OENO IVAS 2019 banner
IVES 9 IVES Conference Series 9 OENO IVAS 9 OENO IVAS 2019 9 Grape and wine microorganisms: diversity and adaptation 9 Application of high-throughput sequencing tools for characterisation of microbial communities during alcoholic fermentation

Application of high-throughput sequencing tools for characterisation of microbial communities during alcoholic fermentation

Abstract

Developments in high-throughput sequencing (HTS) technologies allow us to obtain large amounts of microbial information from wine and must samples. Thus approaches, that are aimed at characterising the microbial diversity during fermentation, can be enhanced, or possibly even replaced, with HTS-based metabarcoding. To reduce experimental biases and increase data reproducibility, we compared 3 DNA extraction methods by evaluating differences in the fungal diversity with Riesling alcoholic fermentation samples at four different vineyards. The fungal diversity profiling was done using the genetic markers ITS2 and D2 using metabarcoding. The extraction methods compared consisted of a commercial kit, a recently published protocol that includes a DNA enhancer, and a protocol based on a buffer containing common inhibitor removal reagents. All methods were able to distinguish vineyard effects on the fungal diversity, but the results differed quantitatively. 

From the results of extraction methods, we applied the chosen methods and further combined the HTS tools of metabarcoding and metagenomics, to characterise how microbial communities of those samples, and their subsequent spontaneously fermented derivatives, vary. We specifically explored microbial community variation related to vineyard level, and during alcoholic fermentation. The vineyard was shown to be strongly influencing the microbial communities. Functional analyses were additionally included to investigate the microbial interactions. An increase in non-Saccharomycetaceae fungal functions and a decrease in bacterial functions were also observed during the early fermentation stage. Overall, our results highlight the importance of standardizing DNA extraction methods when characterising fungal diversity from wine and related samples, and showcase how metagenomic functional analysis offer possibilities to improve our insights into the wine alcoholic fermentation process, including highlighting microbe interactions.

DOI:

Publication date: June 23, 2020

Issue: OENO IVAS 2019

Type: Article

Authors

Sarah Siu Tze Mak, Kimmo Sirén, Christian Carøe, Ulrich Fischer, M. Thomas P. Gilbert 

Section for Evolutionary Genomics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark 
Institute for Viticulture and Oenology, Dienstleistungszentrum Ländlicher Raum Rheinpfalz, Neustadt an der Weinstraße, Germany 

Contact the author

Keywords

Riesling, Metabarcoding, Metagenomics, DNA extraction

Tags

IVES Conference Series | OENO IVAS 2019

Citation

Related articles…

Grapevine sugar concentration model in the Douro Superior, Portugal

Increasingly warm and dry climate conditions are challenging the viticulture and winemaking sector. Digital technologies and crop modelling bear the promise to provide practical answers to those challenges. As viticultural activities strongly depend on harvest date, its early prediction is particularly important, since the success of winemaking practices largely depends upon this key event, which should be based on an accurate and advanced plan of the annual cycle. Herein, we demonstrate the creation of modelling tools to assess grape ripeness, through sugar concentration monitoring. The study area, the Portuguese Côa valley wine region, represents an important terroir in the “Douro Superior” subregion. Two varieties (cv. Touriga Nacional and Touriga Franca) grown in five locations across the Côa Region were considered. Sugar accumulation in grapes, with concentrations between 170 and 230 g l-1, was used from 2014 to 2020 as an indicator of technological maturity conditioned by meteorological factors. The climatic time series were retrieved from the EU Copernicus Service, while sugar data were collected by a non-profit organization, ADVID, and by Sogrape, a leading wine company. The software for calibrating and validating this model framework was the Phenology Modeling Platform (PMP), version 5.5, using Sigmoid and growing degree-day (GDD) models for predictions. The performance was assessed through two metrics: Roots Mean Square Error (RMSE) and efficiency coefficient (EFF), while validation was undertaken using leave-one-out cross-validation. Our findings demonstrate that sugar content is mainly dependent on temperature and air humidity. The models achieved a performance of 0.65

Upscaling the integrated terroir zoning through digital soil mapping: a case study in the Designation of Origin Campo de Borja

homogeneous zones by intersecting several partial zonings of major factors that influence vineyard growth. Each of them follows specific process from their corresponding disciplines. Soil zoning specifically refers to a Soil Resource Inventory map that has traditionally been generated by conventional soil mapping methods. These methods have shortcomings in reaching fine cartographic and categorical details and involve significant expenses, which undermines their applicability. A new framework named Digital Soil Mapping has introduced quantitative models by statistical techniques to establish soil-landscape relationships and is able to provide intensive scale cartography.

In the present study, a microzoning at 1:10.000 scale is generated from an initial zoning, where the conventional soil map with polytaxic map units is replaced by a new one from digital techniques that disaggregates them. The comparison between the zonings considers a quantitative evaluation of capability for each Homogeneous Terroir Unit by means of the Viticultural Quality Index and its categorization based on its distribution by map. The spatial intersection of both maps gives rise to a confusion matrix in which the flows of class variations after the substitution are assessed.

The results show a five-fold increase in the number of Homogeneous Terroir Units identified and a larger differentiation among them, evidenced by a wider range in the capability index distribution. Both elements are accompanied by an increase in the detection of areas of higher potential within previously undervalued uniform zones.These features are a direct effect of the improvements brought by Digital Soil Mapping techniques and would verify the advantages of their implementation in the Integrated Terroir zoning. Eventually, such new highly detailed terroir units would benefit precision viticulture and sustainable management practices.

