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The use of yeast-derived volatile organic compounds (VOCs) represents a promising strategy for the biological control of various plant pathogens, including mycotoxin-producing fungi. Previous studies demonstrated the efficacy of the low-fermenting yeast Candida intermedia isolate 253 in reducing growth, sporulation, and ochratoxin A biosynthesis by Aspergillus carbonarius MPVA566. This study aimed to investigate whether the inhibitory effect of the yeast volatilome is solely attributable to 2-phenylethanol, its major component, or if a synergistic effect of all volatilome components is required to achieve an effective control of the fungal growth and metabolism. Microbiological methods, HPLC measurements and a UPLC-MS/MS approach were used to investigate the metabolic profile of A. carbonarius MPVA566 at different growing conditions: standard incubation (control), exposed to C. intermedia 253 volatilome, and incubation in the presence of 2-phenylethanol. Both yeast volatilome and 2-phenylethanol succeeded in the macroscopic inhibition of the radial mycelial growth, along with a significant reduction of ochratoxin A production. Functional classification of the fungal proteome identified in the diverse growing conditions revealed a different impact of both yeast VOCs and 2-phenylethanol exposure on the fungal proteome. Yeast VOCs target an array of metabolic routes of fungal system biology, including a marked reduction in protein biosynthesis, proliferative activity, mitochondrial metabolism, and particularly in detoxification of toxic substances. Exposure to 2-phenylethanol only partially mimicked the metabolic effects observed by the whole yeast volatilome, with protein biosynthesis and proliferative activity being reduced when compared with the control samples, but still far from the VOCs-exposed condition. This study represents the first investigation on the effects of yeast-derived volatilome and 2-phenylethanol on the metabolism of a mycotoxigenic fungus by means of proteomics analysis. Chemical compounds studied or used in this article: 2-Phenylethanol (PubChem CID: 6054); ochratoxin-A (PubChem CID: 442530); sodium dodecyl sulfate (PubChem CID: 3423265); dithiothreitol (PubChem CID: 446094); phenylmethylsulfonyl fluoride (PubChem CID: 4784); iodoacetamide (PubChem CID: 3727); ammonium bicarbonate (PubChem CID: 14013); acetic acid (PubChem CID: 176); and acetonitrile (PubChem CID: 6342). - 2019 The Authors This publication was made possible by NPRP grant # 8-392-4-003 from the Qatar National Research Fund (a member of Qatar Foundation). The findings achieved herein are solely the responsibility of the authors. Scopus

Many foods and beverages in temperate and tropical regions are prone to contamination by ochratoxin A (OTA), one of the most harmful mycotoxins for human and animal health. Aspergillus ochraceus and Aspergillus carbonarius are considered among the main responsible for OTA contamination. We have previously demonstrated that four low or non- fermenting yeasts are able to control the growth and sporulation of OTA-producing Aspergilli both in vitro and on detached grape berries: the biocontrol effect was partly due to the release of volatile organic compounds (VOCs). Aiming to further characterise the effect of VOCs produced by biocontrol yeast strains, we observed that, beside vegetative growth and sporulation, the volatile compounds significantly reduced the production of OTA by two A. carbonarius and A. ochraceus isolates. Exposure to yeast VOCs also affected gene expression in both species, as confirmed by downregulation of polyketide synthase, non-ribosomal peptide synthase, monooxygenase, and the regulatory genes laeA and veA. The main compound of yeast VOCs was 2-phenylethanol, as detected by Headspace-Solid Phase Microextraction-Gas Chromatography-Tandem Mass Spectrometry (HS-SPME-GC?MS) analysis. Yeast VOCs represent a promising tool for the containment of growth and development of mycotoxigenic fungi, and a valuable aid to guarantee food safety and quality. This publication was made possible by NPRP grant # 8-392-4-003 from the Qatar National Research Fund (a member of Qatar Foundation). The findings achieved herein are solely the responsibility of the authors. We acknowledge constructive comments provided by anonymous reviewers. Scopus

This review summarises the genetic methods used for chemotype determination of the main Fusarium type B-trichothecene producing species. Literature on Fusarium chemotype epidemiology over the last 15 years is reviewed in order to describe temporal and spatial chemotype distribution of these fungi worldwide. Genetic approaches used for chemotype determination are also reviewed and discussed, highlighting successes and potential pitfalls of the technique. Results from both genetic and chemical approaches are summarised to compare reliability, advantages and limitations of the two methods. Potential applications of genetic chemotyping to toxigenic Fusarium species are evaluated in the light of improving food safety of agricultural products. The use of chemotype determination in population studies, toxin prediction as well as for breeding purpose is described.

Aspergillus spp. infection of grape may lead to ochratoxin A (OTA) contamination in processed beverages such as wine and grape juice. The aim of the current study was to evaluate the biocontrol potential of two non-fermenting (Cyberlindnera jadinii 273 and Candida friedrichii 778) and two low-fermenting (Candida intermedia 235 and Lachancea thermotolerans 751) yeast strains against the pathogenic fungus and OTA-producer Aspergillus carbonarius, and their ability to remove OTA from grape juice. Two strains, 235 and 751, showed a significant ability to inhibit A. carbonarius both on grape berries and in in vitro experiments. Neither their filtrate nor their autoclaved filtrate culture broth was able to prevent consistently pathogen growth. Volatile organic compounds (VOCs) produced by all four selected yeasts were likely able to consistently prevent pathogen sporulation in vitro. VOCs produced by the non-fermenting strain 778 also significantly reduced A. carbonarius vegetative growth. Three yeast strains (235, 751, and 778) efficiently adsorbed artificially spiked OTA from grape juice, while autoclaving treatment improved OTA adsorption capacity by all the four tested strains. Biological control of A. carbonarius and OTA-decontamination using yeast is proposed as an approach to meet the Islamic dietary laws concerning the absence of alcohol in halal beverages.

The aim of this study was to test the suitability of the RT-PCR (reverse transcription-polymerase chain reaction) technique to differentiate aflatoxin-producing from aflatoxin-non-producing strains of Aspergillus flavus and Aspergillus parasiticus. Total RNAs of 13 strains grown under inducing yeast extract-sucrose (YES) and non-inducing yeast extract-peptone (YEP) media, respectively, were analyzed by using specific primers based on the conserved regions of nine structural genes (aflD, aflG, aflH, aflI, aflK, aflM, aflO, aflP, and aflQ) and two regulatory genes aflS and aflR of the aflatoxin B1 biosynthetic pathway. Transcription was confirmed by the expression of the beta-tubulin gene. The expression of the majority aflatoxin biosynthetic genes including aflR and aflS of all strains varied with regard to the aflatoxin-producing ability and the growth conditions. Nonetheless, we found that the expression profile of the three genes aflD, aflO, and aflP was consistently correlated with a strain's ability to produce aflatoxins or not in YES as well as the inability to produce aflatoxins in YEP. The devised RT-PCR profiling method reflects aflatoxin concentrations ranging from 0.1 to 60 microg/ml of the culture filtrates as determined by fluorescence HPLC. The results are discussed in relation to the suitability of RT-PCR as well as cDNA-based array techniques in diagnostic laboratory settings where individual isolates are being tested for potential toxin production to identify toxigenic isolates of Aspergillus species.