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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.) 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 FB 1 . 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 FB 1 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 FB 1 reduction. Laccases included in EEs remained stable at different alcoholic degrees in maize steep liquor (MSL), but no significant FB 1 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 FB 1 , 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 (AFB 1 and AFB 2 ) 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 AFB 1 and 26.6% for AFB 2. 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.) 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 B 1 (AFB 1 ) 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 (t 0 ), 3 and 6 months (t 3 and t 6 ) the following parameters were evaluated: percentage of damaged kernels, moisture content, water activity, Aspergillus section Flavi incidence, relative humidity, O 2 and CO 2 levels into the silo bags, and AFB 1 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 t 0 control plots showed 10.9 ± 0.4 μg/kg of AFB 1 while no AFs were detected in all the treatments. Along the storage assay AFB 1 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 AFB 1 accumulation. Both single and mixed inocula were effective to control AFB 1 accumulation in maize kernels during 3 and 6 months. AFB 1 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 AFB 1 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 AFB 1 accumulation during storage in Argentina., Competing Interests: Declaration of competing interest None., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The effect of water activity (a W ; 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 a W (0.99) and 30 °C, with growth decreasing as the a W of the medium was reduced. Maximum levels of aflatoxins were produced at 0.99 a W and 25 °C after 5 days of incubation for two strains, and at 25 °C and 0.96 a W after 21 days of incubation for the third strain. The aflatoxin concentrations varied considerably depending on the a W and temperature interactions assayed. Two-dimensional profiles of a W 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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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 × 200 nm, ~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 °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 FB 1 accumulation by F. proliferatum RCFP 5033 (p < 0.05). Similar reduction of growth and FB 1 (%) was observed at 0.8 and 8 g L -1 ZnO-NPs under photoperiod or darkness incubation. FB 1 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 a W in darkness at 25 °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 a W . 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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

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.

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.

Aims: The aims of this study were to determine the occurrence of Fusarium graminearum species complex (FGSC) on soybean pods, seeds and roots, including rhizoplane, during the period of soybean crop in rotation with wheat and to evaluate the FGSC dynamics on wheat and soybean residues during two soybean growing seasons in rotation with wheat, particularly F. graminearum sensu stricto (FGss)., Methods and Results: Soybean roots, pods and seeds were analysed during 2012/13 and 2013/14 seasons. The morphological identification of FGSC and mycotoxin analysis was done. Crop residues were taken in both soybean season in wheat rotation and FGss were quantificated by real-time PCR. The results showed that Fusarium species, mainly FGSC, survive in a soybean crop in rotation with wheat. Isolation frequency of these species was higher on soybean pods than on seeds at R6 stage. Deoxynivalenol contamination on soybean seeds was higher in the 2013/14 season in comparison with the 2012/13 season. Low isolation levels of Fusarium species and species that did not belong to FGSC were observed in soybean root, whereas in rhizoplane a higher level was observed. Fusarium species inoculum on residues remained stable during crop succession and the FGSC were recovered from both wheat and soybean residues. Real time PCR data showed a higher DNA concentration of FGss in wheat residues in the first developmental stages of soybean plants, being the levels more significant during 2012/13 season. With regard to soybean residues collected during the wheat growing stages, an increase in DNA from anthesis until wheat harvest was observed., Conclusions: In a no-till production system, the populations of FGSC can colonize wheat and soybean residues to become an inoculum source., Significance and Impact of the Study: This study provides new data on the occurrence of FGSC populations in soybean plant and FGss on residues in soybean-wheat rotation, a cultural practice commonly used in in Argentina., (© 2020 The Society for Applied Microbiology.)

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., (Copyright © 2020 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

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. IMPORTANCE Fusarium 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., (Copyright © 2020 Fumero et al.)

This study examined the effect of interacting conditions of water activity (a W , 0.995, 0.98 and 0.95) and temperature (15, 25 and 30 °C) on growth rate of two Fusarium thapsinum and one F. andiyazi strains isolated from sorghum in Argentina. In addition, the effect of interacting conditions (a W  × temperature × incubation time (7, 14, 21 and 28 days)) on mycotoxin production (moniliformin (MON), fusaric acid (FA) and fusarin C (FUS C)) on a sorghum grain substrate was evaluated. Statistical analysis showed that a W and temperature significantly affected growth of both species, mainly the a W . Incubation time significantly influenced mycotoxin production by both species as well, mostly for FA. Maximum growth rates of the F. thapsinum strains were obtained at the highest a W (0.995) and 25 °C and growth rate decreased as a W and temperature were reduced. The same growth profile was observed for F. andiyazi RCFA09 (maximum growth rates at 0.995-25 °C). Mycotoxin production by both species was detected at the highest a W levels whereas at 0.95 a W only low amounts of MON were produced by F. thapsinum. Maximum MON and FUS C production by both F. thapsinum strains was observed at 0.995 a W and 25-30 °C after 28 days of incubation. Also, F. thapsinum strains showed maximum FA production at the highest a W and temperature but after 14 days; after this incubation time toxin levels significantly decreased. The responses to a W and temperature of F. andiyazi were similar to that of F. thapsinum strains in relation to FA and FUS C production. Maximum levels of FA were detected at the highest a W after 14 days of incubation at 25-30 °C. Fusarin C was produced at all assayed temperatures but maximum levels were detected at 30 °C and 0.995 a W after 28 days of incubation. Two-dimensional profiles on the interactions of a W by temperature were developed from these data to identify conditions that indicate a significant risk from MON, FA and FUS C accumulation on sorghum grains. The results of this study suggest that sorghum grains could be colonized by these species and toxin production can occur, especially during development stages under field conditions at high water activity of grains or during grain storage if the drying process is slow or deficient. To our knowledge, this study described for the first time FUS C production by F. thapsinum and F. andiyazi under interacting conditions of a W , temperature and incubation time on sorghum grains., Competing Interests: Declaration of competing interest None., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The objectives of the present study were to determine the combined effects of chitosan and water activity (a W ) 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 a W 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.

