A proteomic investigation of Aspergillus carbonarius exposed to yeast volatilome or to its major component 2-phenylethanol reveals major shifts in fungal metabolism

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 AuthorsThis 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.Scopu

Dietary L-carnitine and vitamin-E; a strategy to combat ochratoxin-A induced immunosuppression

This study aimed to evaluate the effect of dietary ochratoxin A (OA), in the presence and absence of L-carnitine (LC) and vitamin E (VE), on the humoral immune responses of White Leghorn cockerels (WLC). One-day old white male Leghorn chicks were divided into 12 groups, having 20 birds each and were offered ration contaminated with OA (1.0 or 2.0 mg/kg feed) alone and concurrently with LC (1.0 g/kg) and/or VE (0.2 g/kg), for 42 days. The humoral immune responses were accessed by lymphoproliferative response to avian tuberculin, in-vivo phagosomes activity to carbon particles and antibody response to the sheep red blood cells (SRBCs). The dietary addition of OA alone suppressed the humoral immune responses, however, the exposure of birds to 1.0 mg/kg OA in the presence of LC and/or VE showed a significant reduction in OA induced immunotoxicity. This protective response was absent in the birds fed 2.0 mg/kg OA in the presence and absence of LC and/or VE. Histopathological and morphometric examination of the bursa of Fabricius exhibited a decrease in the severity and frequency of OA induced lesions in the presence of dietary LC and/or VE. The use of LC and VE as dietary supplement, can effectively overcome OA (≤1.0 mg/kg) induced immunosuppression.This work has been extracted from the M. Phil thesis research in toxicopathology laboratory of Department of Pathology Faculty of Veterinary Science University of Agriculture Faisalabad Pakistan. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors

Impact of dietary Trichosporon mycotoxinivorans on ochratoxin A induced immunotoxicity; In vivo study

Ochratoxin A (OA), the secondary metabolite of certain Aspergillus and Penicillium species, is one of the potent biological immune-suppressor. The present study was designed to explore the in-vivo efficacy of Trichosporon mycotoxinivorans (TR); yeast strain isolated from the hindgut of the termite Mastotermes darwiniensis, against the immunotoxicity of OA in broiler birds. For this purpose, broiler chicks were offered diet added with TR (0.5, 1.0 or 2.0 g/kg feed) and/or OA (0.15, 0.3 or 1.0 mg/kg feed) for 42 days. Dietary OA at all levels, resulted in significant reduction (p ? 0.05) in the immune response of broiler birds as recorded by vacuolation and darkly stained pyknotic nuclei in bursa of Fabricius and thymus, humoral immune responses to sheep red blood cells (SRBC), in-vivo lymphoproliferative response to Phytohemagglutinin-P (PHA-P) and mononuclear phagocytic system function assay. Addition of TR in broiler diet significantly reduced (p ? 0.05) the immunotoxicity of OA at 0.15 and 0.30 mg/kg; however, against higher dietary level of OA (1.0 mg/kg), a partial protection was observed. Feeding TR alone had no immunomodulatory effect at any of tested level. Dietary addition of TR is proposed as an approach to combat the OA mediated immunological damages in broiler birds. - 2019The authors highly acknowledge the Higher Education Commission of Pakistan for funding this study to first author under HEC Indigenous PhD fellowship program batch VII. Appendix AScopu

Detection of toxigenic mycobiota and mycotoxins in cereal feed market

The present study was designed to investigate the occurrence of mycotoxins and toxigenic mycobiota in cereals used as animal feed in a Qatari market. Aflatoxins (AFs) and ochratoxin A (OTA) were detected in 94 and 44% of mixed cereals, 70 and 40% of maize, 40 and 60% of wheat and wheat bran samples, respectively. Among these positive samples, 20 and 14% (of mixed cereals), 29 and 50% (of maize) were found to be contaminated with levels higher than the European Union (EU) permissible limits for AFs and OTA, respectively. The co-contamination analysis showed that 44, 40 and 50% of the mixed cereals, maize and barley samples were concurrently contaminated with AFs and OTA. In all the tested samples, Aspergillus was the most predominant genus with isolation frequency ranging from 67 to 100%. Likewise, the relative density (occurrence compared to the other genera) of Aspergillus was 42–71% in the different cereals samples. Mixed cereals showed the maximum fungal load with 3 × 103 colony forming units/g. Based on species-specific PCR primers, among Aspergillus species, A. flavus, A. parasiticus A. niger, A. carbonarius, A. ochraceus and A. westerdijkiae were the most predominant. Most of the isolates presented the key genes for either the aflatoxigenic (omt-1, ver-1 and apa-2) or ochratoxigenic (pks) biosynthesis pathways. In spite of possessing mycotoxigenic genes, 10, 20, 17 and 50% of A. flavus, A. parasiticus, A. carbonarius and A. niger isolates, respectively, did not produce any AFs or OTA in culture media. Our findings demonstrate that cereals used as animal feeds are generally contaminated with variable levels of mycotoxigenic fungi and mycotoxins, with maximum levels being present in mixed cereals and maize samples.This publication was made possible by NPRP grant # NPRP8-392-4-003 from the Qatar National Research Fund (a member of Qatar Foundation). The statements made herein are solely the responsibility of the authors. Appendix AScopu

