Aflatoxin contamination of maize by Aspergillus section Flavi constitutes a major health and economic concern. While biological control using non-toxigenic strains has proven effective, the increasing global food demand underscores the need for alternative carrier materials to replace seeds and grains. The aims of the present study were (1) to develop an innovative macroporous starch polymer in which the biocontrol agent can grow and be transported to fields where the bioformulate is applied, and (2) to evaluate the effectiveness of this new formulate in reducing AF contamination in maize kernels in field trials, in comparison with the traditional formulate based on long-grain rice as a substrate. Several methods and different starch sources were tested, and the formulation consisting of 10% maize starch, 0.5% citric acid, 3% sucrose, 0.3% urea, and distilled water was the most effective. Furthermore, this bioformulate demonstrated a performance comparable to that of the traditional long-grain rice-based formulation, reducing AF accumulation by up to 81% in maize kernels under field conditions. The implementation of this macroporous starch polymer-based formulation, in combination with the biological control agent A. flavus AFCHG2, would not only reduce aflatoxin contamination in maize kernels but also minimise the use of food-grade seeds and grains for industrial purposes, thereby preserving their availability for human and animal nutrition. Consequently, this development could enhance the availability of these substrates for food and feed use, thereby contributing to improved safety and food security.

Aflatoxins (AFs) are secondary metabolites produced by Aspergillus section Flavi, mainly Aspergillus flavus and Aspergillus parasiticus. These species may infect food and feed along entire agri-food chains, synthesizing the toxin when environmental conditions are favorable. The scenario of climate change that the world will have to face in the next decades is expected to increase the levels of AF contamination in cereals, nuts, spices, and legumes, among other important crops around the world. This situation will have a great impact on human and animal health, as well as economic losses due to the rejection of contaminated products during commercialization. Several mitigation strategies have been implemented to counteract this problem. Some of them are intended to prevent AF synthesis, while others are designed to decontaminate the pre-formed toxin. In addition, several pre- and post-harvest strategies have shown effectivity. Besides physical and chemical tools, biological control based on different microorganisms and the use of enzymes have been proposed as promissory approaches to mitigate the risk of AF contamination. In this chapter, detailed information about two developments is presented: biocontrol based on prevention by a non-toxigenic A. flavus strain and the use of fungal laccases to decontaminate AFs. The integration of these tools will enhance the quality of food and feed, contributing to food safety in agri-food chains.

Barley (Hordeum vulgare L.) is the second winter crop in Argentina. In the national market, grains are mainly destined to produce malt for beer manufacture. Fusarium species are common, causing Fusarium Head Blight (FHB) in barley, which generates yield and quality losses, as well as mycotoxin occurrence. The aims of this study were to determine (a) the incidence of the main species causing FHB in different locations of the barley-growing region of Argentina, (b) their ability to produce mycotoxins, and (c) the levels of deoxynivalenol (DON) and nivalenol (NIV) natural occurrence in grains at the harvest stage. Additionally, a strain of Bacillus velezensis was studied as a biocontrol agent in order to control F. graminearum sensu stricto and mycotoxin accumulation during the malting process, with the final objective being to reduce DON contamination in the beer manufacture chain. Fusarium graminearum ss was the most prevalent species causing FHB, with Fusarium poae being less distributed. Both species produced several mycotoxins, including NX-2 and NX-3, which is the first report of their production by strains isolated from barley in Argentina. Deoxynivalenol contamination was found in 95% of barley grains during the 2016 harvest season (mean: 0.4 mg/kg), while NIV contamination was present in 29% of samples (mean: 0.49 mg/kg). In the 2017 harvest season, 53.6% of grains were contaminated with DON (mean: 0.42 mg/kg), and 21% with NIV (mean: 0.8 mg/kg). Quantification of F. graminearum ss by real-time PCR during the micro-malting process showed that application of the biocontrol agent before the germination stage was the most effective treatment, with a 45% reduction in fungal DNA levels. Reduction in DON contamination (69.3–100%) in artificially infected grains with F. graminearum ss, was also observed. The present work contributes to the knowledge of FHB in Argentina and to the development of a strategy to control this disease and mycotoxin contamination in barley, promoting at the same time food security.

