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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.)

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.)

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.

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 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 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.)

Biocontrol by competitive exclusion has been developed as the most promising means of controlling aflatoxins in peanuts. A 2-year study was carried out to determine the efficacy of an Aspergillus flavus strain as biocontrol agent to reduce aflatoxin production in peanuts under field conditions in Argentina. The competitive strain used was a nontoxigenic A. flavus (AFCHG2) naturally occurring in peanut from Córdoba, Argentina. The inoculum was produced through solid-state fermentation on long grain rice and applied at rate of 50kg inoculum/ha. The incidence of the released strain within the A. flavus communities in soil and peanuts was determined using the shift in the ratio toxigenic:nontoxigenic and VCG analysis. During the 2009/2010 growing season, treatments produced significant reductions in the incidence of toxigenic isolates of A. flavus/Aspergillus parasiticus in soil and peanuts. However, no preharvest aflatoxin contamination was observed. In the 2010/2011 growing season, plants were exposed to late season drought conditions that were optimal for aflatoxin contamination. Significant reductions in aflatoxin levels averaging 71% were detected in treated plots with different inoculation treatments. The results suggest that using the strategy of competitive exclusion A. flavus AFCHG2 can be applied to reduce aflatoxin contamination in Argentinean peanuts., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Ochratoxin A (OTA) is a mycotoxin commonly present in cereals, grapes, coffee, spices, and cocoa. Even though the main objective of the food and feed chain processors and distributors is to avoid the extended contamination of plant-derived foods and animal feeds with mycotoxins, until now, complete OTA removal from foods and feedstuffs is not feasible. Prevention through pre-harvest management is the best method for controlling mycotoxin contamination. However, in the case that the contamination occurs after this stage, the hazards associated with OTA must be managed through post-harvest strategies. Due to the increasing number of fungal strains resistant to chemical fungicides and the impact of these pesticides on the environment and human health, maximum levels of chemical residues have been regulated in many products. Alternative methods are necessary to substitute or complement treatments with fungicides to control fungi under field or storage conditions. Yeasts are considered one of the most potent biocontrol agents due to their biology and non-toxic properties. Epiphytic yeasts are the major component of the microbial community on the surface of grape berries and they are evolutionarily adapted to this ecological niche. Nowadays, several yeast species included in different genera are considered as potential biocontrol agents to control both, growth of ochratoxigenic Aspergillus species and OTA accumulation.

The efficacy of two strains of Kluyveromyces thermotolerans in preventing the growth and ochratoxin A (OTA) accumulation of ochratoxigenic fungi both "in vitro" and "in situ" was evaluated. The data from this study showed that both yeast strains were able to control Aspergillus carbonarius and A. niger aggregate species growth and ochratoxin A accumulation. The inhibitory effects were dependent on the ochratoxigenic species, yeast strains, a(w) and temperature evaluated and their interactions. Over all conditions assayed, ochratoxin A accumulation was reduced from 3% to 100% and the growth rate from 11% to 82.5%, depending on conditions. These results are promising for future development of a bio-pesticide., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.)

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.)

In this study, ochratoxin A (OTA) occurrence in Argentinean musts, wines and dried vine fruits was evaluated, alongside with the performance of OchraStar(TM) columns for OTA extraction. In all the three matrices analyzed, the OchraStar(TM) columns showed good performance. The analysis of natural occurrence of OTA in the red must and the red wine samples showed low incidence with low levels of mean OTA contamination (0.12 ng/mL and 0.37 ng/mL, respectively), while 60% of the dried vine fruit samples were contaminated with OTA, in levels ranging from 0.26 to 20.28 ng/g.

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.

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.

The aim of this work was to evaluate the fate of ochratoxin A (OTA) content from must to wine during the red wine making process in a pilot scale vinification. The study was done using musts obtained from two red grape varieties (Bonarda and Tempranillo) artificially contaminated with two OTA levels. A duplicate set of tanks of 100 I each was established for each must (Bonarda and Tempranillo). The fermentations were initiated by inoculation of two Saccharomyces spp. strains having different fermentation performance. The must from the Tempranillo variety was spiked with 6 microg/I of OTA while that from the Bonarda variety with 0.3 microg/I of the toxin. Samples were collected at different stages of the process. Performance of the alcoholic and malolactic fermentations was monitored. Titratable and volatile acidity, pH, ethanol, sugar and SO2 concentrations were determined following standard methods proposed by the Office International de la Vigne et du Vin (OIV). OTA analysis was done by HPLC. Detection and quantification limits were 0.01 and 0.1 ng/ml, respectively. The OTA levels during the vinification trials dropped to an average of about 86.5%. The type of Saccharomyces strains used showed no effect on toxin reduction.

Aims: The objectives of this study were: (i) to evaluate genetic relatedness among Aspergillus section Flavi strains isolated from soil and peanut seeds in Argentina; (ii) to determine if AFLP molecular markers could be useful to identify isolates up to species level, and to correlate these markers with the isolates' toxigenic potentials and/or vegetative compatibility group (VCG) affiliations., Methods and Results: Amplified fragment length polymorphism (AFLPs) analysis was applied to compare 82 isolates of Aspergillus section Flavi. Cluster analysis showed a clear separation of A. flavus and A. parasiticus, and comparison of fingerprints revealed several specific markers for each group of isolates. AFLP analysis indicates that no genotypical differences can be established between aflatoxigenic and nonaflatoxigenic producers in both species analysed. In addition, candidate AFLP markers associated with a particular VCG were not found., Conclusions: There was a concordance between morphological identification and separation up to species level using molecular markers. The findings of specific bands for A. flavus and A. parasiticus may be useful for the design of specific PCR primers in order to differentiate these species and detect them in food., Significance and Impact of the Study: The present study provides new data on molecular characterization of Aspergillus section Flavi in Argentina.

Aims: The aims of this work were to identify potential sources of Aspergillus parasiticus inoculum and to evaluate the sclerotial and toxigenic profiles of this species from the peanut agroecosystem in Argentina. Likewise, the genetic diversity of A. parasiticus population was analysed using vegetative compatibility group (VCG) analysis., Methods and Results: The A. parasiticus strains were isolated from soil, debris and peanut seeds in Córdoba Province, Argentina. A. parasiticus was recovered from the three sources analysed. Only 11 of 185 A. parasiticus isolates (5.9%) did not produce aflatoxins, while 57% produced sclerotia. Twenty-four VCG were identified from 63 isolates. The VCG diversity index for A. parasiticus, expressed as the number of groups divided by the total number of isolates, was 0.31. In general, there were significant differences among VCG in aflatoxin production., Conclusions: The presence of aflatoxigenic strains of A. parasiticus in the three substrates suggests that they may be an important source of aflatoxin in Argentina's peanut agroecosystem. The A. parasiticus population shows a low genetic diversity., Significance and Impact of the Study: The present study showed data on inoculum distribution, aflatoxin and sclerotia production and genetic diversity in an A. parasiticus population isolated from the peanut agroecosystem in Argentina.