Molecular Diagnosis of Brettanomyces bruxellensis' Sulfur Dioxide Sensitivity Through Genotype Specific Method

Abstract

The yeast species Brettanomyces bruxellensis is associated with important economic losses due to red wine spoilage. The most common method to prevent and/or control B. bruxellensis spoilage in winemaking is the addition of sulfur dioxide into must and wine. However, recently, it was reported that some B. bruxellensis strains could be tolerant to commonly used doses of SO₂. In this work, B. bruxellensis response to SO₂ was assessed in order to explore the relationship between SO₂ tolerance and genotype. We selected 145 isolates representative of the genetic diversity of the species, and from different fermentation niches (roughly 70% from grape wine fermentation environment, and 30% from beer, ethanol, tequila, kombucha, etc.). These isolates were grown in media harboring increasing sulfite concentrations, from 0 to 0.6 mg.L^-1 of molecular SO₂. Three behaviors were defined: sensitive strains showed longer lag phase and slower growth rate and/or lower maximum population size in presence of increasing concentrations of SO₂. Tolerant strains displayed increased lag phase, but maximal growth rate and maximal population size remained unchanged. Finally, resistant strains showed no growth variation whatever the SO₂ concentrations. 36% (52/145) of B. bruxellensis isolates were resistant or tolerant to sulfite, and up to 43% (46/107) when considering only wine isolates. Moreover, most of the resistant/tolerant strains belonged to two specific genetic groups, allowing the use of microsatellite genotyping to predict the risk of sulfur dioxide resistance/tolerance with high reliability (>90%). Such molecular diagnosis could help the winemakers to adjust antimicrobial techniques and efficient spoilage prevention with minimal intervention.

Keywords: Brettanomyces bruxellensis; resistance; spoilage yeast; sulfur dioxide; tolerance; wine.

FIGURE 1

Dendrogram tree showing the 145 phenotyped B. bruxellensis isolates. The dendrogram tree includes 1488 isolates, and was built using 12 microsatellite markers, Bruvo’s distance, and NJ clustering, as described previously (Avramova et al., 2018). The 145 isolates used in this work are represented by colored circles. The six different colors correspond to the main genetic groups identified and were named from one isolate (e.g., L0308-like means genetic group close to L0308 strain).

FIGURE 2

Examples of B. bruxellensis sensitive, tolerant, and resistant behavior at four mSO₂ concentrations. Strains B002-14 T14 7, 12AVB1, and 2OT14_02 represent sensitive strains. VP1545, AWRI 1606, and AWRI 1605 are tolerant isolates and VP1503, Merlot_329_CM_1, and L0615 are examples of resistant strains. Each curve is built using the mean of three to four replicates, and error bars represent standard deviations and curve colors correspond to increasing SO₂ concentration (light pink 0 mg/L mSO₂ to dark pink 0.6 mg/L mSO₂). The estimated growth parameters (lag phase, maximal growth rate, and maximal OD) are shown below each curve, with mean ± standard deviation.

FIGURE 3

Violin plots for three growth parameters and six genetic groups of B. bruxellensis. Three growth parameters were represented: lag phase (h), maximum growth rate (division per hour), and maximum OD (600 nm). For each genetic group, numeric values (corresponding to the different strains) are represented as diamonds, the corresponding probability densities are represented as plain traits, means, and standard errors are represented by black circles and segments, respectively. Increasing SO₂ concentrations are represented by the same coloring (pink shades, light pink corresponding to 0 mg/L and darker color representing increasing SO₂ concentrations) as in Figure 2. The plots were obtained using ggplot2 package (R). Top letters represent significance groups as defined by Kruskal–Wallis test (agricolae package, p-value < 0.05). Absence of top letters indicates non-significantly different sulfur conditions.