Antifungal activity of non-conventional yeasts against Botrytis cinerea and non -Botrytis grape bunch rot fungi

Abstract

Grapes harbour a plethora of non-conventional yeast species. Over the past two decades, several of the species have been extensively characterised and their contribution to wine quality is better understood. Beyond fermentation, some of the species have been investigated for their potential as alternative biological tools to reduce grape and wine spoilage. However, such studies remain limited to a few genera. This work aimed to evaluate the antagonistic activity of grape must-derived non-conventional yeasts against Botrytis cinerea and non-Botrytis bunch-rotting moulds and to further elucidate mechanisms conferring antifungal activity. A total of 31 yeast strains representing 21 species were screened on different agar media using a dual culture technique and liquid mixed cultures, respectively. Pichia kudriavzevii was the most potent with a minimum inhibitory concentration of 10² cells/mL against B. cinerea but it had a narrow activity spectrum. Twelve of the yeast strains displayed broad antagonistic activity, inhibiting three strains of B. cinerea (B05. 10, IWBT FF1 and IWBT FF2), a strain of Aspergillus niger and Alternaria alternata. Production of chitinases and glucanases in the presence of B. cinerea was a common feature in most of the antagonists. Volatile and non-volatile compounds produced by antagonistic yeast strains in the presence of B. cinerea were analysed and identified using gas and liquid chromatography mass spectrometry, respectively. The volatile compounds identified belonged mainly to higher alcohols, esters, organosulfur compounds and monoterpenes while the non-volatile compounds were cyclic peptides and diketopiperazine. To our knowledge, this is the first report to demonstrate inhibitory effect of the non-volatile compounds produced by various yeast species.

Keywords: Wickerhamomyces anomalus; antagonistic yeasts; biological control; cell wall lytic enzymes; volatile organic compounds.

Figure 1

A representation of the dual culture assay showing yeast antifungal activity against Botrytis cinerea and how the inhibition was measured. R_c represents the longest distance of fungal mycelium growth from the inoculated fungal plug and R_exp is the horizontal distance from the inoculated fungal plug towards the yeast colony.

Figure 2

Headspace vial with YPD-L agar for automated sampling of volatile organic compounds produced by antagonistic yeasts in the presence of Botrytis cinerea IWBT FF1.

Figure 3

Yeast inhibition of Botrytis cinerea B05. 10, IWBT FF1 and IWBT FF2 mycelial growth. Bars represent average inhibitions of three replicates with standard deviation. Different superscript letters (^{a, b, c}) show significant differences according to Tukey and Dunnett test (p <  0.05) when comparing the mycelium inhibition of each B. cinerea strain by different yeasts. Strains with no superscript letters indicate those with no significant difference.

Figure 4

Relative fold changes coloured from blue (lowest) to yellow (highest) of volatile organic compounds produced by Botrytis cinerea (control) and yeast strains co-cultured with various yeasts strains. Compounds were identified using Anisole d8 (Std), comparison with mass spectra from MS NIST05 spectral library.

Figure 5

Principal component analysis score plots obtained from the non-volatile organic compounds profile of Botrytis cinerea and Botrytis cinerea co-cultured with various yeast species.