Modulation of Aflatoxin B1 production by Aspergillus flavus

Mycotoxins are toxic contaminants of foodstuffs produced by a wide range of fungal species. Aflatoxins are the only mycotoxins carcinogenic for humans. They are mainly produced by the Aspergillus genus and can be found at each step of the agrofood chain (e.g. field, storage, process). Due to climate changes, France is starting to be exposed to aflatoxins. In order to limit the consumer exposure, many prevention or decontamination techniques have been developed. To this aim, we started the development of a biocontrol against aflatoxins accumulation for maize field application. Actinomycetes, are soil-borne bacteria that has already been commercialized as biocontrol. In Petri dishes, we studied the in vitro interaction between some actinomycetes and Aspergillus flavus, the main aflatoxins producer. We revealed that the interaction reduced the aflatoxins content (monitored by HPLC). Moreover, some bacterial isolates were able to reduce pure-aflatoxin B1 added in the medium. To understand this mechanism, adsorption tests has been conducted. Otherwise, RT-qPCR methodology was used to study the impact of Streptomyces-Aspergillus sp. on aflatoxin gene expression. Finally, the current knowledge of the impact of environmental factors (temperature, water activity and incubation time) on aflatoxins production was supplemented

Natural Compounds That Modulate the Development of the Fungus Botrytis cinerea and Protect Solanum lycopersicum

Botrytis cinerea is the causal agent of gray mold disease and is responsible for the loss of millions of dollars in crops in worldwide. Currently, this pathogen exhibits increasing resistance to conventional fungicides; therefore, better control methods and novel compounds with a more specific mechanism of action but without biocidal e ects, are required. In this work, several natural compounds to control B. cinerea were analyzed in vitro. Detected e ects were dependent on the stage of fungus development, and 3-phenyl-1-propanol displayed the most potent inhibition of in vitro germination, germ tube development, and sporulation. However, it had lower protection of leaves and postharvest fruit in plant infection. Isoeugenol and 1-phenylethanol exhibited lower inhibition of in vitro germination and sporulation, but at the highest concentrations, they inhibited germ tube elongation. Although the lowest rates of foliage infection were recorded using isoeugenol and 3-phenyl-1-propanol, 1-phenylethanol significantly decreased the disease in postharvest tomato fruit, with an e cacy like Mancozeb, but at 18 times lower micromolar concentration. All compounds resulted in high cell viability after spores were removed from the treatment solution exhibited high cell viability, suggesting a non-biocidal e ect. The diversity of in vitro and in-plant e ects seems to indicate a di erent mechanism of action

Modulation of Aflatoxin B1 production by Aspergillus flavus

Les mycotoxines sont des molécules toxiques produites par de nombreuses espèces fongiques. Les seules mycotoxines avérées aujourd’hui cancérigènes pour l’homme sont les aflatoxines. Elles sont produites par le genre Aspergillus principalement et sont retrouvées tout au long de la chaine alimentaire (champs, stockage, transformation, etc.). A cause du réchauffement climatique, la France devient de plus en plus exposée à la présence de ces mycotoxines. Afin de limiter l’exposition des consommateurs, de nombreuses stratégies de prévention ou de décontamination sont développées. Dans ce contexte, nous avons recherché à mettre au point un système de lutte biologique permettant de prévenir la production d’aflatoxines sur le maïs au champ. Pour cela, nous avons choisi des bactéries issues du sol et déjà connues pour être commercialisées pour la lutte biologique, les actinomycètes. Nous avons étudié l’interaction in vitro sur boites de Pétri entre Aspergillus flavus, principal producteur d’aflatoxines, et certains actinomycètes. Nous avons démontré que l’interaction peut réduire la concentration en aflatoxines mesurée par HPLC. De plus, certains isolats bactériens sont aussi capables de réduire, en culture pure, la concentration d’aflatoxine B1 dans le milieu. Des premiers tests d’adsorption ont été réalisés pour comprendre la nature de ce mécanisme. Par ailleurs, une étude approfondie via RT-qPCR sur 6 souches bactériennes du genre Streptomyces sp. A montré que celles-ci étaient capables d’impacter l’expression de différents gènes impliqués dans la voie de biosynthèse chez A. flavus et A. parasiticus. Enfin, nous avons complété les données déjà existantes sur l’impact de facteurs environnementaux (température, disponibilité en eau et du temps d’incubation) sur la production d’aflatoxines. ABSTRACT : Mycotoxins are toxic contaminants of foodstuffs produced by a wide range of fungal species. Aflatoxins are the only mycotoxins carcinogenic for humans. They are mainly produced by the Aspergillus genus and can be found at each step of the agrofood chain (e.g. field, storage, process). Due to climate changes, France is starting to be exposed to aflatoxins. In order to limit the consumer exposure, many prevention or decontamination techniques have been developed. To this aim, we started the development of a biocontrol against aflatoxins accumulation for maize field application. Actinomycetes, are soil-borne bacteria that has already been commercialized as biocontrol. In Petri dishes, we studied the in vitro interaction between some actinomycetes and Aspergillus flavus, the main aflatoxins producer. We revealed that the interaction reduced the aflatoxins content (monitored by HPLC). Moreover, some bacterial isolates were able to reduce pure-aflatoxin B1 added in the medium. To understand this mechanism, adsorption tests has been conducted. Otherwise, RT-qPCR methodology was used to study the impact of Streptomyces-Aspergillus sp. on aflatoxin gene expression. Finally, the current knowledge of the impact of environmental factors (temperature, water activity and incubation time) on aflatoxins production was supplemented