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 (a w ) 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 a w . All evaluated strains were able to produce MON at all a w values tested, but MON production was higher at 0.995-0.982 a w . 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 a w 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., (© 2018 Society of Chemical Industry.)

Fusarium meridionale has been frequently isolated from soybean in Argentina and showed similar pathogenicity as F. graminearum sensu stricto. However, no data on their growth and mycotoxin production under different environmental conditions are yet available. The aims of this study were: to determine the effect of temperature, water activity (a W ) and strain on growth of F. meridionale and to evaluate deoxynivalenol (DON) and nivalenol (NIV) production in a soybean based medium. The results showed that optimal conditions for F. meridionale growth were at 25 °C and 0.98-0.99 a W . Deoxynivalenol production was favored at 25 °C and 0.96 a W while NIV production was strain-dependent, being 30 °C and 0.98 a W optimal conditions for F. meridionale B2300 strain and 20 °C and 0.98 a W for F. meridionale F5043 and F. meridionale 5048 strains. These conditions are similar to those observed at pre-harvest stage in soybean crop, thus control strategies need to be considered to reduce the risk of the occurrence of DON and NIV in harvested grains., (Copyright © 2018. Published by Elsevier B.V.)

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 B 1 (AFB 1 ) 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 AFB 1 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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Fusarium head blight (FHB) caused by Fusarium graminearum species complex is a devastating disease that causes extensive yield and quality losses to wheat around the world. Fungicide application and breeding for resistance are among the most important tools to counteract FHB. Biological control is an additional tool that can be used as part of an integrated management of FHB. Bacillus velezensisRC 218, Brevibacillus sp. RC 263 and Streptomyces sp. RC 87B were selected by their potential to control FHB and deoxynivalenol production. The aim of this work was to test the tolerance of these biocontrol agents to triazole-based fungicides such as prothioconazole, tebuconazole and metconazole. Bacterial growth was evaluated in Petri dishes using the spread plating technique containing the different fungicides. Bacillus velezensisRC 218 and Streptomyces sp. RC 87B showed better tolerance to fungicides than Brevibacillus sp. RC 263. Complete growth inhibition was observed at concentrations of 20 μg ml -1 for metconazole, 40 μg ml -1 for tebuconazole and 80 μg ml -1 for prothioconazole. The results obtained indicate the possibility of using these biocontrol agents in combination with fungicides as part of an integrated management to control FHB of wheat., Significance and Impact of the Study: This study evaluates the possibility to use biocontrol agents (Bacillus velezensisRC 218, Brevibacillus sp. RC 263 and Streptomyces sp. RC 87B) in combination with triazole-based fungicides to control Fusarium head blight in wheat. The evaluation of biocontrol agents' growth under in vitro conditions was carried out in Petri dishes containing either prothioconazole, tebuconazole or metconazole. Viability studies demonstrated that B. velezensisRC 218 and Streptomyces sp. RC 87B were more tolerant to the fungicides evaluated. Results obtained reflect the possibility to use fungicides at low doses combined with biocontrol agents., (© 2018 The Society for Applied Microbiology.)

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., Competing Interests: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript or in the decision to publish the results.

Bacillus subtilis RC 218 was originally isolated from wheat anthers as a potential antagonist of Fusarium graminearum, the causal agent of Fusarium head blight (FHB). It was demonstrated to have antagonist activity against the plant pathogen under in vitro and greenhouse assays. The current study extends characterizing B. subtilis RC 218 with a field study and genome sequencing. The field study demonstrated that B. subtilis RC 218 could reduce disease severity and the associated mycotoxin (deoxynivalenol) accumulation, under field conditions. The genome sequencing allowed us to accurately determine the taxonomy of the strain using a phylogenomic approach, which places it in the Bacillus velezensis clade. In addition, the draft genome allowed us to use bioinformatics to mine the genome for potential metabolites. The genome mining allowed us to identify 9 active secondary metabolites conserved by all B. velezensis strains and one additional secondary metabolite, the lantibiotic ericin, which is unique to this strain. This study represents the first confirmed production of ericin by a B. velezensis strain. The genome also allowed us to do a comparative genomics with its closest relatives and compare the secondary metabolite production of the publically available B. velezensis genomes. The results showed that the diversity in secondary metabolites of strains in the B. velezensis clade is driven by strains making different antibacterials., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Fusarium graminearum and 21 related species comprising the F. sambucinum species complex lineage 1 (FSAMSC-1) are the most important Fusarium Head Blight pathogens of cereal crops world-wide. FSAMSC-1 species typically produce type B trichothecenes. However, some F. graminearum strains were recently found to produce a novel type A trichothecene (NX-2) resulting from functional variation in the trichothecene biosynthetic enzyme Tri1. We used a PCR-RFLP assay targeting the TRI1 gene to identify the NX-2 allele among a global collection of 2515 F. graminearum. NX-2 isolates were only found in southern Canada and the northern U.S., where they were observed at low frequency (1.8%), but over a broader geographic range and set of cereal hosts than previously recognized. Phylogenetic analyses of TRI1 and adjacent genes produced gene trees that were incongruent with the history of species divergence within FSAMSC-1, indicating trans-species evolution of ancestral polymorphism. In addition, placement of NX-2 strains in the TRI1 gene tree was influenced by the accumulation of nonsynonymous substitutions associated with the evolution of the NX-2 chemotype, and a significant (P<0.001) change in selection pressure was observed along the NX-2 branch (ω=1.16) in comparison to other branches (ω=0.17) in the TRI1 phylogeny. Parameter estimates were consistent with positive selection for specific amino-acid changes during the evolution of NX-2, but direct tests of positive selection were not significant. Phylogenetic analyses of fourfold degenerate sites and intron sequences in TRI1 indicated the NX-2 chemotype had a single evolutionary origin and evolved recently from a type B ancestor. Our results indicate the NX-2 chemotype may be indigenous, and possibly endemic, to southern Canada and the northern U.S. In addition, we demonstrate that the evolution of TRI1 within FSAMSC-1 has been complex, with evidence of trans-species evolution and chemotype-specific shifts in selective constraint., (Published by Elsevier Inc.)

