Diversity and dynamics of fungi during spontaneous fermentations and association with unique aroma profiles in wine

Highlights

• Fungal microbiota showed geographic distribution patterns at small geographic scales.

• Associations between fungal species and wine metabolites were elucidated.

• Geographic differentiation of S. cerevisiae was evident at a distance of 12 km.

• Structural equation modelling revealed the importance of S. cerevisiae to aroma.

Abstract

Microbial ecology is an integral part of an agricultural ecosystem and influences the quality of agricultural commodities. Microbial activity influences grapevine health and crop production, conversion of sugar to ethanol during fermentation, thus forming wine aroma and flavour. There are regionally differentiated microbial patterns in grapevines and must but how microbial patterns contribute to wine regional distinctiveness (terroir) at small scale (<100 km) is not well defined. Here we characterise fungal communities, yeast populations, and Saccharomyces cerevisiae populations during spontaneous fermentation using metagenomics and population genetics to investigate microbial distribution and fungal contributions to the resultant wine. We found differentiation of fungi, yeasts, and S. cerevisiae between geographic origins (estate/vineyard), with influences from the grape variety. Growth and dominance of S. cerevisiae during fermentation reshaped the fungal community and showed geographic structure at the strain level. Associations between fungal microbiota diversity and wine chemicals suggest that S. cerevisiae plays a primary role in determining wine aroma profiles at a sub-regional scale. The geographic distribution at scales of less than 12 km supports that differential microbial communities, including the dominant fermentative yeast S. cerevisiae can be distinct in a local setting. These findings provide further evidence for microbial contributions to wine terroir, and perspectives for sustainable agricultural practices to maintain microbial diversity and optimise fermentation function to craft beverage quality.

Introduction

Wine grapes (Vitis vinifera) are an economically and culturally important agricultural commodity for which microbial activity plays key roles in grape and wine production and quality (Barata et al., 2012; Swiegers et al., 2005). The grapevine harbours complex and diverse microbiota, such as bacteria, filamentous fungi, and yeasts (Barata et al., 2012; Liu and Howell, 2020; Stefanini and Cavalieri, 2018), which substantially modulate vine health, growth, and crop productivity (Berg et al., 2014; Gilbert et al., 2014; Müller et al., 2016). Grapevine-associated microbiota can be transferred to the grape must/juice and have an influence on wine composition, aroma, flavour, and quality (Barata et al., 2012; Ciani et al., 2010; Morrison-Whittle and Goddard, 2018). Wine fermentation is a complex and multispecies process, involving numerous transformations by fungi and bacteria to sculpt chemical and sensory properties of the resulting wines (Swiegers et al., 2005; Verginer et al., 2010). While these consortia all contribute to wine flavour formation, the fermentation process is principally driven by diverse populations of Saccharomyces cerevisiae (Fleet, 2003; Goddard, 2008; Howell et al., 2006).

Microbial biogeography contributes to regional distinctiveness of agricultural products, known as “terroir” in viticulture [reviewed by Liu et al., 2019]. Biogeographical patterns in the microbiota associated with the grape and must have been demonstrated for both fungi and bacteria at a regional scale (Bokulich et al., 2014; Mezzasalma et al., 2018; Pinto et al., 2015; Taylor et al., 2014), which are conditioned by multiple factors, such as cultivar, climate and vintage, topography, and soil properties (Bokulich et al., 2014; Liu et al., 2019; Miura et al., 2017; Portillo et al., 2016; Zarraonaindia et al., 2015). Bokulich et al. (2016) showed that the bacterial and fungal consortia correlated with metabolites in finished wines, highlighting the importance of fermentative yeasts (for example, S. cerevisiae, Hanseniaspora uvarum, Pichia guilliermondii) and lactic acid bacteria (Leuconostocaceae) on the abundance of regional aromatic signatures. Our previous research revealed that wine-related fungal communities structured and distinguished vineyard ecosystems by impacting the flavour and quality of wine, and weather with a contribution by soil properties affected soil and must fungal communities and thus the composition of wines across six winegrowing regions in southern Australia (Liu et al., 2020). However, whether grape-associated microbiota exhibit distinct patterns of distribution at smaller geographic scales (for example individual vineyards) and their associations with wine aroma profiles are not well understood.

Geographic differentiation of S. cerevisiae populations is evident at global (Legras et al., 2007; Liti et al., 2009) and regional scales (Gayevskiy and Goddard, 2012; Knight and Goddard, 2015), revealing a picture of distinctive populations at large scales more than ~100 km. Drumonde-Neves et al. (2018) showed higher genetic divergence among S. cerevisiae populations between rather than within islands/regions (~1.5–260 km scale) and suggested a prevailing role of geography over ecology (grape varieties and agricultural cultivation) in shaping diversification, as previously reported (Goddard et al., 2010). At small scales, several studies characterised significant genetic differences between S. cerevisiae populations residing in different vineyards within the same region [Börlin et al., 2016; <10 km] and different sites within a vineyard [Schuller and Casal, 2007; 10–400 m]. Knight et al. (2015) experimentally demonstrated that regional strains of S. cerevisiae produce distinct wine chemical compositions, suggesting a prominent route by which regional S. cerevisiae shape wine terroir. While few studies have investigated how S. cerevisiae differentiation can affect wine aroma, flavour, and characteristics, and none have considered S. cerevisiae, fermentative yeasts, and the global fungal communities simultaneously to quantify their contributions to the resultant wine.

To investigate these questions, we sampled microbial communities associated with Pinot Noir and Chardonnay grape must and juice from three wine estates with 8–12 km pairwise distances to include grapes from 11 vineyards in the Mornington Peninsula wine region of Victoria, Australia. Using culture-independent sequencing to characterise fungal communities, we disentangled the influences of geographic origin (estate/vineyard), grape variety, and (spontaneous) fermentation stage on the diversity, structure, and composition of the fungal communities. Yeast populations were isolated during spontaneous wine fermentation and taxonomically identified, and the S. cerevisiae populations differentiated using microsatellite analysis. To identify the volatiles that differentiated the wine estates we used headspace solid-phase microextraction gas-chromatographic mass-spectrometric (HS-SPME–GC–MS) for metabolite profiling of the resultant wines. Associations between fungal communities and wine metabolites were elucidated with partial least squares regression (PLSR) and structural equation model (SEM). We demonstrate that the grape/wine microbiota and metabolites are geographically distinct, identify multiple layers of fungal microbiota that correlate with wine aroma profiles, and demonstrate that distinctive S. cerevisiae exert the most powerful influences on wine quality and style at small geographic scales.