Solute and matric potential stress on Penicillium verrucosum : Impact on growth, gene expression and ochratoxin A p production

Funding Information: S.A. is grateful to the British Council and the Newton Musharraf Programme for financial support. Publisher Copyright: © 2020 Wageningen Academic Publishers. All rights reserved. Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Penicillium verrucosum survives in soil and on cereal debris. It colonises grain during harvesting, drying and storage. There is no information on the relative tolerance of P. verrucosum to solute and matric stress in terms of colonisation, or on the biosynthetic toxin gene clusters or ochratoxin A (OTA) production. The objectives were to examine the effect of ionic and non-ionic solute and matric potential stress on (a) growth, (b) expression of two toxin biosynthetic genes otapksPV and otanrpsPV, and (c) OTA production by a strain of P. verrucosum. Optimum growth and OTA production were at-7.0 MPa (= 0.95 water activity, aw) and-1.4 MPa (= 0.99 aw), respectively, regardless of whether solute (Ψs) or matric (Ψm) stress was imposed. P. verrucosum was more sensitive to ionic solute stress (NaCl) with no growth at-19.6 MPa (=0.86 aw) while growth still occurred in the non-ionic solute (glycerol) and matric stress treatments. Relative gene expression of the biosynthetic genes using PCR (RT-qPCR) showed that the otapksPV gene was expressed over a wide range of ionic/non-ionic solute stress conditions (-1.4 to-14.0 MPa; = 0.99-0.90 aw). The highest expression was in the non-ionic Ψs stress treatments at-7.0 MPa (= 0.95 aw). However, the otanrpsPV gene was significantly up regulated under Ψm stress, especially with freely available water (-1.4 MPa = 0.99 aw). OTA production was significantly decreased as Ψs or Ψm stress were imposed. Limited OTA production occurred in the driest treatments under Ψs and Ψm stress respectively. The impact of these two types of stresses on the growth of P. verrucosum was quite different from that for OTA production. The results are discussed in the context of the life cycle and ecological characteristics of this species in contaminating cereals with OTA in the post-harvest phase of the cereal chain.Peer reviewedFinal Accepted Versio

Proof of concept: could snake venoms be a potential source of bioactive compounds for control of mould growth and mycotoxin production

© 2020 The Authors. This is an open access article under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits use, distribution and reproduction in any medium, provided the original work is properly cited.The objective was to screen 10 snake venoms for their efficacy to control growth and mycotoxin production by important mycotoxigenic fungi including Aspergillus flavus, Aspergillus westerdijkiae, Penicillium verrucosum, Fusarium graminearum and F. langsethiae. The Bioscreen C rapid assay system was used. The venoms from the Viperidae snake family delayed growth of some of the test fungi, especially F. graminearum and F. langsethiae and sometimes A. flavus. Some were also able to reduce mycotoxin production. The two most potent crude snake venoms (Naja nigricollis and N. siamensis; 41 and 43 fractions, respectively) were further fractionated and 83/84 of these fractions were able to reduce mycotoxin production by >90% in two of the mycotoxigenic fungi examined. This study suggests that there may be significant potential for the identification of novel fungistatic/fungicidal bioactive compounds as preservatives of raw and processed food commodities post-harvest from such snake venoms.Peer reviewedFinal Published versio