Biological control is one of the most promising strategies for preventing aflatoxin contamination in peanuts at field stage. A population of 46 native Aspergillus flavus nonaflatoxin producers were analysed based on phenotypic, physiological and genetic characteristics. Thirty-three isolates were characterized as L strain morphotype, 3 isolates as S strain morphotype, and 10 isolates did not produce sclerotia. Only 11 of 46 non-aflatoxigenic isolates did not produce cyclopiazonic acid. The vegetative compatibility group (VCG) diversity index for the population was 0.37. For field trials we selected the non-aflatoxigenic A. flavus AR27, AR100G and AFCHG2 strains. The efficacy of single and mixed inocula as potential biocontrol agents in Northern Argentina was evaluated through a 2-year study (2014-2015). During the 2014 peanut growing season, most of the treatments reduced the incidence of aflatoxigenic strains in both soil and peanut kernel samples, and no aflatoxin was detected in kernels. During the 2015 growing season, there was a reduction of aflatoxigenic strains in kernel samples from the plots treated with the potential biocontrol agents. Reductions of aflatoxin contamination between 78.36% and 89.55% were observed in treated plots in comparison with the un-inoculated control plots. This study provides the first data on aflatoxin biocontrol based on competitive exclusion in the peanut growing region of Northern Argentina, and proposes bioproducts with potential use as biocontrol agents., (Copyright © 2016. Published by Elsevier B.V.)

The aim of this study was to evaluate the occurrence of several fungal metabolites, including mycotoxins in natural grasses (Poaceae) intended for grazing cattle. A total number of 72 and 77 different metabolites were detected on 106 and 69 grass samples collected during 2011 and 2014, respectively. A total of 60 metabolites were found across both years. Among the few mycotoxins considered toxic for ruminants, no samples of natural grasses were contaminated with aflatoxins, ochratoxin A, ergot alkaloids, and gliotoxin, among others. However, we were able to detect important metabolites (toxic to ruminants) such as type A trichothecenes, mainly T-2 toxin and HT-2 toxin (up to 5000 µg/kg each), and zearalenone (up to 2000 µg/kg), all at very high frequencies and levels. Other fungal metabolites that were found to be prevalent were other Fusarium metabolites like beauvericin, equisetin and aurofusarin, metabolites produced by Alternaria spp., sterigmatocystin and its precursors and anthrachinone derivatives. It is important to point out that the profile of common metabolites was shared during both years of sampling, and also that the occurrence of important metabolites is not a sporadic event. Considering that this area of temperate grassland is used for grazing cattle all year long due to the richness in palatable grasses (Poaceae), the present work represents a starting point for further studies on the occurrence of multi-mycotoxins in natural grasses in order to have a complete picture of the extent of cattle exposure. Also, the present study shows that the presence of zeranol in urine of beef cattle may not be a consequence of illegal use of this banned substance, but the product of the natural occurrence of zearalenone and α-zearalenol in natural grasses intended for cattle feeding.