Maize (Zea mays L.) is one of the most widely planted crops globally with Argentina leading world production and exportation. Santiago del Estero province, east of Tucumán and north of Córdoba encompasses eight agro-climatic zones in the Chaco Semi-arid region, agro-ecologically characterized by a wide temperature range and frequent drought periods that expose the crop to pathogens, particularly Aspergillus flavus. This pathogen is responsible for ear rot and grain contamination with mycotoxins such as aflatoxin B1 and cyclopiazonic acid. This study obtained fungal isolates from ears of maize and characterized them according to toxigenic capability and morphotype of sclerotia (S < 400 μm, associated with high levels of aflatoxins and L > 400 μm, related to variable levels of aflatoxins). In addition, those not producing aflatoxins were studied to determine phylogenetic relationships based on sequences of a segment of the CaM gene. Fifty-eight isolates were obtained in eight localities representing each agro-climatic zone, 30 of which were non-aflatoxigenic, 28 aflatoxigenic, and all producers of ciclopiazonic acid. Six isolates did not produce sclerotia, 51 were L and only one was S, the latter being a non-producer of aflatoxins. The number of sclerotia was positively correlated with the production of aflatoxin B1, while size was negatively correlated. The CaM gene sequences corroborated that the isolates belonged to the A. flavus clade and the high nucleotide similarity among them (99.4% to 100%) revealed almost zero genetic diversity in this geographic region. No significant differences were observed in the proportion of isolates between growing seasons or among agroclimatic districts. This research revealed characteristics of fungus populations in this agricultural region of north Argentina. Highlights: Aspergillus flavus isolates from ears of maize were characterized according to toxigenic capability, morphotype of sclerotia and phylogenetic relationships. Fifty-eight isolates were obtained, 30 non-aflatoxigenic, 28 aflatoxigenic, and all producers of ciclopiazonic acid. Six isolates did not produce sclerotia, 51 were L and only one was S, the latter being a non-producer of aflatoxins. All isolates belonged to the flavus clade. High nucleotide similarity among the isolates (99.4% to 100%) revealed almost zero genetic diversity in the region.

The presence of Aspergillus section Flavi and aflatoxins (AFs) in nut chains are of great concern due to the toxic effects of AFs on human and animal health as well as the economic losses during commercialisation. Extreme events related to climate change like increased temperature, severe rainfalls and droughts are modulating factors of fungal growth and mycotoxin production. The Expert Working Group (EWG) on nuts that worked under the frame of the Horizon 2020 Mycokey Project prepared this review based on discussions about the situation of contamination with AFs in nuts and the available strategies to reduce the impact of these toxins in the nut chains. The EWG selected as examples peanuts, pistachios and Brazil nuts, and discussed in the review the importance of nut value chains in the production, commercialisation and regulations that establish maximum levels for AFs. Also data on the Rapid Alert System for Food and Feed (RASFF) was considered and showed several notifications for mycotoxins, mainly AFs for nuts and nut products, such as pistachio nuts and dried mix nuts. Under a scenario of climate change, we elaborated strategies to prevent AFs contamination with emphasis on biocontrol and decision support systems. Also, the relevance of strategies to prevent and reduce both fungal infection and AFs accumulation in peanuts, Brazil nuts and pistachio are included as guidelines.

Maize (Zea mays L.) is an important crop in Argentina. Aspergillus section Flavi can infect this crop at the pre-harvest stage, and the harvested grains can be contaminated with aflatoxins (AFs). During the production of bioethanol from maize, AF levels can increase up to three times in the final co-products, known as, dry and wet distiller’s grain with solubles (DDGS and WDGS), intended for animal feed. Fungal enzymes like laccases can be a useful tool for reducing AF contamination in the co-products obtained from this process. The aim of the present study was to evaluate the ability of laccase enzymes included in enzymatic extracts (EE) produced by different species in the Basidiomycota phylum to reduce AF (AFB1 and AFB2) accumulation under the conditions of in vitro assays. Four laccase activities (5, 10, 15, and 20 U/mL) exerted by nine isolates were evaluated in the absence and presence of vanillic acid (VA), serving as a laccase redox mediator for the degradation of total AFs. The enzymatic stability in maize steep liquor (MSL) was confirmed after a 60 h incubation period. The most effective EE in terms of reducing AF content in the buffer was selected for an additional assay carried out under the same conditions using maize steep liquor obtained after the saccharification stage during the bioethanol production process. The highest degradation percentages were observed at 20 U/mL of laccase enzymatic activity and 1 mM of VA, corresponding to 26% for AFB1 and 26.6% for AFB2. The present study provides valuable data for the development of an efficient tool based on fungal laccases for preventing AF accumulation in the co-products of bioethanol produced from maize used for animal feed.

