Macrowine 2021
IVES 9 IVES Conference Series 9 Characterization of Brettanomyces bruxellensis biofilm, a resistance strategy to persist in wine-related environments

Characterization of Brettanomyces bruxellensis biofilm, a resistance strategy to persist in wine-related environments

Abstract

AIM: Biofilm is a resistance mechanism deployed by microorganisms to adapt to stresses, leading to their persistence in the environment. In the case of Brettanomyces bruxellensis, a wine spoilage yeast, knowledge about its capacity to form biofilm remains limited although this potential strategy could explain its recurring presence in cellars. In this study, we propose to investigate the capacity of strains of B. bruxellensis to form biofilm according to different conditions and to characterize its structure.

METHODS: Sixty-five isolates of B. bruxellensis were sampled from a cellar and discriminated into genetic groups by microsatellite analysis. The capacity of selected strains to adhere and form biofilm has been investigated in different conditions of media and supports, related to wine environment. The structure of the biofilm and its components were explored using several microscopic techniques as confocal laser scanning microscopy, electronic microscopy and epifluorescence microscopy.

RESULTS: Twelve strains among the 65 isolates were selected and have showed the capacity to form biofilm on polystyrene surface. Microscopic observations of the biofilm revealed microcolonies, filamentous cells and extracellular polymeric substances despite a small thickness. Then, the study of the impact of wine on B. bruxellensis biofilm revealed biofilm cell released and growth of these released biofilm cells, probable contamination source of the wine. Finally, for both planktonic and biofilm lifestyles in wine, a new chlamydospore-like element was observed for B. bruxellensis, described as a resistance form in other fungi.

CONCLUSIONS: This study validates the capacity of B. bruxellensis to form biofilm and highlights structural element of this mode of life. Moreover, an additional resistance strategy was suggested through the description of the chlamydospore-like elements. New insights into the persistence of B. bruxellensis during the winemaking process and in wineries have emerged.

DOI:

Publication date: September 3, 2021

Issue: Macrowine 2021

Type: Article

Authors

Manon Lebleux, Hany ABDO, Louise BASMACIYAN, Julie LAURENT, Chloé ROULLIER-GALL, Hervé ALEXANDRE, Michèle GUILLOUX-BENATIER, Stéphanie WEIDMANN, Sandrine ROUSSEAUX

Univ. Bourgogne Franche-Comté, AgroSup Dijon, PAM UMR A 02.102, VAlMiS Laboratory-IUVV, Dijon, France

Contact the author

Keywords

Brettanomyces bruxellensis, biofilm, wine spoilage

Citation

Related articles…

The effects of alternative herbicide free cover cropping systems on soil health, vine performance, berry quality and vineyard biodiversity in a climate change scenario in Switzerland

There is an urgent need in viticulture to adopt alternative herbicide-free soil management strategies to mitigate climate change, increase biodiversity, reduce plant protection products and improve soil quality while minimizing detrimental effects on grapevine’s stress tolerance and fruit quality. To propose sustainable solutions, adapted to different pedoclimatic conditions in Switzerland, we developed a multidisciplinary 4-year project, started in 2020. Objectives of the project are to a) evaluate the impact of green covers (spontaneous flora, winter cover crop and permanent ground cover) on environmental and agronomic parameters and b) develop subsequently innovative strategies for different viticultural contexts of Switzerland. The project is divided into 3 phases: 1) diagnosis, 2) on-farm and 3) on-station experiments. Phase 1) consisted in an assessment of 30 commercial vineyards all over Switzerland, where growers already use different herbicide-free soil management strategies. The most promising practices identified in this exploratory phase will be replicated in commercial vineyards across Switzerland (“on-farm”) as well as in a classical randomized block design in an experimental plot (“on-station”). For phase 1), measurements consisted in evaluation of soil status (compaction, structure, roots development), soil microbial diversity (metagenomics), plant diversity and biomass, vine physiology (water stress, vigor, leaf nitrogen) and berry quality (acidity, sugar, available nitrogen). Interestingly, the permanent ground cover resulted in a higher Shannon index thus a higher biodiversity as compared to the other itineraries. The winter cover crop increased vine nitrogen and vigor while deteriorating soil quality, leaving the soil more exposed and compacted likely due to more frequent tillage. The spontaneous flora led to higher berry sugar accumulation, less nitrogen and higher malic acid concentration putatively due to a higher water retention of the flora in a particularly wet vintage. Phases 2) and 3) are required to confirm those tendencies, over the 3 next vintages and different climatic conditions.

Combining effect of leaf removal and natural shading on grape ripening under two irrigation strategies in Manto negro (Vitis vinifera L.)

The increasingly frequent heat waves during grape ripening pose challenges for high quality wine grape production. Defoliation is a common practice that can improve the control of diseases in bunches, but also it increases the exposure to sunlight. Grapes exposed to solar radiation reach temperatures over the optimum for berry development and maturation. This makes the development of irrigation and canopy management techniques of great importance to maximize yield and grape quality. A field experiment was carried out during 2021 using Manto negro wine grapes to study the effect of applied irrigation and different light exposure levels on grape quality. Two irrigation treatments were imposed based on the frequency and amount of water doses in a four-block experimental vineyard at Bodega Ribas (Mallorca). Three light exposure treatments were randomly applied in each irrigation plot. The light treatments included exposed clusters from pea size, non-exposed clusters, and shaded clusters after softening. Leaf area index and canopy porosity was estimated every 2 weeks. Midday leaf water potential was measured weekly. Additionally, apparent electrical conductivity was measured between rows to estimate the soil water content variability. Light and temperature sensors were installed at the bunch level to quantify the differences in bunch temperature and light intensity among treatments. The effect of irrigation and cluster light exposure on berry weight, TSS, TA, malic acid, tartaric acid, K+, and pH were analysed at 5 moments along grape ripening. During different heat waves, the natural shading technique decreased the maximum bunch temperature around 10 °C respect to the exposed bunches in both irrigation strategies. The combination of defoliation and shading techniques after softening decreased TSS at harvest and affected most of the quality parameters during the last stages of ripening, showing an interesting technique to delay ripening in warm viticulture areas.