Climate change impacts on Douro Region viticulture and adaptation measures

Climate has a significant impact in the success of any agricultural system, with a direct influence on the crops suitability to a given region, interfering on yield and quality and also with the economic sustainability of the productive activity. In the Douro Demarcated Region (RDD), as in most regions of the Mediterranean climate, the scarce precipitation (33% has less than 600 mm per year), and your high variability, associated with high rates of evapotranspiration during the summer, is usually one of the fundamental factors that limit the grapevine development, as well as the production and quality of the harvest. Thus, facing the scenario in temperature changes for the next decades (1.5-2.5°C) and confirming the predictions of precipitation decreases and/or great variability in the occurrence of heat waves and intense rainfall, the consequences for slope stability in mountain viticulture and sustainability of all operations involved, are risks to be taken into account. In this way, a deepest and sustained knowledge regarding the adaptation measures to adverse environmental conditions is of a crucial importance, enabling a more efficient adaptation of plant growth conditions and the optimization of production and quality of the grapevines. The development of this work, carried out in two commercial vineyards, one located in Soutelo do Douro, São João da Pesqueira, Cima Corgo sub-region, and another located in Numão, Vila Nova de Foz Côa, Douro Superior sub-region, it seeks to establish a relationship between climatic elements and physiological, productive and qualitative parameters, as well as to evaluate the effectiveness of adaptation measures, including different types of deficit irrigation (2002-2019) and the application of shading nets (2019-2020) in the physiological, viticultural and oenological behavior in the Touriga Nacional and Moscatel Galego Branco varieties, respectively. The results showed that the application of deficit irrigation allowed to significantly reduce the impact of the adverse weather conditions at key moments in the development of the grapevine, particularly in the period immediately before veráison and maturation, reducing the negative effects on the physiological processes and productivity, without compromise the must quality parameters. On the other hand, the application of shading nets significantly reduced de leaves temperature, allowing to increase the water potential, stomatal conductance and photosynthetic rate of grapes, which was reflected in the yield increase in the 2nd year of the study. For the maturation indicators, higher levels of total acidity, malic acid and assimilable nitrogen were obtained. The last measure presents a huge potential, being essential to carry out more years of trials to obtain stronger conclusions in terms of production parameters, but also in characteristics as important as the grape ripening components and the organoleptic characteristics of wines.

Grapevine yield estimation in a context of climate change: the GraY model

Grapevine yield is a key indicator to assess the impacts of climate change and the relevance of adaptation strategies in a vineyard landscape. At this scale, a yield model should use a number of parameters and input data in relation to the information available and be able to reproduce vineyard management decisions (e.g. soil and canopy management, irrigation). In this study, we used data from six experimental sites in Southern France (cv. Syrah) to calibrate a model of grapevine yield limited by water constraint (GraY). Each yield component (bud fertility, number of berries per bunch, berry weight) was calculated as a function of the soil water availability simulated by the WaLIS water balance model at critical phenological phases. The model was then evaluated in 10 grapegrowers’ plots, covering a diversity of biophysical and technical contexts (soil type, canopy size, irrigation, cover crop). We identified three critical periods for yield formation: after flowering on the previous year for the number of bunches and berries, around pre-veraison and post-veraison of the same year for mean berry weight. Yields were simulated with a model efficiency (EF) of 0.62 (NRMSE = 0.28). Bud fertility and number of berries per bunch were more accurately simulated (EF = 0.90 and 0.77, NRMSE = 0.06 and 0.10, respectively) than berry weight (EF = -0.31, NRMSE = 0.17). Model efficiency on the on-farm plots reached 0.71 (NRMSE = 0.37) simulating yields from 1 to 8 kg/plant. The GraY model is an original model estimating grapevine yield evolution on the basis of water availability under future climatic conditions.  It allows to evaluate the effects of various adaptation levers such as planting density, cover crop management, fruit/leaf ratio, shading and irrigation, in various production contexts.

Grapevine varietal diversity as mitigation tool for climate change: Agronomic and oenologic potential of 14 foreign varieties grown in Languedoc region (France)

Climate change effects in Languedoc include an expected rise in temperatures, increased evapotranspiration as well as more severe and frequent climatic hazards, such as frost, drought periods and heat waves. For winegrowers theses phenomena impact both yield and quality, resulting in more frequent unbalanced wines. Research on identified mitigation tools for vineyard management is necessary to improve resilience of grapevine agrosystems. Varietal assortment is one of them. This study focuses on agronomic and oenologic potential of 14 foreign varieties grown in Languedoc French region. Fourteen grapevine varieties were monitored during 2021 from June until harvest on eight different sites, some of which occurring on more than one site adding up to 21 different modalities: 7 white varieties Alvarinho B, Assyrtiko B (2), Malvasia Istriana B, Parellada B, Verdejo B, Verdelho B, Xarello B, and 7 black varieties Saperavi N (2), Touriga nacional N, Baga N, Aleatico N, Montepulciano N (2), Primitivo N (3), Calabrese N (3). Varietals were compared through the following parameters: phenology was assessed by using the information collected in the Database Network of French Vine Conservatories (INRAE-SupAgro-IFV, 2005-2015). The number of inflorescences for shoots from secondary buds and bourillons and suckers were observed to assess post-bud break frost tolerance potential. Grapevine water status was studied through stem water potential measurement, observation of foliage symptoms of drought, and 𝛿13C on must. Frequencies and intensities of downy mildew, powdery mildew, and black rot attacks were estimated before harvest on leaves and clusters and botrytis at harvest to assess disease susceptibilities. Berry composition was monitored from end of veraison until harvest. Yield and mean bunch weight were also calculated. Varieties were then ranked on a 1-4 scale for each parameter and compared through PCA. Forty two stations of the Mediterranean basin were compared by PCA with the Multicriteria Climatic Classification indicators in order to confront the collected information during 2021 campaign to the hypothesis that plants coming from dry and hot regions are genetically adapted to such climatic conditions.