Maize (Zea mays L.) may be infected by Fusarium verticillioides and F. proliferatum, and consequently contaminated with fumonisins (FBs), as well as the co-products of bioethanol intended for animal feed. Laccase enzymes have a wide industrial application such as mycotoxin degradation. The aims were to isolate and identify fungal laccase-producing strains, to evaluate laccase production, to determine the enzymatic stability under fermentation conditions, and to analyse the effectiveness in vitro of enzymatic extracts (EEs) containing laccases in degrading FB1. Strains belonging to Funalia trogii, Phellinus tuberculosus, Pleurotus ostreatus, Pycnoporus sanguineus and Trametes gallica species showed laccase activity. Different isoforms of laccases were detected depending on the evaluated species. For the FB1 decontamination assays, four enzymatic activities (5, 10, 15 and 20 U/mL) were tested, in the absence and presence of vanillic acid (VA) and 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) as redox mediators (1 and 10 mM). Trametes gallica B4-IMICO-RC EE was the most effective strain in buffer, achieving a 60% of FB1 reduction. Laccases included in EEs remained stable at different alcoholic degrees in maize steep liquor (MSL), but no significant FB1 reduction was observed under the conditions evaluated using MSL. This study demonstrate that although laccases could be good candidates for the development of a strategy to reduce FB1, further studies are necessary to optimise this process in MSL.

Fusarium head blight (FHB) is one of the most common diseases in Argentina, affecting the quality and yield of barley grains. Fusarium graminearum sensu stricto (ss) and Fusarium poae are causal agents of FHB and potential sources of mycotoxin contamination in barley. Conventional management strategies do not lead to a complete control of FHB; therefore, biological control emerges as an eco-friendly alternative in the integrated management of the disease. In the present work, Bacillus velezensis, Bacillus inaquosorum, Bacillus nakamurai and Lactobacillus plantarum were evaluated as potential biocontrol agents against F. graminearum ss and F. poae on barley-based media. Bacillus velezensis RC218 was selected to carry out greenhouse and field trials in order to reduce FHB and mycotoxin accumulation. This strain was able to control growth of both Fusarium species and reduced deoxynivalenol (DON) and nivalenol (NIV) production by 66 % and 79 %, respectively. Bacillus inaquosorum and B. nakamurai were more effective in controlling F. poae growth, and the mean levels of reduction in DON accumulation were 50 and 38 %, and 93 and 26 % for NIV, respectively. Lactobacillus plantarum showed variable biocontrol capacity depending on the strain, with no significant mycotoxin reduction. The biocontrol on incidence and severity of FHB in the greenhouse and field trials was effective, being more efficient against F. graminearum ss and DON accumulation than against F. poae and NIV occurrence. This study provides valuable data for the development of an efficient tool based on biocontrol agents to prevent FHB-producing Fusarium species development and mycotoxin occurrence in barley, contributing to food safety.

Tan spot is a devastating foliar disease of wheat caused by Pyrenophora tritici - repentis , a necrotrophic ascomycete, which causes considerable yield losses worldwide. To control this disease, an integrated management plan must be implemented by the combination of different tools such as the use of wheat varieties with resistance to Tan spot, crop rotation, tillage systems, balanced fertilization and fungicide application. However, there are increasing worldwide restrictions related to the use of chemical products due to environmental and human risks, aggravated by the fact that consumers are demanding food produced in a more environmentally-friendly way. These facts encouraged producers and scientists to exploit the use of native beneficial microorganisms to control phytopathogens of wheat, that can be used as a tool to control Tan spot. This review focuses on the combination of microorganisms with different management practices to produce wheat in a more sustainable way.

