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The aim of this study was to evaluate the interactions between wheat plant (spikelets and straws), a strain of mycotoxigenic pathogen Fusarium graminearum and commercial biocontrol agents (BCAs). The ability of BCAs to colonize plant tissue and inhibit the pathogen or its toxin production was observed throughout two phases of the life cycle of pathogens in natural conditions (colonization and survival). All evaluated BCAs showed effective reduction capacities of pathogenic traits. During establishment and the expansion stage, BCAs provoked an external growth reduction of F. graminearum (77–93% over the whole kinetic studied) and mycotoxin production (98–100% over the whole kinetic studied). Internal growth of pathogen was assessed with digital droplet polymerase chain reaction (ddPCR) and showed a very strong reduction in the colonization of the internal tissues of the spikelet due to the presence of BCAs (98% on average). During the survival stage, BCAs prevented the formation of conservation perithecia of the pathogen on wheat straw (between 88 and 98% of perithecia number reduction) and showed contrasting actions on the ascospores they contain, or perithecia production (−95% on average) during survival form. The mechanisms involved in these different interactions between F. graminearum and BCAs on plant matrices at different stages of the pathogen’s life cycle were based on a reduction of toxins, nutritional and/or spatial competition, or production of anti-microbial compounds.

The aim of this study was to develop a set of experiments to screen and decipher the mechanisms of biocontrol agents (BCAs), isolated from commercial formulation, against two major mycotoxigenic fungi in cereals, Fusarium graminearum and Fusarium verticillioides. These two phytopathogens produce mycotoxins harmful to human and animal health and are responsible for the massive use of pesticides, for the protection of cereals. It is therefore essential to better understand the mechanisms of action of alternative control strategies such as the use of BCAs in order to optimize their applications. The early and late stages of interaction between BCAs and pathogens were investigated from germination of spores to the effects on perithecia (survival form of pathogen). The analysis of antagonist activities of BCAs revealed different strategies of biocontrol where chronological, process combination and specialization aspects of interactions are discussed. Streptomyces griseoviridis main strategy is based on antibiosis with the secretion of several compounds with anti-fungal and anti-germination activity, but also a mixture of hydrolytic enzymes to attack pathogens, which compensates for an important deficit in terms of spatial colonization capacity. It has good abilities in terms of nutritional competition. Trichoderma asperellum is capable of activating a very wide range of defenses and attacks combining the synthesis of various antifungal compounds (metabolite, enzymes, VOCs), with different targets (spores, mycelium, mycotoxins), and direct action by mycoparasitism and mycophagy. Concerning Pythium oligandrum, its efficiency is mainly due to its strong capacity to colonize the environment, with a direct action via microbial predation, stimulation of its reproduction at the contact of pathogens and the reduction of perithecia formation.

Wild yeasts isolated from the surface of apples were screened for antagonistic activity against Penicillium expansum, the main producer of the mycotoxin patulin. Three antagonistic yeasts (Y33, Y29 and Y24) from a total of 90 were found to inhibit P. expansum growth. Identification by ITS region sequence and characterization showed that three selected isolates of yeast should be different strains of Metschnikowia pulcherrima. Several concentrations of the selected yeasts were used to study their in vitro antifungal effectivity against P. expansum on Petri dishes (plates with 63.6 cm2 surface) whereas their potential activity on patulin reduction was studied in liquid medium. Finally, the BCA that had the best in vitro antifungal capacity against P. and the best patulin degradation capacity was selected to be assessed directly on apples. All the selected strains demonstrated antifungal activity in vitro but the most efficient was the strain Y29. Isolated strains were able to reduce patulin content in liquid medium, Y29 being the only strain that completely reduced patulin levels within 120 h. The application of Y29 as biocontrol agent on the surface of apples inoculated with P. expansum, inhibited fungal growth and patulin production during storage. Therefore, the results shown that this yeast strain could be used for the reduction of P. expansum and its mycotoxin in apples or apple-based products by adapting the procedure application., The authors acknowledge the financial support of the Agence Nationale de la Recherche (France) (PATRISK project: ANR-17-CE21-0008) and the financial support of the European Union (Erasmus grant attributed to Laura Settier-Ramírez).