Essential oils modulate gene expression and ochratoxin a production in Aspergillus carbonarius

Ochratoxin A (OTA) is a mycotoxin, mainly produced on grapes by Aspergillus carbonarius, that causes massive health problems for humans. This study aims to reduce the occurrence of OTA by using the ten following essential oils (E.Os): fennel, cardamom, anise, chamomile, celery, cinnamon, thyme, taramira, oregano and rosemary at 1 µL/mL and 5 µL/mL for each E.O. As a matter of fact, their effects on the OTA production and the growth of A. carbonarius S402 cultures were evaluated, after four days at 28 C on a Synthetic Grape Medium (SGM). Results showed that A. carbonarius growth was reduced up to 100%, when cultured with the E.Os of cinnamon, taramira, and oregano at both concentrations and the thyme at 5 µL/mL. As for the other six E.Os, their effect on A. carbonarius growth was insignificant, but highly important on the OTA production. Interestingly, the fennel E.O at 5 µL/mL reduced the OTA production up to 88.9% compared to the control, with only 13.8% of fungal growth reduction. We further investigated the effect of these E.Os on the expression levels of the genes responsible for the OTA biosynthesis (acOTApks and acOTAnrps along with the acpks gene) as well as the two regulatory genes laeA and vea, using the quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) method. The results revealed that these six E.Os reduced the expression of the five studied genes, where the ackps was downregulated by 99.2% (the highest downregulation in this study) with 5 µL/mL of fennel E.O. As for the acOTApks, acOTAnrps, veA and laeA, their reduction levels ranged between 10% and 96% depending on the nature of the E.O and its concentration in the medium

Resilience of biocontrol for aflatoxin minimisation strategies: climate change abiotic factors may affect control in non-GM and GM-maize cultivars

here has been significant interest in the development of formulations of non-toxigenic strains of Aspergillus flavus for control of toxigenic strains to reduce the aflatoxin B1 (AFB1) contamination of maize. In the future, climate change (CC) abiotic conditions of temperature (+2–4°C), CO2 (existing levels of 400 vs. 800–1,200 ppb), and drought stress will impact on the agronomy and control of pests and diseases. This study has examined (1) the effect of two-way interacting factors of water activity × temperature on colonization and AFB1 contamination of maize cobs of different ripening ages; (2) the effect of non-toxigenic strains of A. flavus (50:50 inoculum ratio) on relative control of toxigenic A. flavus and AFB1 contamination of ripening cobs; (3) post-harvest control of AFB1 by non-toxigenic strains of A. flavus in non-GM and isogenic GM maize cultivars using the same inoculum ratio; and (4) the impact of three-way interacting CC factors on relative control of AFB1 in maize cobs pre-harvest and in stored non-GM/GM cultivars. Pre-harvest colonization and AFB1 production by a toxigenic A. flavus strain was conserved at 37°C when compared with 30°C, at the three ripening stages of cob development examined:milk ripe (R3), dough (R4), and dent (R5). However, pre-harvest biocontrol with a non-toxigenic strain was only effective at the R3 and R4 stages and not at the R5 stage. This was supported by relative expression of the aflR regulatory biosynthetic gene in the different treatments. When exposed to three-way interacting CC factors for control of AFB1 pre-harvest, the non-toxigenic A. flavus strain was effective at R3 and £4 stages but not at the R5 stage. Post-harvest storage of non-GM and GM cultivars showed that control was achievable at 30°C, with slightly better control in GM-cultivars in terms of the overall inhibition of AFB1 production. However, in stored maize, the non-toxigenic strains of A. flavus had conserved biocontrol of AFB1 contamination, especially in the GM-maize cultivars under three-way interacting CC conditions (37°C × 1,000 ppm CO2 and drought stress). This was supported by the relative expression of the aflR gene in these treatments. This study suggests that the choice of the biocontrol strains, for pre- or post-harvest control, needs to take into account their resilience in CC-related abiotic conditions to ensure that control of AFB1 contamination can be conserved

Three-Dimensional Study of F. graminearum Colonisation of Stored Wheat: Post-Harvest Growth Patterns, Dry Matter Losses and Mycotoxin Contamination

