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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.

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Abstract 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.

Resumen Las micotoxinas son metabolitos secundarios producidos por diferentes especies fúngicas pertenecientes, principalmente, a los géneros Aspergillus, Fusarium, Penicillium y Alternaria. Dichos microorganismos pueden crecer en una gran variedad de cultivos, entre los que se incluyen cereales, oleaginosas y frutas. La presencia de micotoxinas en alimentos y subproductos no sólo afecta la salud humana y animal, sino que también causa pérdidas importantes en los mercados nacionales e internacionales. Esta revisión proporciona datos sobre la prevalencia de especies fúngicas toxigénicas y de micotoxinas en diferentes cultivos y productos cosechados en Argentina. Dicha información será relevante para establecer estrategias de manejo adecuadas para reducir la entrada de las micotoxinas en las cadenas alimentarias del hombre y de los animales, así como para establecer futuras legislaciones sobre los niveles máximos permitidos de dichos metabolitos.

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This study examined the effect of interacting conditions of water activity (aW, 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 (aW × 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 aW and temperature significantly affected growth of both species, mainly the aW. 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 aW (0.995) and 25 °C and growth rate decreased as aW 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 aW levels whereas at 0.95 aW 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 aW and 25-30 °C after 28 days of incubation. Also, F. thapsinum strains showed maximum FA production at the highest aW and temperature but after 14 days; after this incubation time toxin levels significantly decreased. The responses to aW 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 aW 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 aW after 28 days of incubation. Two-dimensional profiles on the interactions of aW 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 aW, temperature and incubation time on sorghum grains.

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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 (aW) 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 aW. Deoxynivalenol production was favored at 25 °C and 0.96 aW while NIV production was strain-dependent, being 30 °C and 0.98 aW optimal conditions for F. meridionale B2300 strain and 20 °C and 0.98 aW 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.

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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.

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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.

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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.

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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.

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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.

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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, ß-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.

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