Diversity of the species Brettanomyces bruxellensis:

genotypic and phenotypic study Marta Avramova1, Alice Cibrario1, Emilien Peltier1, Monika Coton2, Emmanuel Coton2, Franck Salin3 , Warren Albertin1, 4, Chris Curtin6, Isabelle Masneuf-Pomarede1, 5

marta.avramova@u-bordeaux.fr

1Univ. Bordeaux, ISVV, Unité de recherche Œnologie EA 4577, USC 1366 INRA, Bordeaux INP, 33140 Villenave d’Ornon, France 2Université de Brest, EA 3882, Laboratoire Universitaire de Biodiversité et Ecologie Microbienne, ESIAB, Technopôle Brest-Iroise, 29280 Plouzané, France 3INRA, UMR Biodiversité Gènes et Ecosystèmes, PlateForme Génomique, 33610 Cestas, France 4ENSCBP, Bordeaux INP, 33600 Pessac, France 5Bordeaux Sciences Agro, 33170 Gradignan, France 6The Australian Wine Research Institute, Glen Osmond, Adelaide, SA, Australia

WHY ARE WE INTERESTED IN BRETTANOMYCES BRUXELLENSIS ? Brettanomyces bruxellensis in wine B. bruxellensis genetic complexity

Brettanomyces bruxellensis (B. bruxellensis) is one of the first causes of wine spoilage. Its development in wine results in a phenol character which often causes consumers’ rejection and therefore economic loss. Nevertheless, there is no efficient method to avoid or prevent contamination.

B. bruxellensis in other beverages

B. bruxellensis is also associated with other fermented products worldwide such as beer, cider, kombucha, tequila, bioethanol and others. While B. bruxellensis is considered as an important contaminating species in some industrial fermentations, it also plays a beneficial role for others such as Belgian lambic beer production.

B. buxellensis species comprises diploid as well as at least two types of triploid strains. There is a diploid core genome and an additional genome in the triploid strains which indicates that at least two hybridization events took place during the history of the species. (Borneman et al., 2014)

ARE B. BRUXELLENSIS FROM VARIOUS SUBSTRATES GENETICALLY DIFFERENT ?

IS THERE A LINK BETWEEN SULPHUR TOLERANCE AND STRAIN GENOTYPE ?

SSR analysis (Simple sequence repeat)

12 microsatellite sequences (Albertin et al., 2014)

Genetic relationship between strains Poppr R Package (Kamvar et al., 2014) Bruvo’s distance (Bruvo et al., 2004)

Neighbour Joining (Paradis et al., 2004)

1241 isolates from diverse substrates and geographical

origins

The strains used in the study were isolated from wine in our laboratory, sent from other laboratories or were already present in CRB oenologie strain collection. Finally, the study was performed with 1231 isolates from 29 countries and 9 different kinds of substrates. DNA extraction was performed from a single fresh colony by treatment with 30 µl of 20 mM NaOH and 99 °C heat for 10 min. After, microsatellite analysis was done by amplifying Simple Sequence Repeat (or SSR) regions as described by Albertin et al., 2014. Microsatellite markers are highly polymorphic and discriminant markers which are suitable for complex populations and therefore suitable for our study of B. bruxellensis. Four additional microsatellites were used in order to improve the robustness of the test. Amplicon sizes were measured by ABI3730 DNA analyzer and GeneMarker software. Then, raw data was treated on R software by the use of Poppr Package (Figure 1).

Figure 1. Materials and Methods

Figure 4: Growth parameters of different B. bruxellensis strains belonging to three different groups indicated on figure 2 : A (8 strains), B (8 strains) et C (17 strains) for growing concentrations of SO2. Kruskal-Wallis test was performed for each parameter and each strain group, the letters a, b, c and d indicate the significantly different average values at 5% threshold .

Small-scale fermentations (see Figure 2) were performed with 33 B. bruxellensis strains belonging to different genetic groups, therefore with different ploidy level and isolated from different substrates. The assay was performed with 4 different concentrations of molecular SO2 (mSO2). Growth and growth parameters were assayed by OD600 measurement and was performed in biological triplicates.

Figure 3. Protocol for evaluating sulphur tolerance

o For Group A the lag phase is slightly but significantly longer

o However, for Group A, once the growth starts, other growth parameters remain the same even at 0.6 mg/l mSO2

Growth in the presence of mSO2 is different between genetic groups

o For Group B and C all growth parameters vary with increasing mSO2 concentrations

o At higher concentrations of mSO2 for Groups B and C, lag phase is longer, growth rate and maximum population are significantly lower

o Group B and C are less tolerant than Group A

Some genetic groups are more tolerant to SO2 than others

B. bruxellensis worldwide

B. bruxellensis have been isolated from all over the world. Therefore, this yeast species has an important impact on different industries (winemaking, brewery, bioethanol, cider and bioethanol production , etc.) all over the word.

References : Albertin, W., Panfili, A., Miot-Sertier, C., Goulielmakis, A., Delcamp, A., Salin, F., Lonvaud-Funel, A., Curtin, C., Masneuf-Pomarede, I., 2014. Development of microsatellite markers for the rapid and reliable genotyping of Brettanomyces bruxellensis at strain level. Food Microbiology 42, 188–195. doi:10.1016/j.fm.2014.03.012; Curtin, C.D., Pretorius, I.S., 2014. Genomic insights into the evolution of industrial yeast species Brettanomyces bruxellensis. FEMS Yeast Res 14, 997–1005. doi:10.1111/1567-1364.12198; Borneman, A.R., Zeppel, R., Chambers, P.J., Curtin, C.D., 2014. Insights into the Dekkera bruxellensis Genomic Landscape: Comparative Genomics Reveals Variations in Ploidy and Nutrient Utilisation Potential amongst Wine Isolates. PLoS Genet 10, e1004161. doi:10.1371/journal.pgen.1004161

Figure 2. Dendrogram representing the genetic relations between 1231 B. bruxellensis strains

The population structure visualized by the dendrogram on figure below suggests that :

Based on the microsatellite analysis, 33 strains from different genetic groups were used in order to perform phenotypic analysis (below) : Group A : triploid strains mostly form wine Group B : distant triploid strains mostly from beer Group C : diploid strains from wine and other substrates

Strains cluster according to the substrate they were isolated

from (beer, wine,

kombucha, etc.)

Strains cluster according to ploidy level

(2n and 3n on Figure 2)

Conclusions : B. bruxellensis strains used in this study cluster according to ploidy level and substrate they were isolated from. There is a phenotypic diversity at intraspecific level -> strains from Group A are more tolerant to mSO2 than strains from Group B and C.

Next : Comparative genomics of the strains used in the phenotypic study will be performed and competition experiments will be held with strains from different genetic groups.