Fungal pathogens are an international concern regarding their impact on numerous crops with diseases such as the Fusarium wilt, grey molds, blue molds or downy mildews that cause colossal yield losses both at pre- and post-harvest stages all over the world. The fungal diseases are usually associated with the infection and accumulation of mycotoxins in plant products and derivate foods. Alternative approaches of biological control that are non-chemical and eco-friendly have been studied such as the crop rotations or the application of organic phyto-protective amendments using living organisms. Many different bacterial families have been identified but accounting for low abundance in the total communities. The participation of the predominant families in 3-day-old soil decreased after 28 days of incubation. The fungal community of the reference soil was more diversified as compared to the fungal communities of the amendments.

Acetic acid bacteria are involved in many food and beverage fermentation processes. They play an important role in cocoa bean fermentation through their acetic acid production. They initiate the development of some of the flavor precursors that are necessary for the organoleptic quality of cocoa, and for the beans’ color. The development of starter cultures with local strains would enable the preservation of the microbial biodiversity of each country in cocoa-producing areas, and would also control the fermentation. This approach could avoid the standardization of cocoa bean fermentation in the producing countries. One hundred and thirty acetic acid bacteria were isolated from three different cocoa-producing countries, and were identified based on their 16S rRNA gene sequence. The predominate strains were grown in a cocoa pulp simulation medium (CPSM-AAB) in order to compare their physiological traits regarding their specific growth rate, ethanol and lactic acid consumption, acetic acid production, and relative preferences of carbon sources. Finally, the intraspecific diversity of the strains was then assessed through the analysis of their genomic polymorphism by (GTG)5-PCR fingerprinting. Our results showed that Acetobacter pasteurianus was the most recovered species in all of the origins, with 86 isolates out of 130 cultures. A great similarity was observed between the strains according to their physiological characterization and genomic polymorphisms. However, the multi-parametric clustering results in the different groups highlighted some differences in their basic metabolism, such as their efficiency in converting carbon substrates to acetate, and their relative affinity to lactic acid and ethanol. The A. pasteurianus strains showed different behaviors regarding their ability to oxidize ethanol and lactic acid into acetic acid, and in their relative preference for each substrate. The impact of these behaviors on the cocoa quality should be investigated, and should be considered as a criterion for the selection of acetic acid bacteria starters.

Ochratoxin A (OTA) is a secondary metabolite produced by fungal pathogens such as Penicillium verrucosum, which develops in food commodities during storage such as cereals, grapes, and coffee. It represents public health concerns due to its genotoxicity, carcinogenicity, and teratogenicity. The objective of this study was to evaluate the ability of actinobacteria and their metabolites to degrade OTA and/or to decrease its production. Sixty strains of actinobacteria were tested for their ability to prevent OTA formation by in vitro dual culture assays or with cell free extracts (CFEs). In dual culture, 17 strains strongly inhibited fungal growth, although it was generally associated with an increase in OTA specific production. Seventeen strains inhibited OTA specific production up to 4% of the control. Eleven actinobacteria CFEs reduced OTA specific production up to 62% of the control, while no substantial growth inhibition was observed except for two strains up to 72% of the control. Thirty-three strains were able to degrade OTA almost completely in liquid medium whereas only five were able to decrease it on solid medium, and two of them reduced OTA to an undetectable amount. Our results suggest that OTA decrease could be related to different strategies of degradation/metabolization by actinobacteria, through enzyme activities and secretion of secondary metabolites interfering with the OTA biosynthetic pathway. CFEs appeared to be ineffective at degrading OTA, raising interesting questions about the detoxification mechanisms. Common degradation by-products (e.g., OT&alpha, or L-&beta, phenylalanine) were searched by HPLC-MS/MS, however, none of them were found, which implies a different mechanism of detoxification and/or a subsequent degradation into unknown products.