Prevalence of Fusarium fungi and their toxins in marketed feed

This study aimed to explore the incidence of Fusarium fungi and their mycotoxins in marketed feed in Qatar. Samples of mixed-grain-cereal, maize, wheat, millet, barley, oat and soybean were tested for the presence of zearalenone (ZEN), fumonisins, T2/HT2 and deoxynevalenol (DON)mycotoxins. PCR-based identification of the Fusarium isolates was followed by investigation into genes involved in trichothecenes (tri gene cluster), fumonisins (fum gene cluster)and ZEN (pks gene)biosynthesis. In-vitro mycotoxin production potential of the isolates was compared with their molecular profiles. Mixed-grain cereals showed highest contamination with Fusarium, followed by maize and wheat. The incidence of ZEN was highest in mixed grain samples (87.5%)followed by maize (50%), millet (42.85%)and wheat (40%). In case of DON; 62.5%, 30%, 40%, 14.28%, 50% and 66.67% of the tested mixed cereal grain, maize, wheat, millet, barley and soybean samples were positive. Based on PCR results, F. verticillioides showed highest frequency distribution (34%), followed by F. graminearum (16%), F. oxysporum (15%), F. proliferatum (13%), F. culmorum (8%), F. solani (7%), F. subglutinans (4%)and F. avenaceum (3%)in the feed cereals. All the isolates of F. verticillioides presented both fum1 and fum13 genes, and produced fumonisin mycotoxins in-vitro. All the isolates of F. graminearum and F. culmorum had the tri5 and tri6 genes and resulted in DON accumulation in rice media. In addition to DON, all the F. culmorum isolates produced ZEN in artificially contaminated media, in line with their molecular profile of having tri5, tri6 and pks13 genes. The findings of present study confirmed the occurrence of toxigenic Fusarium and their mycotoxins in animal feed, all below the maximum limit set by EU. Further, the in-vitro mycotoxin production potential of the isolates was in agreement with their tri, fum and pks genetic profiles. - 2019 Elsevier LtdThis publication was made possible by the NPRP Grant '8-392-4-003 ' from the Qatar National Research Fund (a member of the Qatar Foundation ). The statements made herein are solely the responsibility of the authors. Appendix AScopu

Evidence of low levels of aflatoxin M1 in milk and dairy products marketed in Qatar

This study was designed to explore the incidence of aflatoxin M1 (AFM1) in milk and dairy products marketed in Qatar. Milk (n = 72), yogurt (n = 21), cheese (n = 46), butter (n = 18) and laban (n = 25) samples were initially screened by competitive enzyme linked immunosorbent assay (ELISA) for the presence of AFM1, followed by confirmation with ultra-high-performance liquid chromatography (UHPLC). AFM1 was detected in 85%, 76%, 85%, 67% and 76% of the milk, yogurt, cheese, butter and laban samples, respectively. The levels of AFM1 in pasteurized vs ultra-high temperature (UHT) milk and full-vs low-fat yogurts showed a non-significant difference. Halloumi and Kashkaval cheese varieties showed a significantly higher AFM1 contents as compared to Mozzarella, Edam, Cheddar, cream and Moshalal cheese. Likewise, unsalted butter demonstrated significantly (p < 0.05) higher AFM1 values than salted butter samples. None of the tested samples presented AFM1 levels above the EU maximum limits of 50 ng/L or kg for milk, yogurt and butter, and 250 ng/kg for cheese. From the finding of present study, it can be concluded that, although high percentage of dairy products marketed in Qatar demonstrated AFM1 contents, but do not represent a public health concern considering the EU maximum limits.This publication was made possible by NPRP grant # NPRP8-392-4-003 from the Qatar National Research Fund (a member of Qatar Foundation). The statements made herein are solely the responsibility of the authors

Effect of yeast volatile organic compounds on ochratoxin A-producing Aspergillus carbonarius and A. ochraceus

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.Scopu