Aflatoxin Decontamination in Maize Steep Liquor Obtained from Bioethanol Production Using Laccases from Species within the Basidiomycota Phylum.

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.

Bacillus velezensis RC218 and emerging biocontrol agents against Fusarium graminearum and Fusarium poae in barley: in vitro, greenhouse and field conditions.

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.

Pre-harvest strategy for reducing aflatoxin accumulation during storage of maize in Argentina.

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.

Influence of abiotic factors (water activity and temperature) on growth and aflatoxin production by Aspergillus flavus in a chickpea-based medium.

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.

Combination of Bacillus velezensis RC218 and Chitosan to Control Fusarium Head Blight on Bread and Durum Wheat under Greenhouse and Field Conditions.

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.

Fusarium graminearum Species Complex: A Bibliographic Analysis and Web-Accessible Database for Global Mapping of Species and Trichothecene Toxin Chemotypes.

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.

Fusarium chaquense, sp. nov, a novel type A trichothecene-producing species from native grasses in a wetland ecosystem in Argentina.

The Chaco wetland is among the most biologically diverse regions in Argentina. In collections of fungi from asymptomatic native grasses (Poaceae) from the wetlands, we identified isolates of Fusarium that were morphologically similar to F. armeniacum, but distinct from it by their production of abundant microconidia. All the isolates had identical, or nearly identical, partial sequences of TEF1 and RPB2. But they were distinct from reference sequences from F. armeniacum and Fusarium species closely related to it. Phylogenetic analysis of 34 full-length housekeeping gene sequences retrieved from whole genome sequences of three Chaco wetland isolates, 29 genes resolved the isolates as an exclusive clade within the F. sambucinum species complex. Based on results of the morphological and phylogenetic analysis, we concluded that the Chaco wetland isolates are a distinct and novel species, herein described as Fusarium chaquense, sp. nov., which is closely related to F. armeniacum. F. chaquense in culture can produce the trichothecenes T-2 and HT-2 toxin, neosolaniol, diacetoxyscirpenol, and monoacetoxyscirpenol, as well as beauvericin and the pigment aurofusarin. Genome sequence analysis also revealed the presence of three previously described loci required for trichothecene biosynthesis. This research represents the first study of Fusarium in a natural ecosystem in Argentina.

Key Global Actions for Mycotoxin Management in Wheat and Other Small Grains.

Mycotoxins in small grains are a significant and long-standing problem. These contaminants may be produced by members of several fungal genera, including Alternaria, Aspergillus, Fusarium, Claviceps, and Penicillium. Interventions that limit contamination can be made both pre-harvest and post-harvest. Many problems and strategies to control them and the toxins they produce are similar regardless of the location at which they are employed, while others are more common in some areas than in others. Increased knowledge of host-plant resistance, better agronomic methods, improved fungicide management, and better storage strategies all have application on a global basis. We summarize the major pre- and post-harvest control strategies currently in use. In the area of pre-harvest, these include resistant host lines, fungicides and their application guided by epidemiological models, and multiple cultural practices. In the area of post-harvest, drying, storage, cleaning and sorting, and some end-product processes were the most important at the global level. We also employed the Nominal Group discussion technique to identify and prioritize potential steps forward and to reduce problems associated with human and animal consumption of these grains. Identifying existing and potentially novel mechanisms to effectively manage mycotoxin problems in these grains is essential to ensure the safety of humans and domesticated animals that consume these grains.

Phylogenomic Analysis of a 55.1-kb 19-Gene Dataset Resolves a Monophyletic Fusarium that Includes the Fusarium solani Species Complex.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option available.

Divergence and Gene Flow Between Fusarium subglutinans and F. temperatum Isolated from Maize 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.

Toxigenic fungal species and natural occurrence of mycotoxins in crops harvested in Argentina.

Mycotoxins are secondary metabolites produced by fungal species that mainly belong to Aspergillus, Fusarium, Penicillium and Alternaria, which can grow in a variety of crops including cereals, oilseeds and fruits. Consequently, their prevalence in foods and by-products not only affects human and animal health but also causes important losses in both domestic and international markets. This review provides data about toxigenic fungal species and mycotoxin occurrence in different crops commonly grown in Argentina. This information will be relevant to establish adequate management strategies to reduce the impact of mycotoxins on human food and animal feed chains and to implement future legislation on the maximum permitted levels of these fungal metabolites.

Fumonisin and Beauvericin Chemotypes and Genotypes of the Sister Species Fusarium subglutinans and Fusarium temperatum.