Fusarium proliferatum is a member of the Fusarium fujikuroi species complex (FFSC) involved in the maize ear rot together with Fusarium verticillioides, which is a very closely related species. Recently, different studies have detected natural fumonisin contamination in wheat kernels and most of them have shown that the main species isolated was F. proliferatum. Fusarium strains obtained from freshly harvested durum wheat samples (2008 to 2011 harvest seasons) from Argentina were characterized through a phylogenetic analysis based on translation elongation factor-1 alpha (EF-1α) and calmodulin (CaM) genes, determination of mating type alleles, and evaluation of fumonisin production capability. The strains were identified as F. proliferatum (72%), F. verticillioides (24%) and other Fusarium species. The ratio of mating type alleles (MAT-1 and MAT-2) obtained for both main populations suggests possible occurrence of sexual reproduction in the wheat fields, although this seems more frequent in F. proliferatum. Phylogenetic analysis revealed greater nucleotide variability in F. proliferatum strains than in F. verticillioides, however this was not related to origin, host or harvest year. The fumonisin-producing ability was detected in 92% of the strains isolated from durum wheat grains. These results indicate that F. proliferatum and F. verticillioides, among the fumonisin producing species, frequently contaminate durum wheat grains in Argentina, presenting a high risk for human and animal health., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Mycotoxins including aflatoxins, deoxynivalenol, fumonisins and ochratoxin A are among the main fungal secondary metabolites detected as natural contaminants in South America in different commodities such as peanuts (aflatoxins), cereals (deoxynivalenol and fumonisins) or grapes (ochratoxin A). Different strategies including crop rotation, tillage practices, fungicide application and planting less susceptible cultivars are used in order to reduce the impact of these mycotoxins in both food and feed chains. The development of fungicide resistance in many fungal pathogens as well as rising of public concern on the risks associated with pesticide use led to the search for alternative environmentally friendly methods. Biological control of plant pathogens and toxigenic fungi offers an alternative that can complement chemical control in the frame of an integrated pest management to reduce the impact of mycotoxins in the food and feed chains. The advances made in Argentina on reducing the impact of toxigenic fungi and mycotoxins in peanut, grapes and cereals using the biocontrol strategy are summarised. Native bacteria, yeasts and filamentous fungi have been selected to evaluate them as potential biocontrol agents. Field trials showed that Bacillus subtilis RC 218 and Brevibacillus sp. RC 263 were effective at reducing deoxynivalenol accumulation in wheat. The application of Clonostachys rosea isolates on wheat stubble reduced Fusarium colonisation on the stubble. Bacillus amyloliquefaciens and Microbacterium oleovorans showed good activity to control both Fusarium verticillioides growth and the accumulation of fumonisins at pre-harvest stage in maize. Control of toxigenic Aspergillus flavus and aflatoxin accumulation in peanuts was achieved using a native atoxigenic Aspergillus flavus strain based on competitive exclusion of the toxigenic strains. Kluyveromyces thermotolerans strains were used as biocontrol agents to reduce the impact of Aspergillus section Nigri and ochratoxin A accumulation in grapes.

Fusarium poae is a relatively weak pathogen with increasing importance in cereal grains, principally due to its capacity to produce several mycotoxins. In this study, we evaluated the pathogenicity and toxin accumulation of individual F. poae isolates on wheat and barley under natural conditions for 3 years. Analysis of variance demonstrated significant differences for year-genotype, year-isolate, genotype-isolate, and year-genotype-isolate interactions for both incidence and disease severity. Based on contrast analysis, 'Apogee' was more susceptible than the other wheat genotypes, wheat genotypes were more susceptible than barley genotypes, durum wheat genotypes were more susceptible than bread wheat genotypes, and barley genotype 'Scarlett' had greater symptom development per spike than the other barley genotypes. Neither HT-2 nor T-2 toxins were detected in the grain samples. However, high levels of nivalenol were found in both wheat and barley samples. The increased reported isolation of F. poae from wheat and barley and the high capacity of this fungus to produce nivalenol underlie the need for more studies on F. poae-host interactions, especially for barley.

Background: Aspergillus species belonging to section Nigri are the main fungi responsible for ochratoxin (OTA) contamination in grapes and wine. These species live as saprophytes in the superficial layer of the vineyard soil. We evaluated the biodiversity of potentially ochratoxigenic strains of Aspergillus section Nigri isolated from vineyard soils from different grapevine growing regions of Argentina. The isolates were characterized by classical and molecular methods. A multiple correspondence analysis was performed to identify the overall correlation of the Aspergillus group distribution with environmental conditions, geographical characteristics and vineyard practices., Results: Aspergillus niger aggregate was the prevalent group (71%) and A. carbonarius made up only 2%. Species discrimination by species-specific primers showed that in A. niger aggregate 89% were A. tubingensis; 97% of the uniseriate were A. japonicus/A. aculeatus. Isolates belonging to these groups were unable to produce OTA. Our results clearly demonstrate a strong association between presence of A. carbonarius, high average temperatures and drip irrigation. Precipitation levels appear as a secondary factor, and altitude, vineyard age, predominant species, grape variety or total fungal count showed no association with A. carbonarius., Conclusion: We demonstrated a low prevalence of ochratoxigenic species in vineyard soil from the grape-growing regions of Argentina., (© 2014 Society of Chemical Industry.)

