Volatile organic compounds (VOCs) produced by Aureobasidium pullulans were investigated for antagonistic actions against Alternaria alternata and Botrytis cinerea. Conidia germination and colony growth of these two phytopathogens were suppressed by A. pullulans Vocs. A novel experimental setup was devised to directly extract VOCs using solid-phase microextraction-gas chromatographymass spectrometry (SPME-GC-MS) from antagonist-pathogen culture headspace. The proposed system is a robust method to quantify microbial VOCs using an internal standard. Multivariate curve resolutionalternating least squares deconvolution of SPME-GC-MS spectra identified fourteen A. pullulans Vocs. 3-Methyl-1-hexanol, acetone, 2-heptanone, ethyl butyrate, 3-methylbutyl acetate and 2-methylpropyl acetate were newly identified in A. pullulans headspace. Partial least squares discriminant analysis models with variable importance in projection and selectivity ratio identified four VOCs (ethanol, 2-methyl-1-propanol, 3-methyl-1-butanol and 2-phenylethanol), with high explanatory power for discrimination between A. pullulans and pathogen. The antifungal activity and synergistic interactions of the four VOCs were evaluated using a Box-Behnken design with response surface modelling. Ethanol and 2-phenylethanol are the key inhibitory A. pullulans VOCs against both B. cinerea and A. alternata. Our findings introduce a novel, robust, quantitative approach for microbial VOCs analyses and give insights into the potential use of A. pullulans Vocs to control B. cinerea and A. alternata.Microbial antagonists have been widely explored as more environmentally friendly disease management alternatives to reduce the excessive use of synthetic fungicides 1,2 . Among a wide array of mechanisms exploited by microbial antagonists, the production of volatile organic compounds (VOCs) with antimicrobial properties 3 has become popular in the recent research due to their low risk of toxic residues, biodegradability 4 and enormous spectrum of activity regardless of a physical contact of the commodity 5 .Aureobasidium pullulans is a yeast-like saprophytic fungus, naturally inhabiting plant and fruit surfaces 6 . This yeast is a well-known biocontrol agent against a range of pathogenic fungi 7,8 and strains of the organism have been formulated as commercial preparations for the practical management of fungal phytopathogens of a range of horticultural crops 9 . Production of antifungal VOCs has been recently identified as a potential mode of action for its biocontrol 10,11 . One of these studies has confirmed the identity of its four VOCs, 2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-1-propanol and 2-phenylethanol 11 . These compounds were shown to suppress conidia germination and mycelium growth of Botrytis cinerea, Colletotrichum acutatum, Penicillium expansum, Penicillium digitatum and Penicillium italicum under both in vitro and in vivo conditions 11 . A similar study identified over 20 VOCs from A. pullulans isolated from grapevine 12 . However, none of these c...

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The combined biochemical impact of carbon, nitrogen and temperature on the biosynthesis of the antifungal volatile organic compounds (VOCs): ethanol, 2-methyl-1-propanol, 3-methyl-1-butanol and 2phenylethanol produced by Aureobasidium pullulans A1 and A3 was investigated using a Box-Behnken experimental design and response surface methodology (RSM). Normalized peak areas derived from solid phase micro extraction-gas chromatography-mass spectrometry (SPME-GC-MS) analysis, indicated that initial carbon content had a significant influence on the biosynthesis of ethanol and alcohols with greater than three carbon atoms. This result suggests a dominant activity of the A. pullulans anabolic pathway to biosynthesize three higher alcohols via de novo biosynthesis of amino acids from sugar metabolism. Low concentrations of carbon (3-13 g l −1 ) with nitrogen as both ammonium and amino acids in the growth medium resulted in a higher number of significant linear and quadratic relationships. Nitrogen availability and growth temperature had significant negative linear and quadratic correlations with VOCs biosynthesis in most instances. Isolate-dependant metabolic response was evident for all abiotic parameters tested on alcohol production. The findings of this study offer new perspectives to improve the production of key antifungal compounds by antagonists in biological control systems.