETA, required the presence of live yeast cells, as did the conversion of MeSH to MTA. In contrast, the reaction from MTA to MeSH and ETA to EtSH was due primarily to chemical degradation. Ultimately, this research unravelled some of the complex interactions and interconversions between VSCs, pinpointing the key biochemical and chemical nodes. These pathways are highly interconnected and showcase the complexity of both the sulfur pathways in yeast and the reactive chemistry of sulfur-containing compounds.

Spoilage potential of brettanomyces bruxellensis strains isolated from Italian wines.

Brettanomyces bruxellensis is an important wine spoilage agent. In this study a population of Brettanomyces strains isolated from Italian wines was thoroughly investigated to evaluate adaptability to wine conditions and spoilage potential. The presumptive isolates of Brettanomyces were identified at species level with 26S rRNA gene sequencing and species-specific PCR, and subsequently subjected to analysis of intra-species variability through the study of intron splice sites (ISS-PCR). Although, some strains were tracked in wines from different regions, extensive genetic biodiversity was observed within the B. bruxellensis population investigated. All strains were evaluated for their growth ability in the presence of ethanol, high sugar content, low pH, different temperatures and sulphur dioxide, using optical density and flow cytometry measurement. The ability of yeasts to produce ethyl phenols in red wines with different chemical compositions was evaluated by means of high performance liquid chromatography with electrochemical detection (HPLC-ECD). The results highlighted wide variability in B. bruxellensis in response to wine limiting factors and in terms of the accumulation of ethyl phenols. As regards this last aspect, the differences found among strains were closely related to chemical composition of wine and strain resistance to environmental stress factors, making a priori evaluation of risk of wine alteration quite difficult. These results suggest that strategies for the control of Brettanomyces should be tailored on the basis of strain distribution and wine characteristics.

Drosophila suzukii (Diptera: Drosophilidae) Contributes to the Development of Sour Rot in Grape.

This research aimed to more clearly describe the interactions of Drosophila suzukii (Matsumura; Diptera: Drosophilidae) with microorganisms that may contribute to spoilage or quality loss of wine grapes during harvest. Experiments were conducted in controlled laboratory experiments and under field conditions to determine these effects. Laboratory trials determined the role of insect contact and oviposition to vector spoilage bacteria onto wine grapes. In the field, the roles of key organoleptic parameters in grape fruit ripening were assessed to determine their relative contribution to oviposition potential as fruit ripened. Finally, field trials determined the relationships of egg and larval infestation to sour rot levels. Non-ovipositional trials indicated elevated levels of microbiota when D. suzukii was present. D. suzukii oviposition exponentially increased the concentration of acetic acid bacteria. Both incised and sound berries showed a significant increase in concentrations of acetic acid bacteria exposed to D. suzukii. Volatile acidity was higher in treatments infested with D. suzukii. Fruit with only eggs did not develop a significant increase of volatile acidity. Larva-infested grape berries in 9.5% of samples developed higher volatile acidity after 14 d. Sound grape berries were less susceptible to the development of microbiota associated with sour rot and spoilage. D. suzukii oviposition and larval development increase risk of spoilage bacteria vectored by D. suzukii adults. Acetic acid bacteria induced fermentation and produced several volatile compounds contributing to spoilage. Spoilage bacteria may create a positive feedback loop that attracts both D. suzukii and other drosophilids, which may contribute to additional spoilage.

The impact of different barrel sanitation approaches on the spoilage microflora and phenols composition of wine.

Careful control of spoilage microflora inside wine containers is a key issue during winemaking. To date, attention has been paid to the development of an effective protocol for the eradication of spoilage agents, especially Brettanomyces, from barrels. Few studies have taken into account the modifications caused by sanitation treatments in wine and wood barrels. In the present study the effects of two sanitation treatments (ozone and sodium hydroxide) on barrel spoilage microflora and the composition of the wine stored inside them were evaluated. The phenols of wine (38 compounds) were characterised using a UHPLC-MS during the first 3 months of wine ageing, to see possible alterations in composition due to the chemical exchange from wood to wine in presence of sanitising agents. With the same scope, a panel of 13 judges carried out sensorial analysis of wines. The results showed that the tested treatments had little effect on the organoleptic characteristics of wines, but underline the different performance of the sanitation treatments in terms of eradicating microorganisms.

Exploring the microbiota of the red-brown defect in smear-ripened cheese by 454-pyrosequencing and its prevention using different cleaning systems.

Red-brown pigmentation can occasionally form in smeared-ripened cheese such as Fontina during the ripening process. This reaction is due to over-development of the typical microbiota present on the rind. Previous studies have demonstrated the relationship between red-brown pigmentation and the traditional utilization of wooden shelves during cheese ripening. The first part of the paper focuses on the characterisation of yeast and bacterial microbiota: plate counts and 454-pyrosequencing were performed in spoiled (n = 6) and non-spoiled cheeses (n = 6) and on the wooden shelves used during ripening. The second part shows different systems tested for cleaning the wooden shelves and avoiding the development of the red-brown defect in cheese: washing with hot water and ozone treatment. Actinobacteria, dominated on the wooden shelves, suggesting to be responsible for the red-brown pigmentation; they were also found in traces in the defected cheese samples. Galactomyces and Debaryomyces were the main species characterizing the yeast population, with Debaryomyces being the most dominant species on the shelves used during ripening of the red-brown defective cheese. Hot water treatment reduced the microbial contamination of shelves, whereas only the ozone treatment ensured complete elimination of both yeast and bacteria, resulting in the cheese rind not having the red-brown defect.

Silicification of wood adopted for barrel production using pure silicon alkoxides in gas phase to avoid microbial colonisation.

The paper presents a new approach, covering wood with silica-based material in order to protect it from spoilage due to microbial colonisation and avoiding the loss of the natural features of the wood. Wood specimens derived from wine barrels were treated with methyltriethoxysilane in gas phase, leading to the deposition of a silica nanofilm on the surface. (29)Si and (13)C solid state Nuclear Magnetic Resonance and Scanning Electron Microscope-Energy Dispersive X-ray analysis observations showed the formation of a silica polymeric film on the wood samples, directly bonding with the wood constituents. Inductively Coupled Plasma-Mass Spectroscopy quantification of Si showed a direct correlation between the treatment time and silica deposition on the surface of the wood. The silica-coated wood counteracted colonisation by the main wine spoilage microorganisms, without altering the migration from wood to wine of 21 simple phenols measured using a HPLC-Electrochemical Coulometric Detection.

Metabolite profiling of grape: Flavonols and anthocyanins.

Flavonols are products of the flavonoid biosynthetic pathway, which also give rise to anthocyanins and condensed tannins in grapes. We investigated their presence in the berry skins of 91 grape varieties (Vitis vinifera L.), in order to produce a classification based on the flavonol profile. The presence of laricitrin 3-O-galactoside and syringetin 3-O-galactoside in red grapes is reported here for the first time. In red grapes, the main flavonol was quercetin (mean = 43.99%), followed by myricetin (36.81%), kaempferol (6.43%), laricitrin (5.65%), isorhamnetin (3.89%), and syringetin (3.22%). In white grapes, the main flavonol was quercetin (mean = 81.35%), followed by kaempferol (16.91%) and isorhamnetin (1.74%). The delphinidin-like flavonols myricetin, laricitrin, and syringetin were missing in all white varieties, indicating that the enzyme flavonoid 3',5'-hydroxylase is not expressed in white grape varieties. The pattern of expression of flavonols and anthocyanins in red grapes was compared, in order to gain information on the substrate specificity of enzymes involved in flavonoid biosynthesis.