Investigating the Influence of Fungicide Usage and Vineyard Disease Management on the Grapevine Microbiome and Wine Quality

Abstract

The grapevine microbiome has recently become recognised as an essential feature in winemaking. As we advance our understanding of the role of these microorganisms in grape growing, wine production, and wine flavour and aroma, research must be redirected to preserving beneficial microbial communities. This is highlighted by the increasing number of wine producers beginning to ferment wines using ‘wild’ grapevine yeasts as opposed to typical inoculations with Saccharomyces cerevisiae (Chapter IV). To effectively characterise these communities and the factors that impact them, first we must improve current microbial harvesting methods, which need more standardisation across the scientific field and second, we need greater capability in differentiating between DNA from viable and non-viable sources. We developed a novel, phyllosphere microbiome harvesting protocol to combat these issues, adopting swabs for more standardised harvesting (Chapter III). In addition, we employed the viability selection dye Propidium Monoazide (PMA), which binds to DNA from non-living sources (relic DNA), preventing it from being amplified by PCR-based methods (Chapter III). This interaction leaves only DNA from viable cells free to be amplified and analysed using PCR based methods such as quantitative PCR (qPCR) or DNA sequencing. With this viable phyllosphere harvesting protocol, we commenced an investigation into the effect of fungicide usage on the phyllosphere microbiome of grapevines used for winemaking (Chapter V). The use of broad-target fungicides can influence both grapevine health and wine quality but is also one factor that may impact how the grapevine microbiome manifests and develops over time. Common anti-fungal sprays such as copper and sulphur do not specifically target pathogenic fungi and, therefore, have the potential to impact the structure of beneficial fungal and bacterial communities. Using the method above for sampling the phyllosphere microbiome, combined with 16S and ITS gene amplicon sequencing, we defined the longitudinal effects that two common fungicides, Copper- and Sulphur-based, have on the structure of live communities within the grapevine microbiome in a semi-controlled environment using potted Shiraz grapevines. From these results, we found little to no effect on the microbial communities caused by the copper- or sulphur-based fungicides. The results demonstrated that despite an immediate, rapid loss of diversity, likely due to the spraying action, the diversity recovered over the course of the following weeks. Following this initial characterisation, a broader examination of the effects of fungicide regimes in commercial vineyards was conducted (Chapter VI). Each of the three vineyards selected utilised a different type of vineyard management practice, conventional, organic, or biodynamic, and thus different disease control. Grape samples were collected from each; a subset was taken for microbial analysis before the rest was used for small-lot wine-making with identical parameters to produce completed wine samples for sensory analysis. Microbial samples were also collected at key time points throughout the fermentation process until the wines were complete. Finished wines underwent chemical and sensory analyses, and both results were compared to the microbial samples' 16S rRNA and ITS gene amplicon sequencing results. Trends were identified between the three different vineyards to understand the varying effects each vineyard disease management method had on the phyllosphere microbiome and the impact these effects have on the resulting wines. We found that the structure of the phyllosphere microbiome directly correlated to the sensory outcomes of the final wines. We also observed some distinct differences between the vineyards, particularly the organic group. This thesis presents an in-depth investigation into the repercussions of common fungicides on the phyllosphere microbiome and its temporal dynamics, concurrently exploring the correlation between these interactions and the sensory outcomes of wines. This aspect has yet to be fully explored. This research expands our fundamental understanding of the grapevine microbiome in the context of grape cultivation and winemaking, offering potential insights into refined phyllosphere characterisation, preservation, and practical utilisation.