In a sustainable development concern, the agricultural practices notably those to control the pathogenic and/or mycotoxigenic fungi that maintain the quality and the abundance of crops with respecting the ecosystems and public health are indispensable. Our study focuses on evaluating the biocontrol potential of the actinomycete microflora of some organic amendments against the phytopathogenic and mycotoxigenic Fusarium verticillioides. The inhibitory effect was assessed in term of growth and mycotoxin production (fumonisin FB1 and FB2) of F. verticillioides in the presence of amended soils and selected antagonist agents from the amendments. Results demonstrated that the amended soils with different organic amendments exhibited a higher inhibition rate of Fusarium verticillioides growth than the reference soil. They were able to inhibit the fungal pigment and spore productions which are the major mean of survival and dispersion of this fungus. The fumonisin production was strongly reduced by the metabolites produced by the amended soils’ microbiota, up to 68.7% and 92.5% for FB1 and FB2 respectively. Actinomycete strains isolated from these amendments were identified as Streptomyces and were also tested for their antifungal potential. The fungal growth was reduced up to more than 60% in the presence of extracellular metabolites of several Streptomyces strains. In confrontation assays, most of the Streptomyces strains inhibited strongly both fungal growth and fumonisin production. The FB1 and FB2 amounts were decreased at best, by up to 87.5% and 98.2% respectively, depending on the actinomycete strains and fungal culture time. These results are promising for the use of these biocontrol agents, and especially some actinomycete strains, as tools to reduce fumonisin contamination in cereal crops and derived products.

International audience; The Streptomyces sp. strain AV05 isolated from an organic amendment was found to impact both growth and fumonisin production of Fusarium verticillioides during in vitro direct confrontation. In order to investigate the interactions between the Streptomyces sp. strain AV05 and F. verticillioides, a metabolomic approach was used. The study of the endometabolomes of the microorganisms was carried out in two different conditions: the microorganisms were cultivated alone or in confrontation. The aim of this study was to examine the modifications of the endometabolome of F. verticillioides in confrontation with the Streptomyces strain. The metabolites involved in these modifications were identified using 2D NMR. Many metabolites were found to be overproduced in confrontation assays with the Streptomyces strain, notably 16 proteinogenic amino acids, inosine, and uridine. This suggested that fungal metabolic pathways such as protein synthesis have been affected due to interaction. Thus, metabolomic studies, as well as proteomics or transcriptomics, are useful for deciphering the mechanisms of interactions between biological control agents and mycotoxigenic fungi. This comprehension is one of the key elements of the improvement of the selection and use of antagonistic agents.

'Kankankan' is a popular spice powder used to season roasted meat in Côte d'Ivoire. However, produced in a traditional way, the conditions of production and storage of kankankan favour the proliferation of mycotoxin-producing fungal strains. The aim of this study was to carry out an inventory of mycotoxin contamination of this spice powder and to assess risk exposure to consumers. In total, 75 samples of kankankan were collected from wholesalers (6), sellers of kankankan in the markets (35) and sellers of roasted meat (34) across three municipalities of Abidjan, the economic capital of Côte d'Ivoire. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used to detect and quantify nine different mycotoxins. Dietary exposure was calculated by using estimated daily intake (EDI), whereas risk characterisation was assessed using the margin of exposure (MOE) approach. Aflatoxins and fumonisins were found in 99% of samples assessed, while contamination with beauvericin was proportionally lowest (28%). At all the three types of actors within the food production chain (wholesalers, kankankan sellers and roasted meat sellers) the mean concentrations of aflatoxin B1 (AFB1) in samples exceeded the European standard for spice mixtures, with concentrations reaching up to 502 µg/kg. The estimated daily intakes of aflatoxins observed in the different populations were above the recommended level of 0.017 ng kg−1 b.w. day−1. The MOES values for adolescents and adults were 8.10 and 12.78, respectively, well below the safe margin of 10,000. The co-occurrence of mycotoxins in kankankan samples together with high aflatoxin exposure to consumers represent a potential risk to public health, calling for immediate risk management and education of kankankan producers and consumers.

