YEAST-PRODUCED VOLATILES IN GRAPE BASED SYSTEM MODEL ACTING AS ANTIFUNGAL BIOAGENTS AGAINST PHYTOPATHOGEN BOTRYTIS CINEREA

Abstract

Botrytis cinerea Pers., the causal agent of grey mould disease, is responsible for substantial economic losses, as it causes reduction of grape and wine quality and quantity. Exploitation of antagonistic yeasts is a promising strategy for controlling grey mould incidence and limiting the usage of synthetic fun- gicides. In our previous studies, 119 different indigenous yeasts were screened for putative multidi- mensional modes of action against filamentous fungus B. cinerea [1]. The most promissing biocontrol yeast was Pichia guilliermondii ZIM624, which exhibited several anatagonistic traits (production of cell wall degrading enzymes, chitinase and β-1,3-glucanase; demonstration of in vitro inhibitory effect on B. cinerea mycelia radial growth; production of antifungal volatiles, assimilation of a broad diversity of carbon sources, contributing to its competitivnes in inhabiting grapes in nature). In addition, P. guilliermondii ZIM 624 possessed interesting enological traits, did not produce off-flavor related H2S and appeared as β-lyase and β-glucosidase producer.

Accordingly, the aim of this researsch was to study the antifungal mechanisms by assessing the volatiles produced by P. guilliermondii ZIM624. Namely, a study was conducted to identify volatile organic com- pounds (4 higher alcohols, 6 volatile phenols, 23 esters and 27 terpenes) produced by antagonistic Pichia guilliermondii strain ZIM624 and to determine the efficacy of the chosen volatiles of P. guilliermondii in suppression of B. cinerea growth and control of Botrytis fruit rot of grape berries. Thereby, a comprehensive assessment of produced volatiles in the process of wine production was achieved using two validated analytical methods (one for terpenes and one for the rest of mentioned volatiles) comprised of automated headspace (HS) solid-phase microextraction (SPME) and gas chromatography coupled with mass spectrometric detection (GC-MS). Both methods were developed based on already published me- thod for determionation of volatiles in wine samples [2]. Among identified volatiles, 13 yeast-produced volatiles were selected and their antifungal activity was tested against B. cinerea in the fumigation bioassay. Terpenes citronellol, geraniol, nerol, α-terpineol and linalool were the most effective against B. cinerea mycelium growth with the EC50 beetwen 6,6 to 32,8 μL/L. 4-Vinyl phenol and isoamyl ace- tate also effectively inhibited mycelial growth of B. cinerea, EC50 being 48,6 and 63,3 μL/L, respectively, followed by eucalyptol (EC50 201,6 μL/L) and ethyl butyrate (EC50 238,4 μL/L). 4-Vinyl guaiacol did not show any inhibitory effect, while the remaining tested compounds showed inhibition against B. cinerea growth, however we were not able to determine EC50 with the selected concentration ranges. Additio- naly, exposure of B. cinerea-infected grape berries to the volatiles from P. guilliermondii cultures also lowered the number of infected grape berries, when applied to in vivo assay.