EDITORIAL article Front. Fungal Biol., 14 November 2023Sec. Fungal Physiology and Metabolism Volume 4 - 2023 | https://doi.org/10.3389/ffunb.2023.1320198

Maize ( Zea mays L.) is one of the most important cereals around the world since it is widely used for food and feed. This cereal is relevant to the economy of several countries due to its volume of production and exportation. During pre-harvest stages this crop is susceptible to infection by species of Aspergillus section Flavi and Fusarium and prone to mycotoxin contamination, mainly aflatoxins and fumonisins. Additionally, the climate change scenario predicted for next decades, including increases in the global temperature, CO 2 levels and periods of hydric stress, would contribute to higher levels of mycotoxins worldwide. In this context, there is a need to find successful, feasible and eco-friendly tools for an effective mycotoxin mitigation in order to guarantee food security and safety. This review summarises the current situation of aflatoxin and fumonisin contamination in maize, highlighting the importance of the evaluation and implementation of biocontrol strategies at pre-harvest stage in the frame of an integrated control management to reduce the impact of mycotoxin accumulation under the risk of climate change expected for the next years.

Fusarium graminearum is ranked among the five most destructive fungal pathogens that affect agroecosystems. It causes floral diseases in small grain cereals including wheat, barley, and oats, as well as maize and rice. We conducted a systematic review of peer-reviewed studies reporting species within the F. graminearum species complex (FGSC) and created two main data tables. The first contained summarized data from the articles including bibliographic, geographic, methodological (ID methods), host of origin and species, while the second data table contains information about the described strains such as publication, isolate code(s), host/substrate, year of isolation, geographical coordinates, species and trichothecene genotype. Analyses of the bibliographic data obtained from 123 publications from 2000 to 2021 by 498 unique authors and published in 40 journals are summarized. We describe the frequency of species and chemotypes for 16,274 strains for which geographical information was available, either provided as raw data or extracted from the publications, and sampled across six continents and 32 countries. The database and interactive interface are publicly available, allowing for searches, summarization, and mapping of strains according to several criteria including article, country, host, species and trichothecene genotype. The database will be updated as new articles are published and should be useful for guiding future surveys and exploring factors associated with species distribution such as climate and land use. Authors are encouraged to submit data at the strain level to the database, which is accessible at https://fgsc.netlify.app .

This study evaluated the antifungal effect of ZnO nanoparticles (ZnO-NPs) on Fusarium proliferatum growth and fumonisin accumulation both on a maize-based medium (in vitro) and on irradiated maize grains (in situ). The ZnO-NPs were obtained by drop-by-drop synthesis without further thermal treatment and characterized by scanning electronic microscopy/ energy dispersive X-ray spectroscopy (SEM/EDS) and X-ray diffraction (XRD). SEM analysis showed them as thin flakes of 200 x 200 nm, similar to 30 nm thickness and its purity were confirmed by XRD. During the in vitro assay ZnO-NPs (0, 0.8; 4, 8 g L-1) were evaluated at 25 degrees C during 21 days under darkness or photoperiod incubation (12/12 h light (cold white and black fluorescent lamps)/darkness) to determine its possible photocatalytic influence. Fumonisins were detected by high performance liquid chromatography coupled to mass spectrometry (HPLC- MS/MS). All ZnO-NPs concentrations significantly affected growth rates and FB1 accumulation by F. proliferatum RCFP 5033 (p < 0.05). Similar reduction of growth and FB1 (%) was observed at 0.8 and 8 g L-1 ZnO-NPs under photoperiod or darkness incubation. FB1 reduction was observed after 14 and 21 days, although the highest reduction occurred after 14 days under photoperiod incubation (84-98%). No clear light enhancing effect on the antifungal and anti-mycotoxin capability of the ZnO-NPs was observed. Morphological alterations in mycelia and conidia were observed by SEM. Under the in situ assay, the effect of the ZnO-NPs (0, 0.4, 0.8, 2 g kg(-1)) on growth rates and fumonisin B-1, B-2 and B-3 accumulation by two F. proliferatum strains was evaluated on irradiated maize grains adjusted to 0.995, 0.98 and 0.97 aW in darkness at 25 degrees C during 21 days. Also, zinc acetate at 0.8 g kg(-1) was included to compare their antifungal effect against the same ZnO-NPs concentration. Growth rates decreased significantly as ZnO-NPs concentrations increased. Higher than 60% of growth reduction was observed for both F. proliferatum strains. Zinc acetate significantly reduced growth, although it was less efficient that the same ZnO-NPs concentration. ZnO-NPs reduced total fumonisins accumulation by 71-99% at 0.8-2 g kg(-1) ZnO-NPs and 0.98-0.995 aW. Moreover, 0.4 g kg(-1) ZnO-NPs also produced significant reduction of the 3 fumonisins. This study showed the application of ZnO-NPs in maize grains could be a low cost and environmental impact strategy to control phytopathogen and toxigenic fungi such as F. proliferatum and to reduce fumonisins accumulation, both during crop development at preharvest stage and during maize storage.