1H-NMR-based Metabolomics to assess the impact of soil type on the chemical composition of Mediterranean red wines

The aim of this study was to evaluate the effects of different soil types on the chemical composition of Mediterranean red wines, through untargeted and targeted 1H-NMR metabolomics. One milliliter of raw wine was analyzed by means of a Bruker Avance II 400 spectrometer operating at 400.15 MHz. The spectra were recorded by applying the NOESYGPPS1D pulse sequency, to achieve water and ethanol signals suppression. No modification of the pH was performed to avoid any chemical alteration of the matrix. The generation of input variables for untargeted analysis was done via bucketing the spectra. The resulting dataset was preprocessed prior to perform unsupervised PCA, by means of MetaboAnalyst web-based tool suite. The identification of compounds for the targeted analysis was performed by comparison to pure compounds spectra by means of SMA plug-in of MNova 14.2.3 software. The dataset containing the concentrations (%) of identified compounds was subjected to one-way analysis of variance (ANOVA) to highlight significant differences among the wines. The untargeted analysis, carried out through the PCA, revealed a clear differentiation among the wines. The fragments of the spectra contributing mostly to the separation were attributed to flavonoids, aroma compounds and amino acids. The targeted analysis leaded to the identification of 68 compounds, whose concentrations were significant different among the wines. The results were related to soils physical-chemical analysis and showed that: 1) high concentrations of flavan-3-ols and flavonols are correlated with high clay content in soils; 2) high concentrations of anthocyanins, amino acids, and aroma compounds are correlated with neutral and moderately alkaline soil pH; 3) low concentrations of flavonoids and aroma compounds are correlated with high soil organic matter content and acidic pH. The 1H-NMR metabolomic analysis proved to be an excellent tool to discriminate between wines originating from grapes grown on different soil types and revealed that soils in the Mediterranean area exert a strong impact on the chemical composition of the wines.

Assessment of climate change impacts on water needs and growing cycle on grapevine in three DOs of NE Spain

This study assessed the suitability of grapevine growing in three DOs (Empordà, Pla de Bages and Penedès) of Catalonia (NE Spain) over the 21st century. For this purpose, an estimation of water needs and agroclimatic and phenological indicators was made. Climate change impacts were estimated at 1 km pixel resolution using temperature and precipitation projections from several general circulation models (GCM) and two climate change scenarios: RCP 4.5 (stabilization scenario) and RCP 8.5 (worst-case scenario). Potential crop evapotranspiration (following FAO procedure) and a daily water balance considering soil water holding capacity were used to estimate actual evapotranspiration of vines and, finally, water needs. Dynamics would be similar in the three DOs studied although the magnitude of impact differs. Water needs would be 2 and 3 times greater (ranging from 0 to more than 1500 m3/ha) than current water needs at both climate change scenarios. Moreover, blooming date would advance from 3 to 6 weeks, harvest date from 1 to 2.5 months, resulting in growing cycles from 10 to 80 days shorter. It should also be noted that frost risk would decrease from 6 to 76%, the number of days with temperatures above 30ºC during ripening would rise from 48 to 500% and tropical nights (minimum temperature >20ºC) at ripening would increase from 28 to 150%, depending on the scenario and the DOs. The impacts of climate change in the three DOs could result in significant limitations for grapevine cultivation and wine production if adaptive strategies are not applied. This result could serve as a basis for the design of specific and particular adaptation strategies to improve and maintain vineyards in the DOs studied and could be extrapolated to similar DOs and regions.

Delaying irrigation initiation linearly reduces yield with little impact on maturity in Pinot noir

When to initiate irrigation is a critical annual management decision that has cascading effects on grapevine productivity and wine quality in the context of climate change. A multi-site trial was begun in 2021 to optimize irrigation initiation timing using midday stem water potential (ψstem) thresholds characterized as departures from non-stressed baseline ψstemvalues (Δψstem). Plant material, vine and row spacing, and trellising systems were concomitant among sites, while vine age, soil type, and pruning systems varied. Five target Δψstem thresholds were arranged in an RCBD and replicated eight times at each site: 0.2, 0.4, 0.6, 0.8, and 1.0 MPa (T1, T2, T3, T4, and T5, respectively). When thresholds were reached, plots were irrigated weekly at 70% ETc. Yield components and berry composition were quantified at harvest. To better generalize inferences across sites, data were analyzed by ANOVA using a mixed model including site as a random factor. Across sites, irrigation was initiated at Δψstem = 0.24, 0.50, 0.65, 0.93, and 0.98 MPa for T1, T2, T3, T4, and T5, respectively. Consistent significant negative linear trends were found for several key yield and berry composition variables. Yield decreased by 12.9, 15.9, 19.5, and 27.4% for T2, T3, T4, and T5, respectively, compared to T1 (p < 0.0001) across sites that were driven by similarly linear reductions in berry weight (p < 0.0001). Comparatively, berry composition varied little among treatments. Juice total soluble solids decreased linearly from T1 to T5 – though only ranged 0.9 Brix (p = 0.012). Because producers are paid by the ton, and contracts simply stipulate a target maturity level, first-year results suggest that there is no economic incentive to induce moderate water deficits before irrigation initiation, regardless of vineyard site. Subsequent years will further elucidate the carryover effects of delaying irrigation initiation on productivity over the long term.