Fusarium subglutinans and Fusarium temperatum are common maize pathogens that produce mycotoxins and cause plant disease. The ability of these species to produce beauvericin and fumonisin mycotoxins is not settled, as reports of toxin production are not concordant. Our objective was to clarify this situation by determining both the chemotypes and genotypes for strains from both species. We analyzed 25 strains from Argentina, 13 F. subglutinans and 12 F. temperatum strains, for toxin production by ultraperformance liquid chromatography mass spectrometry (UPLC-MS). We used new genome sequences from two strains of F. subglutinans and one strain of F. temperatum, plus genomes of other Fusarium species, to determine the presence of functional gene clusters for the synthesis of these toxins. None of the strains examined from either species produced fumonisins. These strains also lack Fum biosynthetic genes but retain homologs of some genes that flank the Fum cluster in Fusarium verticillioides None of the F. subglutinans strains we examined produced beauvericin although 9 of 12 F. temperatum strains did. A complete beauvericin (Bea) gene cluster was present in all three new genome sequences. The Bea1 gene was presumably functional in F. temperatum but was not functional in F. subglutinans due to a large insertion and multiple mutations that resulted in premature stop codons. The accumulation of only a few mutations expected to disrupt Bea1 suggests that the process of its inactivation is relatively recent. Thus, none of the strains of F. subglutinans or F. temperatum we examined produce fumonisins, and the strains of F. subglutinans examined also cannot produce beauvericin. Variation in the ability of strains of F. temperatum to produce beauvericin requires further study and could reflect the recent shared ancestry of these two species.IMPORTANCEFusarium subglutinans and F. temperatum are sister species and maize pathogens commonly isolated worldwide that can produce several mycotoxins and cause seedling disease, stalk rot, and ear rot. The ability of these species to produce beauvericin and fumonisin mycotoxins is not settled, as reports of toxin production are not concordant at the species level. Our results are consistent with previous reports that strains of F. subglutinans produce neither fumonisins nor beauvericin. The status of toxin production by F. temperatum needs further work. Our strains of F. temperatum did not produce fumonisins, while some strains produced beauvericin and others did not. These results enable more accurate risk assessments of potential mycotoxin contamination if strains of these species are present. The nature of the genetic inactivation of BEA1 is consistent with its relatively recent occurrence and the close phylogenetic relationship of the two sister species.

Preliminary Study on the Use of Chitosan as an Eco-Friendly Alternative to Control Fusarium Growth and Mycotoxin Production on Maize and Wheat.

The objectives of the present study were to determine the combined effects of chitosan and water activity (aW) on growth and mycotoxin production in situ on the two most important Fusarium species (F. proliferatum and F. verticillioides) present on maize, and on F. graminearum, the main pathogen causing Fusarium head blight on wheat. Results showed that low-molecular-weight chitosan with more than 70% deacetylation at the lowest dose used (0.5 mg/g) was able to reduce deoxynivalenol (DON) and fumonisin (FBs) production on irradiated maize and wheat grains. Growth rates of F. graminearum also decreased at the lowest chitosan dose used (0.5 mg/g), while F. verticillioides and F. proliferatum growth rates were reduced at 0.98 aW at the highest chitosan dose used (2 mg/g). Since mycotoxins are unavoidable contaminants in food and feed chains, their presence needs to be reduced in order to minimize their effects on human and animal health and to diminish the annual market loss through rejected maize and wheat; in this scenario, pre- and post-harvest use of chitosan could be an important alternative.

Fusarium species and moniliformin occurrence in sorghum grains used as ingredient for animal feed in Argentina.

BACKGROUND: A survey on Fusarium species and moniliformin (MON) occurrence in sorghum grains collected from one of the main sorghum-producing areas of Argentina was conducted. Also, growth of F. thapsinum, one of the main sorghum pathogens, and MON production under different water activity (aw ) conditions on a sorghum-based medium were determined. RESULTS: Infection of sorghum grains by Fusarium species ranged from 82.5 to 99%; closely related species F. verticillioides, F. thapsinum and F. andiyazi were the most frequently recovered, followed by F. proliferatum and F. subglutinans. By sequencing a portion of the translation elongation factor-1α (TEF-1α) gene and by maximum parsimony analysis, F. verticillioides and closely related species were identified as F. thapsinum, F. andiyazi and F. verticillioides. Species within the F. graminearum species complex (FGSC) were isolated in high frequency. Maximum growth rates of 12 F. thapsinum strains were obtained at 0.995 aw . All evaluated strains were able to produce MON at all aw values tested, but MON production was higher at 0.995-0.982 aw . MON was detected in 41% of the samples at levels ranging from 363.2 to 914.2 µg kg-1 . CONCLUSION: This study provides new data on the occurrence of Fusarium species in sorghum grains destined for animal consumption in Argentina. The production of MON at different aw values showed that the toxin can be produced under field conditions. The risk to livestock exposed to daily low levels of MON associated with the toxin occurrence in the sorghum grains analyzed is unknown. © 2018 Society of Chemical Industry.