The current study assesses the natural occurrence of mycotoxins and fungal metabolites in Tunisian Pearl millet. Investigations using a multi-mycotoxin liquid chromatography/electrospray ionization-tandem mass spectrometric (LC/ESI–MS/MS) method were carried out on a total of 220 pearl millet samples collected from farmers (n = 144) located in four different agroecological zones in Tunisia and from retail shops (n = 76). Results revealed fifty-seven regulated and emerging metabolites contaminating 91.4% of grain samples. Among major mycotoxins, both aflatoxin B1 (AFB1) and ochratoxin A (OTA) were the most prevalent at rate of 8.6% each and occurring at an average level of 106 and 69.4 μg/kg, respectively. All positives samples were significantly exceeding the European thresholds (5 and 3 μg/kg, respectively). Lower incidence (5.9%) of Zearalenone (ZEA) contaminated samples at levels below the authorized limits (100 μg/kg) was recorded. Pearl millet grains from the south coastal zone (CT.S) had significantly the highest mean AFB1 contamination (p < 0.05). Meanwhile, OTA was encountered only in CT.S zone. Furthermore, commercial samples showed higher OTA and ZEA mean concentrations than farmers samples (p < 0.05). As regards emerging mycotoxins, Fusarium mycotoxins including beauvericin (BEA), equisetin (EQUS), monoacetoxyscirpenol (MAS) and diacetoxyscirpenol (DAS) were detected in 43.2%, 43.2%, 11.4% and 10.5% of positive samples, respectively. Furthermore, 12.7%, 50.9%, 53.2% and 14.1% of grains were contaminated with Alternaria mycotoxins namely alternariol (AOH), alternariolmonomethyl ether (AME), tentoxin (TEN) and tenuazonic acid (TA), respectively. The majority of emerging Fusarium and Alternaria mycotoxins mean levels were significantly higher in grains from north coastal area (p < 0.01). The present study highlighted a wide range of mycotoxins in Tunisian pearl millet with a different mycotoxin patterns within the agroecological zones.

The actinobacteria Streptomyces sp. AV05 appears to be a potential biocontrol agent (BCA) against mycotoxigenic fungi. It was found to significantly inhibit F. verticillioides growth and mycotoxin production during their co-cultivation. F. verticillioides growth was durably affected while the decrease of the toxin production levels was reversible, suggesting different BCA actions. The study of both transcriptomes brought useful information on the microbial interaction. RNA-seq data indicated that the dual interaction modified genetic expression of both microorganisms as 18.5 % of the genes were differentially expressed for the fungus against 3.8 % for the actinobacteria. Fungal differentially expressed genes (DEGs) were equally up and down regulated while bacterial ones were mainly upregulated. We especially focused the analysis of DEGs on fungal defense reaction to bacterial attack. For example, if this potential BCA implements a strategy of antibiosis with the over expression of 'siderophore-interacting protein' linked to the production of bacteriocins, the fungus in a state of stress is able to adapt its metabolism by up-regulation of amidase. It could correspond to the induction of resistance gene clusters and suggest a detoxification process. Moreover fumonisins-related pathway appears underexpressed in the presence of Streptomyces that explain the reduction of fumonisin accumulation observed.

Phytopathogenic and/or mycotoxigenic filamentous fungi are involved in a great number of plant diseases that cause yield and quality losses of crops. Besides the economic damages, these fungi produce mycotoxins that present health risks for humans and animals who consume contaminated foods. The most dangerous mycotoxins in the agriculture and the food industry that are regulated in European Union (trichothecenes especially deoxynivalenol, fumonisins, aflatoxins and ochratoxin A) are produced by three main fungal genera ( Fusarium, Aspergillus and Penicillium ). Many approaches have been applied to prevent and manage the phytopathogenic and/or mycotoxigenic fungi. However, these methods involve the use of chemical inputs that are harmful for humans, animals and environment. In a concern of sustainable development, the application of biocontrol has been considered for addressing this problem in a more environmentally friendly way. This review considered the incidence of the three fungal genera and their mycotoxins in crops and in foodstuff. The impacts of the fungal contamination and the toxin accumulation were reported. Besides, the biological control means against these pathogens were reviewed. Among them, organic amendments showed to be effective in both producing antifungal activities and reinforcing plant health.