The effect of water activity (aW; 0.995, 0.99, 0.98, 0.96, 0.94, 0.92, and 0.90), temperature (15, 25, and 30°C), incubation time (7, 14, 21 and 28days), and their interactions on mycelial growth and fumonisin production on wheat-based medium by three Fusarium proliferatum strains isolated from wheat in Argentina was evaluated. Maximum growth rates were obtained at the highest aW (0.995) and 30°C, with growth decreasing as the aW of the medium was reduced. Maximum amounts of total fumonisins (FB1, FB2 and FB3) were produced at 0.99 aW and 25°C after 21 and 28days of incubation for 2 strains, and at 15°C and 0.98 aW after 28days of incubation for the third strain. The fumonisin concentrations varied considerably depending on the aW and temperature interactions assayed. The studied strains had different fumonisin production profiles. F. proliferatum ITEM 15661 and ITEM 15664 produced FB1 and FB2 whereas F. proliferatum ITEM 15654 was able to produce FB1, FB2 and FB3. Interestingly, fumonisin production profiles for each particular strain were related to incubation temperatures. Fumonisins were produced from 15 to 30°C and at aW values of 0.92 to 0.995 after 21 to 28days of incubation. However at 7 and 14days of incubation small amounts of fumonisin were produced at aW lower than 0.94. Two-dimensional profiles of aW by temperature interactions were developed from these data to identify areas where conditions indicate a significant risk from fumonisin accumulation on wheat. Temperature and aW conditions that resulted in fumonisin production are those found during wheat grain development (especially milk and dough stages) in the field. This study provides useful base line data on conditions representing a high and a low risk for contamination of wheat by fumonisins which is becoming of greater concern because this cereal is destined mainly for human consumption., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Aspergillus section Nigri are described as the main source of ochratoxin A (OTA) contamination in grapes and wine worldwide. The knowledge of the factors affecting grape contamination by species included in this section and OTA production is essential to be able to reduce their presence, not only to improve wine quality, but also to maintain their safety. Therefore, the aims of this study were to determine the incidence of Aspergillus section Nigri species harvested in different grape-growing regions from Argentina, their ability to produce OTA, to correlate with meteorological conditions and geographical coordinates with their prevalence and to evaluate the OTA natural occurrence in grapes and wines. The morphological identification showed that Aspergillus niger aggregate species were the most prevalent ones, followed by Aspergillus carbonarius and Aspergillus uniseriate. These populations were confirmed through using AFLP markers and sequencing and, Aspergillus tubingensis was separated from A. niger aggregate. Climatic factors, altitude, longitude and latitude have influenced on the distribution of species included in the section. A. carbonarius and A. niger were OTA producers but differed in their OTA producing ability. Temperature was the factor which influenced the most over the highest incidence of A. carbonarius in La Rioja and San Juan regions. The trellis system in vineyards and drip irrigation also influenced the species isolation. The OTA levels detected in grapes and wines were low, but grape variety was more important in susceptibility to fungal infection and OTA levels., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The objectives of this study were to evaluate the ability to produce alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) by A. alternata and A. infectoria strains recovered from wheat kernels obtained from one of the main production area in Argentina; to confirm using AFLPs molecular markers the identify of the isolates up to species level, and to evaluate the intra and inter-specific genetic diversity of these two Alternaria species. Among all the Alternaria strains tested (254), 84% of them were able to produce mycotoxins. The most frequent profile of toxin production found was the co-production of AOH and AME in both species tested. TA was only produced by strains of A. alternata. Amplified fragment polymorphism (AFLPs) analysis was applied to a set of 89 isolates of Alternaria spp (40 were A. infectoria and 49 were A. alternata) in order to confirm the morphological identification. The results showed that AFLPs are powerful diagnostic tool for differentiating between A. alternata and A. infectoria. Indeed, in the current study the outgroup strains, A. tenuissima was consistently classified. Characteristic polymorphic bands separated these two species regardless of the primer combination used. Related to intraspecific variability, A. alternata and A. infectoria isolates evaluated seemed to form and homogeneous group with a high degree of similarity among the isolates within each species. However, there was more scoreable polymorphism within A. alternata than within A. infectoria isolates. There was a concordance between morphological identification and separation up to species level using molecular markers. Clear polymorphism both within and between species showed that AFLP can be used to asses genetic variation in A. alternata and A. infectoria. The most important finding of the present study was the report on AOH and AME production by A. infectoria strains isolated from wheat kernels in Argentina on a semisynthetic media for the first time. Also, specific bands for A. alternata and A. infectoria have been identified; these may be useful for the design of specific PCR primers in order to differentiate these species and to detect them in cereals.

Aims: The objective of this work was to design an amplified fragment length polymorphism (AFLP)-derived specific primer for the detection of Fusarium solani aetiological agent of peanut brown root rot (PBRR) in plant material and soil., Methods and Results: Specific primers for the detection of the pathogen were designed based on an amplified region using AFLPs. The banding patterns by AFLPs showed that isolates from diseased roots were clearly distinguishable from others members of the F. solani species complex. Many bands were specific to F. solani PBRR, one of these fragments was selected and sequenced. Sequence obtained was used to develop specific PCR primers for the identification of pathogen in pure culture and in plant material and soil. Primer pair FS1/FS2 amplified a single DNA product of 175 bp. Other fungal isolates occurring in soil, included F. solani non-PBRR, were not detected by these specific primers. The assay was effective for the detection of pathogen from diseased root and infected soils., Conclusions: The designed primers for F. solani causing PBRR can be used in a PCR diagnostic protocol to rapidly and reliably detect and identify this pathogen., Significance and Impact of the Study: These diagnostic PCR primers will aid the detection of F. solani causing PBRR in diseased root and natural infected soils. The method developed could be a helpful tool for epidemiological studies and to avoid the spread of this serious disease in new areas., (© 2013 The Society for Applied Microbiology.)