The effect of water activity (aW; 0.87, 0.90, 0.92, 0.94, 0.96, 0.98 and 0.99), temperature (15, 25, and 30 °C), incubation time (5, 10, 14, and 21 days), and their interactions on mycelial growth and aflatoxin production in a chickpea-based medium by three Aspergillus flavus strains isolated from chickpea grains in Argentina was evaluated. Maximum growth rates were obtained at the highest aW (0.99) and 30 °C, with growth decreasing as the aW of the medium was reduced. Maximum levels of aflatoxins were produced at 0.99 aW and 25 °C after 5 days of incubation for two strains, and at 25 °C and 0.96 aW after 21 days of incubation for the third strain. The aflatoxin concentrations varied considerably depending on the aW and temperature interactions assayed. Two-dimensional profiles of aW by temperature interactions were developed from these data to identify areas where conditions indicate a significant risk from aflatoxin accumulation on chickpea. This study provides useful baseline data on conditions representing a high and a low risk for contamination of chickpea by aflatoxins which is of greater concern because this pulse is destined mainly for human consumption.

Fusarium graminearum sensu stricto is, worldwide, the main causal agent of Fusarium head blight in small cereal crops such as wheat, barley, and oat. The pathogen causes not only reductions in yield and grain quality but also contamination with type-B trichothecenes such as deoxynivalenol. Prevention strategies include the use of less susceptible cultivars through breeding programs, cultural practices, crop rotation, fungicide application, or a combination of them through an integrated pest management. Additionally, the use of more eco-friendly strategies by the evaluation of microorganisms and natural products is increasing. The effect of combining Bacillus velezensis RC218 and chitosan on Fusarium Head Blight (FHB) and deoxynivalenol accumulation under greenhouse and field conditions in bread and durum wheat was evaluated. Under greenhouse conditions, both B. velezensis RC218 and chitosan (0.1%) demonstrated FHB control, diminishing the severity by 38 and 27%, respectively, while the combined treatment resulted in an increased reduction of 54% on bread wheat. Field trials on bread wheat showed a biocontrol reduction in FHB by 18 to 53%, and chitosan was effective only during the first year (48% reduction); surprisingly, the combination of these active principles allowed the control of FHB disease severity by 39 and 36.7% during the two harvest seasons evaluated (2017/18, 2018/19). On durum wheat, the combined treatment showed a 54.3% disease severity reduction. A reduction in DON accumulation in harvested grains was observed for either bacteria, chitosan, or their combination, with reductions of 50.3, 68, and 64.5%, respectively, versus the control.