The Mycotox Charter: Increasing Awareness of, and Concerted Action for, Minimizing Mycotoxin Exposure Worldwide.

Mycotoxins are major food contaminants affecting global food security, especially in low and middle-income countries. The European Union (EU) funded project, MycoKey, focuses on “Integrated and innovative key actions for mycotoxin management in the food and feed chains” and the right to safe food through mycotoxin management strategies and regulation, which are fundamental to minimizing the unequal access to safe and sufficient food worldwide. As part of the MycoKey project, a Mycotoxin Charter (charter.mycokey.eu) was launched to share the need for global harmonization of mycotoxin legislation and policies and to minimize human and animal exposure worldwide, with particular attention to less developed countries that lack effective legislation. This document is in response to a demand that has built through previous European Framework Projects—MycoGlobe and MycoRed—in the previous decade to control and reduce mycotoxin contamination worldwide. All suppliers, participants and beneficiaries of the food supply chain, for example, farmers, consumers, stakeholders, researchers, members of civil society and government and so forth, are invited to sign this charter and to support this initiative.

Characterization and competitive ability of non-aflatoxigenic Aspergillus flavus isolated from the maize agro-ecosystem in Argentina as potential aflatoxin biocontrol agents.

Aspergillus flavus is an opportunistic pathogen and may produce aflatoxins in maize, one of the most important crops in Argentina. A promising strategy to reduce aflatoxin accumulation is the biological control based on competitive exclusion. In order to select potential biocontrol agents among isolates from the maize growing region in Argentina, a total of 512 A. flavus strains were isolated from maize kernels and soil samples. Thirty-six per cent of the isolates from maize kernels did not produce detectable levels of aflatoxins, while 73% of the isolates from soil were characterized as non-aflatoxin producers. Forty percent and 49% of the isolates from maize kernels and soil samples, respectively, were not producers of cyclopiazonic acid (CPA). Sclerotia morphology was evaluated using Czapek Dox media. Eighty-six per cent of the isolates from maize kernels and 85% of the isolates from soil samples were L sclerotia morphotype (average diameter > 400 µm). The remaining isolates did not produce sclerotia. All isolates had MAT 1-1 idiomorph. The competitive ability of 9 non aflatoxigenic strains, 4 CPA(+) and 5 CPA(-), was evaluated in co-inoculations of maize kernels with an aflatoxigenic strain. All evaluated strains significantly (p < 0.05) reduced aflatoxin contamination in maize kernels. The aflatoxin B1 (AFB1) reduction ranged from 6 to 60%. The strain A. flavus ARG5/30 isolated from maize kernels would be a good candidate as a potential biocontrol agent to be used in maize, since it was characterized as neither aflatoxin nor CPA producer, morphotype L, MAT 1-1 idiomorph, and reduced AFB1 content in maize kernels by 59%. This study showed the competitive ability of potential aflatoxin biocontrol agents to be evaluated under field trials in a maize agro-ecosystem in Argentina.

MycoKey Round Table Discussions of Future Directions in Research on Chemical Detection Methods, Genetics and Biodiversity of Mycotoxins.

MycoKey, an EU-funded Horizon 2020 project, includes a series of "Roundtable Discussions" to gather information on trending research areas in the field of mycotoxicology. This paper includes summaries of the Roundtable Discussions on Chemical Detection and Monitoring of mycotoxins and on the role of genetics and biodiversity in mycotoxin production. Discussions were managed by using the nominal group discussion technique, which generates numerous ideas and provides a ranking for those identified as the most important. Four questions were posed for each research area, as well as two questions that were common to both discussions. Test kits, usually antibody based, were one major focus of the discussions at the Chemical Detection and Monitoring roundtable because of their many favorable features, e.g., cost, speed and ease of use. The second area of focus for this roundtable was multi-mycotoxin detection protocols and the challenges still to be met to enable these protocols to become methods of choice for regulated mycotoxins. For the genetic and biodiversity group, both the depth and the breadth of trending research areas were notable. For some areas, e.g., microbiome studies, the suggested research questions were primarily of a descriptive nature. In other areas, multiple experimental approaches, e.g., transcriptomics, proteomics, RNAi and gene deletions, are needed to understand the regulation of toxin production and mechanisms underlying successful biological controls. Answers to the research questions will provide starting points for developing acceptable prevention and remediation processes. Forging a partnership between scientists and appropriately-placed communications experts was recognized by both groups as an essential step to communicating risks, while retaining overall confidence in the safety of the food supply and the integrity of the food production chain.