Aflatoxins are produced by Aspergillus flavus and A. parasiticus in oil-rich seed and grain crops and are a serious problem in agriculture, with aflatoxin B₁ being the most carcinogenic natural compound known. Sexual reproduction in these species occurs between individuals belonging to different vegetative compatibility groups (VCGs). We examined natural genetic variation in 758 isolates of A. flavus, A. parasiticus and A. minisclerotigenes sampled from single peanut fields in the United States (Georgia), Africa (Benin), Argentina (Córdoba), Australia (Queensland) and India (Karnataka). Analysis of DNA sequence variation across multiple intergenic regions in the aflatoxin gene clusters of A. flavus, A. parasiticus and A. minisclerotigenes revealed significant linkage disequilibrium (LD) organized into distinct blocks that are conserved across different localities, suggesting that genetic recombination is nonrandom and a global occurrence. To assess the contributions of asexual and sexual reproduction to fixation and maintenance of toxin chemotype diversity in populations from each locality/species, we tested the null hypothesis of an equal number of MAT1-1 and MAT1-2 mating-type individuals, which is indicative of a sexually recombining population. All samples were clone-corrected using multi-locus sequence typing which associates closely with VCG. For both A. flavus and A. parasiticus, when the proportions of MAT1-1 and MAT1-2 were significantly different, there was more extensive LD in the aflatoxin cluster and populations were fixed for specific toxin chemotype classes, either the non-aflatoxigenic class in A. flavus or the B₁-dominant and G₁-dominant classes in A. parasiticus. A mating type ratio close to 1∶1 in A. flavus, A. parasiticus and A. minisclerotigenes was associated with higher recombination rates in the aflatoxin cluster and less pronounced chemotype differences in populations. This work shows that the reproductive nature of the population (more sexual versus more asexual) is predictive of aflatoxin chemotype diversity in these agriculturally important fungi.

The natural occurrence of alternariol (AOH) and alternariol monomethyl ether (AME) in soya beans harvested in Argentina was evaluated. Both toxins were simultaneously detected by using HPLC analysis coupled with a solid phase extraction column clean-up. Characteristics of this in-house method such as accuracy, precision and detection and quantification limits were defined by means of recovery test with spiked soya bean samples. Out of 50 soya bean samples, 60% showed contamination with the mycotoxins analyzed; among them, 16% were only contaminated with AOH and 14% just with AME. Fifteen of the positive samples showed co-occurrence of both mycotoxins analyzed. AOH was detected in concentrations ranging from 25 to 211 ng/g, whereas AME was found in concentrations ranging from 62 to 1,153 ng/g. Although a limited number of samples were evaluated, this is the first report on the natural occurrence of Alternaria toxins in soya beans and is relevant from the point of view of animal public health.

A survey was carried out to determine Fusarium species and fumonisin contamination in 55 durum wheat (Triticum turgidum L. var. durum) samples collected during two harvest seasons (2007 and 2008) using HPLC and further LC-MS/MS confirmation. All samples showed Fusarium contamination with infection levels ranging from 8 to 66%, F. proliferatum being the species most frequently isolated during 2007 and the second most frequently isolated one during the 2008 harvest season, respectively. Natural contamination with fumonisins was found in both harvest seasons. In 2007, 97% of the samples showed total fumonisin (FB(1) + FB(2)) levels ranging from 10.5 to 1245.7 ng/g, while very low levels of fumonisins were detected in samples collected during 2008. These results could be explained by differences in the amount of rainfall during both periods evaluated. A selected number (n = 48) of F. proliferatum isolates showed fumonisin production capability on autoclaved rice. This is the first report of the presence of natural fumonisins in durum wheat grains.

Aspergillus section Nigri populations isolated from seven growing regions from Argentina were characterized by sequencing in order to identify species responsible for production of ochratoxin A (OTA) and fumonisins (FB(s)). Sequences of genes encoding calmodulin, β-tubulin, the second largest subunit of RNA polymerase II and translation elongation factor 1 alpha were analysed. The phylogenetic analysis showed the presence of six lineages: A. carbonarius, A. tubingensis, A. niger, A. japonicus, A. homomorphus and A. foetidus grouped in four major clusters. The molecular tools used allowed the identification for the first time of A. homomorphus from vineyards. OTA production confirmed the importance of A. carbonarius as the main ochratoxigenic species isolated and, to a variable degree, of A. niger and A. tubingensis, which were by far the most commonly occurring species on grapes in Argentina. The only strains able to produce OTA and fumonisins (B(2)-B(4)) belong to the A. niger cluster., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The aim of this study was to determine the effects of water activity (a(w)) (0.99-0.90), temperature (15, 25 and 30°C) and their interactions on growth and alternariol (AOH) and alternariol monomethyl ether (AME) production by Alternaria alternata on irradiated soya beans. Maximum growth rates were obtained at 0.980 a(w) and 25°C. Minimum a(w) level for growth was dependent on temperature. Both strains were able to grow at the lowest a(w) assayed (0.90). Maximum amount of AOH was produced at 0.98 a(w) but at different temperatures, 15 and 25°C, for the strains RC 21 and RC 39 respectively. Maximum AME production was obtained at 0.98 a(w) and 30°C for both strains. The concentration range of both toxins varied considerably depending on a(w) and temperature interactions. The two metabolites were produced over the temperature range 15 to 30°C and a(w) range 0.99 to 0.96. The limiting a(w) for detectable mycotoxin production is slightly greater than that for growth. Two-dimensional profiles of a(w)× temperature were developed from these data to identify areas where conditions indicate a significant risk from AOH and AME accumulation on soya bean. Knowledge of AOH and AME production under marginal or sub-optimal temperature and a(w) conditions for growth can be important since improper storage conditions accompanied by elevated temperature and moisture content in the grain can favour further mycotoxin production and lead to reduction in grain quality. This could present a hazard if the grain is used for human consumption or animal feedstuff., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Fusarium species can produce fumonisins (FBs), fusaric acid, beauvericin (BEA), fusaproliferin (FUS) and moniliformin. Data on the natural occurrence of FBs have been widely reported, but information on BEA and FUS in maize is limited. The aims of this study were to establish the occurrence of Fusarium species in different maize hybrids in Mexico, to determine the ability of Fusarium spp. isolates to produce BEA, FUS and FBs and their natural occurrence in maize. Twenty-eight samples corresponding to seven different maize hybrids were analyzed for mycobiota and natural mycotoxin contamination by LC. Fusarium verticillioides was the dominant species (44-80%) followed by F. subglutinans (13-37%) and F. proliferatum (2-16%). Beauvericin was detected in three different hybrids with levels ranging from 300 to 400 ng g(-1), while only one hybrid was contaminated with FUS (200 ng g(-1)). All samples were positive for FB1 and FB2 contamination showing levels up to 606 and 277 ng g(-1), respectively. All F. verticillioides isolates were able to produce FB1 (13.8-4,860 μg g(-1)) and some also produced FB2 and FUS. Beauvericin, FUS, FB1 and FB2 were produced by several isolates including F. proliferatum and F. subglutinans and co-production was observed. This is the first report on the co-occurrence of these toxins in maize samples from Mexico. The analysis of the presence of multiple mycotoxins in this substrate is necessary to understand the significance of these compounds in the human and animal food chains.