The effects of light at different wavelengths and photoperiod on growth and ochratoxin A production of Aspergillus carbonarius and Aspergillus westerdijkiae were studied: far-red (740 nm), red (625 nm), blue (445 nm), and UV-A (366 nm). Fungal growth was not significantly affected by photoperiod or light wavelength; the only exception was A. westerdijkiae which showed reduced growth under UV-A light (366 nm). Short-wavelength blue light (445 nm) and UV-A light caused a reduction in ochratoxin A production of both fungal species. However, long-wavelength red light (625 nm) and far-red light (740 nm) reduced ochratoxin A production only in A. westerdijkiae but not in A. carbonarius. It is believed that this difference in reactivity to light is due to differences in the melanin content of the two fungal species: A. carbonarius is a black fungus with higher melanin content than A. westerdijkiae, a yellow fungus. Other possible explanations for the reduction of ochratoxin A production by light were also discussed.

International audience; A method was developed to analyze neutral lipids through the use of three triglycerides, four free fatty acids, six di- and four mono-glycerides standards by high performance liquid chromatography (HPLC) normal phase coupled with either with evaporative light scattering detector (ELSD) or with mass spectrometry (MS) operating in atmospheric pressure chemical ionization (APCI) mode. The method was applied to the determination of the neutral lipid fraction from a Botryococcus braunii race A (B. braunii) culture. This method led us to identify neutral lipids synthesized by B. braunii in a single analysis within 45min through HPLC-APCI-MS/MS technique.

The fungus Fusarium langsethiae, exclusively described in Europe at present, seems to have taken the place of other Fusarium species in barley fields over the last 5 years. It has proved to be a highly toxic type-A trichothecene producer (T-2 and HT-2 toxins). The aim of this work was to study the ecotoxinogenesis of this fungus the better to identify and manage the health risk it may pose during the beer manufacturing process. The influence of temperature and water activity on its growth rate and production of toxins are particularly assessed from a macroscopic point of view. Different cultures were grown on sterilized rehydrated barley with a water activity between 0.630 and 0.997 and a temperature ranging from 5 to 35 degrees C. Biomass specific to F. langsethiae and T-2 and HT-2 toxins were quantified by real-time polymerase chain reaction and liquid chromatography-mass spectrometry, respectively. It appears that the optimal temperature and water activity for F. langsethiae toxinogenesis are 28 degrees C and 0.997. This fungus was able to produce 2.22 g kg(-1) of these toxins in 16 days on barley in optimal production conditions. The malting process seems to be a critical step because, in its temperature range, specific production was six times higher than under optimal temperatures for fungus growth. In the short-term, this work will help redefine the process conditions for malting. In the medium-term, the results will contribute to the development of a molecular tool to diagnose the presence of this contaminant and the detection of the toxins in barley, from fields to the end product.

Development of bacterial resistance to antibiotics has lead to investigations of rare bacteria, which produce new bioactive molecules. Saccharothrix algriensis has been isolated from the desert Maghreb. It produces dithiolopyrrolones, some of which were newly identified. In order to optimize and control production of dithiolopyrrolones, investigation regarding microorganism metabolism was required. Growth on semisynthetic medium containing 2 g· l −1 of yeast extract was complicated because it was performed on several substrates. Moreover, because development of this bacterium on minimum medium was difficult, its composition was optimized by screening of different compounds led by yeast extract. Uracil added to the minimum medium allowed a maximum biomass production of 1.35 g· l −1 compared to 0.32 g· l −1 without uracil. Moreover, nonpolar amino acids and trace metal elements stimulated Saccharothrix algeriensis growth.

One strategy to increase the stability of proteins is to reduce the area of water-accessible hydrophobic surface. In order to test it, we replaced 14 solvent-exposed hydrophobic residues of acetylcholinesterase by arginine. The stabilities of the resulting proteins were tested using denaturation by high temperature, organic solvents, urea and by proteolytic digestion. Altough the mutational effects were rather small, this strategy proved to be successful since half of the mutants showed an increased stability. This stability may originate from the suppression of unfavorable interactions of nonpolar residues with water or from addition of new hydrogen bonds with the solvent. Other mechanisms may also contribute to the increased stability observed with some mutants. For example, introduction of a charge at the surface of the protein may provide a new coulombic interaction on the protein surface.