Abstract
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 chromatography-mass 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 resolution-alternating 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.
Highlights
Microbial antagonists have been widely explored as more environmentally friendly disease management alternatives to reduce the excessive use of synthetic fungicides[1,2]
A. pullulans A1 Volatile organic compounds (VOCs) resulted in the highest inhibition of B. cinerea TN080, whereas B. cinerea DAR69764 and A. alternata were highly inhibited by A. pullulans A2 VOCs
Among the four A. pullulans isolates examined in this study, A. pullulans A3 headspace inhibited the growth of B. cinerea TN080, B. cinerea DAR69764 and A. alternata the least,(Fig. 2a–c)
Summary
Microbial antagonists have been widely explored as more environmentally friendly disease management alternatives to reduce the excessive use of synthetic fungicides[1,2]. All four isolates have been previously www.nature.com/scientificreports proven to have antifungal properties against Greeneria uvicola in a dual culture system which reduced the radial growth of the pathogen compared to controls[8]. Their ability to produce VOCs has not been investigated. The current study proposes a method to introduce an IS to the antagonist-pathogen culture system without disturbing the headspace and thereby perform quantitative sampling of VOCs. Multivariate chemometrics which uses a combination of mathematical and statistical approaches[21], have become important in data resolution obtained from analytical platforms such as gas chromatography-mass spectrometry (GC-MS)[22]. Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS), one of the popular chemometric methods used in the resolution of multiple component responses[23], was employed in this study in an association with untargeted, SPME-GC-MS analysis, to characterise A. pullulans VOCs
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