The understanding of mycotoxins transfer to biological fluids is challenged by the difficulties in performing and replicating in vivo experiments as well as the lack of suitable methods of analysis to detect simultaneously a range of chemically different metabolites at trace levels. LC-MS/MS has been used herein to study the urinary excretion profile of the mycotoxin deoxynivalenol in human and Wistar rat. Deoxynivalenol and deoxynivalenol glucuronide were found in both human and rat urines, whereas de-epoxydeoxynivalenol and its glucuronide conjugate were only detected in rat urine. The presence of two deoxynivalenol glucuronide isomers in Wistar rat urine has been shown for the first time. Structure confirmation of the detected metabolites was provided by the analysis of fragmentation patterns. A solid phase extraction clean up procedure allowing recoveries in the range 72-102% for deoxynivalenol, de-epoxydeoxynivalenol, and their glucuronide conjugates was optimized. A multiple reaction monitoring method for the simultaneous determination of all investigated metabolites was elaborated allowing the direct detection of deoxynivalenol metabolites without the hydrolysis step. Deoxynivalenol urinary levels in the range 0.003-0.008 μg/ml were detected in healthy human subjects, whereas deoxynivalenol and de-epoxynivalenol levels between 1.9-4.9 μg/ml and 1.6-5.9 μg/ml, respectively were found in administered rat urine. These findings emphasize the relevance of the highly selective and sensitive LC-MS/MS technique for the direct detection and characterization of deoxynivalenol metabolites in complex biological matrices., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Argentina is the fourth largest exporter of wheat in the world. The main pathogen associated with Fusarium Head Blight (FHB) of wheat in Argentina is Fusarium graminearum lineage 7 also termed F. graminearum sensu stricto in the F. graminearum species complex, which can produce the Type B trichothecenes, usually deoxynivalenol (DON) and its acetylated forms (3-ADON and 15-ADON) or nivalenol (NIV). We used a multiplex PCR assay of Tri3, Tri7, and Tri13 to determine the trichothecene genotype of 116 strains F. graminearum collected from three locations in Argentina and then verified the chemotype by chemical analysis. PCR assays and chemical analyses gave the same results for all strains that produced trichothecenes. Most strains (> 92%) had the 15-ADON genotype, with the remaining strains having the DON/NIV genotype. We observed neither the NIV nor the 3-ADON genotypes amongst the strains evaluated. The nine strains with the DON/NIV genotype produced DON when analyzed chemically. Thus, the Argentinean populations of F. graminearum are similar to those from wheat elsewhere in the world, in that all the strains produced DON/15-ADON and belong to lineage 7. However approximately 8% of the strains tested were incorrectly diagnosed as DON/NIV producers with the current multiplex PCR and were only DON producers by chemical analysis., (Copyright © 2011. Published by Elsevier B.V.)

Aims: The objective of this study was to evaluate the biodiversity of Aspergillus section Nigri populations from Argentinean vineyards by morphological, toxigenic and AFLP analysis., Materials and Methods: Five hundred and thirty-eight strains were isolated from grapes during 2006/07 and 2007/08 vintages. The morphological identification and toxigenic profile for all strains isolated were performed. Eighty-eight strains were selected for characterization at species level by AFLP markers. Cluster analysis showed a clear separation into four main groups: A. carbonarius, A. tubingensis, A. niger'aggregate' and Aspergillus'uniseriate'. A. carbonarius strains constituted a homogeneous group, while a high degree of genetic diversity was found within the A. niger'aggregate' and 'A. uniseriate' clusters. The A. tubingensis cluster was the most prevalent group and was clearly separated from A. niger'aggregate'. Ten strains showed 45% homology with A. tubingensis FRR 5720 ex-type strain and were considered as 'atypical' or a closely related species. AFLP results indicate that no genotypical differences can be established between ochratoxigenic and nonochratoxigenic strains., Conclusions: Aspergillus section Nigri populations on grapes were represented mainly by four groups. A. tubingensis species were separated from A. niger'aggregate' group and some of their strains produced OTA., Significance and Impact of the Study: This study provides new data on molecular characterization of Aspergillus section Nigri populations in Argentina., (© 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.)