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).Fusarium causes significant post-harvest quality losses and mycotoxin contamination instored wheat but the colonisation dynamics of the grain and how this may be affected by the initialinoculum position in the grain mass is poorly understood. This study examined the 3D growthkinetics and mycotoxin production (deoxynivalenol and zearalenone) by F. graminearum duringhyphal colonisation from different initial inoculum positions in wheat microcosms (top-centre,bottom-centre, and bottom-side) maintained at two water activities (aw; 0.95 and 0.97). Clear jarswere used to visually follow the colonisation dynamics. Fungal respiration and associated drymatter loss (DML) and ergosterol were also quantified. Colonisation dynamics was shown to beaffected by the inoculation position. At the end of the colonisation process, fungal respiration andDML were driven by the inoculation position, and the latter also by the prevailing aw. Fungalbiomass (ergosterol) was mainly affected by the aw. The initial inoculum position did not affect therelative mycotoxin production. There was a positive correlation between respiration and ergosterol,and between mycotoxin production and colonisation indicators. We suggest that spatially explicitpredictive models can be used to better understand the colonisation patterns and mycotoxincontamination of stored cereal commodities and to aid more effective post-harvest management.Peer reviewedFinal Published versio

Unveiling the effect of interacting forecasted abiotic factors on growth and aflatoxin B1 production kinetics by Aspergillus flavus

© 2020 Elsevier Ltd. All rights reserved. This manuscript is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International Licence http://creativecommons.org/licenses/by-nc-nd/4.0/.The aim was to decipher the temporal impact of key interacting climate change (CC) abiotic factors of temperature (30 vs 37 °C), water activity (aw; 0.985 vs 0.930) and CO2 exposure (400 vs 1000 ppm) on (a) growth of Aspergillus flavus and effects on (b) gene expression of a structural (aflD) and key regulatory gene (aflR) involved in aflatoxin B1 (AFB1) biosynthesis and (c) AFB1 production on a yeast extract sucrose medium over a period of 10 days. A. flavus grew and produced AFB1 very early with toxin detected after only 48 h. Both growth and toxin production were significantly impacted by the interacting abiotic factors. The relative expression of the aflD gene was significantly influenced by temperature; aflR gene expression was mainly modulated by time. However, no clear relationship was observed for both genes with AFB1 production over the experimental time frame. The optimum temperature for AFB1 production was 30 °C. Maximum AFB1 production occurred between days 4–8. Exposure to higher CO2 conditions simulating forecasted CC conditions resulted in the amount of AFB1 produced in elevated temperature (37 °C) being higher than with the optimum temperature (30 °C) showing a potential for increased risk for human/animal health due to higher accumulation of this toxin.Peer reviewedFinal Accepted Versio

Impact of predicted climate change environmental conditions on the growth of Fusarium asiaticum strains and mycotoxins production on a wheat-based matrix

Fusarium asiaticum is a predominant fungal pathogen causing Fusarium Head Blight (FHB) in wheat and barley in China and is associated with approximately £201 million in annual losses due to grains contaminated with mycotoxins. F. asiaticum produces deoxynivalenol and zearalenone whose maximum limits in cereals and cereals-derived products have been established in different countries including the EU. Few studies are available on the ecophysiological behaviour of this fungal pathogen, but nothing is known about the impact of projected climate change scenarios on its growth and mycotoxin production. Therefore, this study aimed to examine the interacting effect of i) current and increased temperature (25 vs 30 °C), ii) drought stress variation (0.98 vs 0.95 water activity; aw) and iii) existing and predicted CO2 concentrations (400 vs 1000 ppm) on fungal growth and mycotoxin production (type B trichothecenes and zearalenone) by three F. asiaticum strains (CH024b, 82, 0982) on a wheat-based matrix after 10 days of incubation. The results showed that, when exposed to increased CO2 concentration (1000 ppm) there was a significant reduction of fungal growth compared to current concentration (400 ppm) both at 25 and 30 °C, especially at 0.95 aw. The multi-mycotoxin analysis performed by LC-MS/MS qTRAP showed a significant increase of deoxynivalenol and 15-acetyldeoxynivalenol production when the CH024b strain was exposed to elevated CO2 compared to current CO2 levels. Zearalenone production by the strain 0982 was significantly stimulated by mild water stress (0.95 aw) and increased CO2 concentration (1000 ppm) regardless of the temperature. Such results highlight that intraspecies variability exist among F. asiaticum strains with some mycotoxins likely to exceed current EU legislative limits under prospected climate change conditions