The Aureobasidium pullulans volatile metabolome and potential antifungal activity against Botrytis cinerea and Alternaria alternata

Plant infections by Botrytis cinerea and Alternaria alternata remain a challenge to horticulture as they cause the devastating fungal diseases of grey and black mould in many crop plants worldwide. The emergence of fungicide resistance, concerns of fungicide persistence in soil and implications for public health due to residues in the environment, have led to the creation of a demand for alternatives to traditional chemically based fungicides to manage pathogenic fungi. Research on biological control agents has been carried out extensively as it provides a potential eco-friendly and effective pathogen suppression tool. Microbial volatile organic compounds (VOCs) with antifungal properties produced by some of these potential biocontrol agents have gained substantial interest as alternatives to chemically based fungicides. This is largely due to perceptions of low toxicity, biodegradability, a reduced possibility of the target pathogen developing resistance to the VOCs and activity not requiring a physical contact with the targeted host.
The yeast-like fungus Aureobasidium pullulans occurs naturally on the phyllosphere of many plant species and is among the microorganisms that could potentially be used as biocontrol agents for plant disease management. This yeast is known for its antifungal properties against various plant pathogenic fungi. Production of VOCs with antifungal properties is one of the multiple mechanisms exhibited by A. pullulans that limit the growth of fungi, including B. cinerea. Little information is available regarding its antifungal volatilome. Previous research involving qualitative studies on the A. pullulans volatilome has confirmed the identity of four antifungal VOCs: 2-methyl-1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol and 2-phenylethanol. However, there is no quantitative information on the chemical composition, expression of antifungal VOCs in response to abiotic factors or on the antifungal mechanisms associated with the A. pullulans volatile metabolome, which limit the efficacy of application of A. pullulans as an antifungal agent utilising its VOCs production. This project was designed to investigate the volatile metabolome of A. pullulans and determine its antifungal properties and associated mechanisms against B. cinerea and A. alternata in vitro.
A double Petri dish method was deployed to assess the suppression of colony growth and conidia germination of B. cinerea and A. alternata by A. pullulans culture headspace. A novel experimental approach that enabled the addition of an internal standard (IS) was devised to directly extract and quantify VOCs from antagonist-pathogen culture headspace using solid-phase microextraction-gas chromatography mass spectrometry (SPME-GC-MS). An untargeted metabolomics approach followed by chemometric analysis identified fourteen VOCs from A. pullulans volatilome. Amongst these, 3-methyl-1-hexanol, acetone, 2-heptanone, ethyl butyrate, 3-methylbutyl acetate and 2-methylpropyl acetate were detected for the first time in the A. pullulans volatile metabolome. Four VOCs: ethanol, 2-methyl-1-propanol, 3-methyl-1-butanol and 2-phenylethanol, were identified by partial least squares-discriminant analysis models as variables with high explanatory power for discrimination between A. pullulans headspace from A. pullulans non-inoculated headspace using variable importance in projection and selectivity ratios. Of these compounds, ethanol (397-524 mg/L) was the most concentrated VOC in the A. pullulans-pathogen interaction headspace. The other three alcohols were present in concentrations in the range of 1.0-3.6 mg/L in the A. pullulans-pathogen interaction headspace. These four compounds were modelled using response surface methodology (RSM) following a Box-Behnken experimental design, to evaluate the combined effects of VOCs to exert an antifungal activity. The results suggested that ethanol and 2-phenylethanol are the key inhibitory A. pullulans VOCs against both B. cinerea and A. alternata.
Production of microbial VOCs is affected by several abiotic factors associated with microbial growth. A Box-Behnken experimental design followed by RSM was deployed to explore the combined effect of nutrient uptake with special focus on carbon and nitrogen, along with temperature, on the production of the four important antifungal VOCs identified from the A. pullulans volatilome. Design of experiments approach and RSM enabled an efficient modelling process by drastically reducing the number of experiments required. Response surface modelling of peak areas of the four VOCs from SPME-GC-MS analysis normalised to IS and A. pullulans biomass, revealed that the initial carbon content exerts the greatest significant influence on the synthesis of the targeted VOCs including the three higher alcohols: 2-methyl-1-propanol, 3-methyl-1-butanol and 2-phenylethanol. These results suggested a dominant involvement of an anabolic pathway to produce the targeted A. pullulans VOCs, where α-keto acids are formed via the de novo biosynthesis of amino acids through carbohydrate metabolism. Further, isolate dependent response variations to all three parameters were observed.
The mechanisms behind the antagonistic interactions between A. pullulans VOCs-B. cinerea or A. alternata were investigated with regards to the accumulation of reactive oxygen species (ROS) and electrolyte leakage of the two pathogens upon exposure to A. pullulans VOCs. A mixture of A. pullulans VOCs: ethanol, 2-methyl-1-propanol, 3-methyl-1-butanol and 2-phenylethanol triggered electrolyte leakage from the mycelia of both pathogens. Fluorescence microscopic analysis indicated an increased accumulation of ROS, particularly the superoxide radical, in pathogen mycelia when exposed to A. pullulans VOCs. It was hypothesised that the enzyme complex I of the mitochondrial respiratory chain (MRC) as a target site for A. pullulans VOCs. To study this, the enzymes of the MRC complex I of B. cinerea and A. alternata were partially inhibited by pre-treatment with rotenone prior to exposure to A. pullulans headspace or a mixture of VOCs from A. pullulans. This pre-treatment reduced fluorescence intensity of mycelia exposed to the VOCs indicating reduced ROS accumulation in pathogen mycelia and increased fungal growth equivalent to that of the control samples, suggesting reduced susceptibility of the two pathogens to A. pullulans VOCs. Scanning electron micrographs revealed altered cell wall structures in exposed B. cinerea and A. alternata mycelia.
The results of this PhD study suggest that isolates of A. pullulans that produce efficacious levels of antifungal VOCs, could potentially serve as biocontrol agents against B. cinerea and A. alternata. This was the first study to introduce a robust quantitative approach for microbial VOCs for SPME-GC-MS, which can be used for screening microbial isolates for VOCs production. It was also the first study to investigate MRC enzyme complexes as potential target sites for antifungal microbial VOCs. Insights from this multidisciplinary research provide knowledge that will assist in quantitative analysis of microbial VOCs in antagonist-pathogen interaction systems, strain selection, elucidation of antifungal mechanisms of microbial VOCs and efficient analysis of combined effects of factors affecting microbial VOCs biosynthesis.