Aim: The aim of this work was to evaluate the effect of Planococcus ficus infection in red wine grapes on Aspergillus section Nigri and ochratoxin A (OTA) contamination., Methods and Results: During 2006/2007 and 2008/2009 vintages, Merlot, Malbec and Cabernet Sauvignon varieties divided into two categories of grape samples (undamaged and damaged by P. ficus) were evaluated. Regardless of the grape variety and the harvest season evaluated, Aspergillus section Nigri incidence and the mean OTA concentration in damaged berries were significantly higher than that in the undamaged ones (P < 0.05; P < 0.001). The Merlot variety showed the highest level of black aspergilli contamination in damaged grapes during the 2006/2007 vintage (53.5% of infection), whereas Malbec presented the highest incidence during the 2008/2009 vintage (57.6% of infection). The Cabernet Sauvignon variety showed the highest OTA levels, ranging from 0.1 to 140 microg kg(-1)., Conclusions: The presence of P. ficus in vineyards increased the risk of OTA occurrence in grapes, suggesting the need to implement insect control at preharvest stage to reduce the entry of OTA in the wine production chain., Significance and Impact of the Study: This study is the first report on the influence of P. ficus on the potential risk of OTA contamination in grapes.

Maize (Zea mays L.) is one of the main cereals as a source of food, forage and processed products for industry. World production is around 790 million tonnes of maize because as a staple food it provides more than one-third of the calories and proteins in some countries. Stored maize is a man-made ecosystem in which quality and nutritive changes occur because of interactions between physical, chemical and biological factors. Fungal spoilage and mycotoxin contamination are of major concern. Aspergillus and Fusarium species can infect maize pre-harvest, and mycotoxin contamination can increase if storage conditions are poorly managed. Prevention strategies to reduce the impact of mycotoxin in maize food and feed chains are based on using a hazard analysis critical control point systems (HACCP) approach. To reduce or prevent production of mycotoxins, drying should take place soon after harvest and as rapidly as feasible. The critical water content for safe storage corresponds to a water activity (a(w)) of about 0.7. Problems in maintaining an adequately low a(w) often occur in the tropics where high ambient humidity make the control of commodity moisture difficult. Damage grain is more prone to fungal invasion and, therefore, mycotoxin contamination. It is important to avoid damage before and during drying, and during storage. Drying maize on the cob before shelling is a very good practice. In storage, many insect species attack grain and the moisture that can accumulate from their activities provides ideal conditions for fungal activity. To avoid moisture and fungal contamination, it is essential that the numbers of insects in stored maize should be kept to a minimum. It is possible to control fungal growth in stored commodities by controlled atmospheres, preservatives or natural inhibitors. Studies using antioxidants, essential oils under different conditions of a(w), and temperature and controlled atmospheres have been evaluated as possible strategies for the reduction of fungal growth and mycotoxin (aflatoxins and fumonisins) in stored maize, but the cost of these treatments is likely to remain prohibitive for large-scale use.

The objective of this study was to determine the effect of water activity (a(w); 0.995, 0.98, 0.96, 0.94, 0.92, and 0.90), temperature (5, 18, 25, and 30 degrees C), incubation time (7 to 35 days), and their interactions on mycelial growth and alternariol (AOH) and alternariol monomethyl ether (AME) production. Two Alternaria alternata strains isolated from soybeans in Argentina were grown on 2% soybean extract agar. Maximum growth rates were obtained at the highest a(w) (0.995) and 25 degrees C, with growth decreasing as the water availability of the medium was reduced. Maximum amount of AOH was produced at 0.98 a(w) and 25 degrees C for both strains. Maximum AME production was obtained for both strains at 30 degrees C but different a(w) values, 0.92 and 0.94, for the strains RC 21 and RC 39, respectively. The concentrations of both toxins varied considerably depending on the a(w) and temperature interactions assayed. The two metabolites were produced from 5 to 30 degrees C and at a(w) values of 0.92 to 0.995. Although at 5 and 18 degrees C little mycotoxin was produced at a(w) lower than 0.94. Two-dimensional profiles of a(w) by temperature interactions were developed from these data to identify areas where conditions indicate a significant risk from AOH and AME accumulation on soybeans. All the conditions of a(w) and temperature that resulted in maximum production of both toxins are those found during soybean development in the field. Thus, field conditions are likely to be conducive to optimum A. alternata growth and toxin production.

Vineyards located in eight grape growing regions of Argentina during the harvest season 2006/07 were evaluated. The aims of this study were to determine the incidence of Aspergillus section Nigri, their ability to produce ochratoxin A (OTA) and to evaluate the OTA natural occurrence in grapes. Bunches of grapes at maturation stage were collected, and grapes (50 per sample) were plated on Petri dishes containing dichloran-glycerol 18% agar (DG18) and dichloran-rose bengal-chloramphenicol agar (DRBC) media. After an incubation period of 7 days at 25 degrees C, the mycoflora belonging to Aspergillus section Nigri was identified. OTA occurrence and the toxicogenic ability of the strains were analyzed by HPLC. A. niger aggregate strains were dominant showing the highest infection percentage (81%), followed by A. carbonarius (11%) and Aspergillus uniseriate (8%). A. carbonarius strains presented the highest percentage of OTA-producer strains (82%) and the highest toxin levels (mean 202 ng/g). A positive correlation between the isolation percentage of A. carbonarius in grapes and temperature was found. The warmest regions showed the highest A. carbonarius incidence. OTA was detected at low levels in grapes during the survey. OTA levels in grapes and rain at harvest time correlated positively.