Understanding Brettanomyces and its adaptation to control measures
Brettanomyces yeast cause wine spoilage by producing 4-ethylphenol and 4-ethylguiacol which are responsible for ‘phenolic’, ‘leather’, ‘sweaty’ and ‘medicinal’ aromas (collectively known as ‘Brett’ character). Previous AWRI research has shown that it is possible for sulfite-resistant Brettanomyces strains to evolve and develop even greater levels of sulfite tolerance (when subjected to directed evolution under laboratory conditions), although the genetic basis for this adaptive response remains to be determined.
New molecular tools, including genetic transformation and gene knockout technology have recently been developed, and these now provide a powerful means to assist in the understanding of the evolution of Brettanomyces both in the laboratory and in the field.
This project will therefore extend the results of previous work by combining a new field survey of Brettanomyces (using both high-throughput phenotyping and whole genome sequencing to determine if further adaptive responses are occurring in the winery environment), with detailed molecular analysis of the genes responsible for resistance to sulfite and the production of the key sensory compounds responsible for Brettanomyces spoilage character (4-ethyl phenol (4-EP) and 4-ethyl guaiacol (4-EG)).
Sulfur dioxide tolerance of new industry isolates
A key question for the Australian wine industry is whether Brettanomyces may be developing tolerance to SO2, as this would severely constrain current control strategies. Previous industry-based population surveys in the early 2000s showed that the strains of Brettanomyces with the highest levels of SO2 resistance were most frequently isolated from Australian wineries (Curtin et al. 2007, Curtin et al. 2012). While this original study alerted Australian winemakers to the importance of SO2 management for Brettanomyces spoilage control, current trends towards higher pH wines and the minimisation of SO2 may provide conditions for Brettanomyces to develop further tolerance.
Initial studies conducted using historical industry isolates sourced from the AWRI Wine Microorganism Culture Collection (including those isolated during the 2007 study by Curtin et al.) and industry isolates sourced from two industry partners in 2016 and 2017 pointed to a shift towards increased SO2 tolerance. Further sampling over 2018 (now from more than ten wineries) indicated a definite shift in SO2 tolerance, with the 2016-2018 average tolerance approximately equal to the highest tolerances observed in isolates from 2000-2004 (Figure 16). Genetic testing will now be used to determine if these more tolerant isolates represent existing known genotypes that are evolving increased tolerance or new genotype(s) not observed previously.
Curtin, C.D., Bellon, J.R., Henschke, P.A., Godden, P.W., de Barros Lopes, M.A. 2007. Genetic diversity of Dekkera bruxellensis yeasts isolated from Australian wineries. FEMS Yeast Res. 7(3): 471–481.
Curtin, C., Kennedy, E., Henschke, P.A. 2012. Genotype-dependent sulphite tolerance of Australian Dekkera (Brettanomyces) bruxellensis wine isolates. Lett. Appl. Microbiol. 55(1): 56-61.