Maize is affected by Aspergillus flavus and by the contamination of its kernels with aflatoxins, of which AFB(1) is the most important. The Chaco Semi-arid is a region of Argentina in which the climate conditions increase this problem and due to the use of resistant cultivars is the best available tactic to reduce aflatoxin accumulation, we analyzed six hybrids against artificial inoculation with native aflatoxin-producing isolates of A. flavus. A multifactorial experiment was conducted in three complete randomized blocks, under Inoculated and Non-Inoculated treatment, in two locations and during two growing seasons, in which yield, kernel infection, and AFB(1) accumulation (using HPLC) were evaluated. P2089VYHR was outstanding during 2018/19 with 11,089 kg ha(-1), coinciding with the growing season with the highest rainfall and 4% decrease in yield (395 kg ha(-1)) was observed between treatments. No yield differences were observed in the hybrids between localities, or in the hybrid x treatment interaction. The level of infection by A. flavus showed differences between localities and growing seasons but not between hybrids. The Inoculated treatment registered kernel infections above 50%, while natural infection was 35% during 2019/20, associated with rainfall below crop requirements during the flowering period. The AFB(1) accumulation analysis identified P2089VYHR and P1366VYHR without AFB(1) accumulation. Yield correlated negatively with the percentage of kernel infection, but not with AFB(1) accumulation, while a positive correlation was observed between these last two parameters. The pathogen causes a decrease in yield in the region and there are hybrids that are resistant to AFB(1).

Maize (Zea mays L.) is an important crop in Argentina. Aspergillus flavus may infect this crop at growing stage and the harvested kernels can be contaminated with aflatoxins (AFs), whose levels may increase during storage. In Argentina, silo bags, a hermetic type of storage system, are widely used. Biocontrol based on competitive exclusion by atoxigenic A. flavus strains is a useful tool for AFs management at pre-harvest stage. The aim of the present study was to evaluate the effect of pre-harvest biocontrol treatments on aflatoxin B1 (AFB1) accumulation in maize stored in silo bags during 3 and 6 months. Three bioformulations based on A. flavus AFCHG2 and ARG5/30 strains were applied during field trials as single and mixed inocula. Harvested kernels were stored in non-hermetic and hermetic silo bags. At initial time (t0), 3 and 6 months (t3 and t6) the following parameters were evaluated: percentage of damaged kernels, moisture content, water activity, Aspergillus section Flavi incidence, relative humidity, O2 and CO2 levels into the silo bags, and AFB1 levels. The biocontrol strains included in the 3 bioformulations were able to infect maize kernels during the field trial and displaced native toxigenic isolates. At t0 control plots showed 10.9 ± 0.4 μg/kg of AFB1 while no AFs were detected in all the treatments. Along the storage assay AFB1 levels varied from not detected (<1 μg/kg) to 20.1 ± 0.8 μg/kg. Hermetic bags were better than non-hermetic bags in preventing AFB1 accumulation. Both single and mixed inocula were effective to control AFB1 accumulation in maize kernels during 3 and 6 months. AFB1 was not detected in kernels from the treatment at field stage with AFCHG2 + ARG5/30 after 6 months of storage into hermetic bags. The application of the biocontrol agents at field stage is an appropriate tool to reduce AFB1 accumulation under storage in hermetic silo bags. This is the first report on biocontrol strategy based on native atoxigenic strains applied at pre-harvest stage to reduce AFB1 accumulation during storage in Argentina.

Fusarium subglutinans and F. temperatum are two important fungal pathogens of maize whose distinctness as separate species has been difficult to assess. We isolated strains of these species from commercial and native maize varieties in Argentina and sequenced >28,000 loci to estimate genetic variation in the sample. Our objectives were to measure genetic divergence between the species, infer demographic parameters related to their split, and describe the population structure of the sample. When analyzed together, over 30% of each species' polymorphic sites (>2,500 sites) segregate as polymorphisms in the other. Demographic modeling confirmed the species split predated maize domestication, but subsequent between-species gene flow has occurred, with gene flow from F. subglutinans into F. temperatum greater than gene flow in the reverse direction. In F. subglutinans, little evidence exists for substructure or recent selective sweeps, but there is evidence for limited sexual reproduction. In F. temperatum, there is clear evidence for population substructure and signals of abundant recent selective sweeps, with sexual reproduction probably less common than in F. subglutinans. Both genetic variation and the relative number of polymorphisms shared between species increase near the telomeres of all 12 chromosomes, where genes related to plant-pathogen interactions often are located. Our results suggest that species boundaries between closely related Fusarium species can be semipermeable and merit further study. Such semipermeability could facilitate unanticipated genetic exchange between species and enable quicker permanent responses to changes in the agro-ecosystem, e.g., pathogen-resistant host varieties, new chemical and biological control agents, and agronomic practices.