Reduction of Fusarium proliferatum growth and fumonisin accumulation by ZnO nanoparticles both on a maize based medium and irradiated maize grains.

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 FB1 accumulation by F. proliferatum RCFP 5033 (p < 0.05). Similar reduction of growth and FB1 (%) was observed at 0.8 and 8 g L-1 ZnO-NPs under photoperiod or darkness incubation. FB1 reduction was observed after 14 and 21 days, although the highest reduction occurred after 14 days under photoperiod incubation (84-98%). No clear light enhancing effect on the antifungal and anti-mycotoxin capability of the ZnO-NPs was observed. Morphological alterations in mycelia and conidia were observed by SEM. Under the in situ assay, the effect of the ZnO-NPs (0, 0.4, 0.8, 2 g kg-1) on growth rates and fumonisin B1, B2 and B3 accumulation by two F. proliferatum strains was evaluated on irradiated maize grains adjusted to 0.995, 0.98 and 0.97 aW in darkness at 25 °C during 21 days. Also, zinc acetate at 0.8 g kg-1 was included to compare their antifungal effect against the same ZnO-NPs concentration. Growth rates decreased significantly as ZnO-NPs concentrations increased. Higher than 60% of growth reduction was observed for both F. proliferatum strains. Zinc acetate significantly reduced growth, although it was less efficient that the same ZnO-NPs concentration. ZnO-NPs reduced total fumonisins accumulation by 71-99% at 0.8-2 g kg-1 ZnO-NPs and 0.98-0.995 aW. Moreover, 0.4 g kg-1 ZnO-NPs also produced significant reduction of the 3 fumonisins. This study showed the application of ZnO-NPs in maize grains could be a low cost and environmental impact strategy to control phytopathogen and toxigenic fungi such as F. proliferatum and to reduce fumonisins accumulation, both during crop development at preharvest stage and during maize storage.

Fusarium graminearum species complex occurrence on soybean and F. graminearum sensu stricto inoculum maintenance on residues in soybean-wheat rotation under field conditions.

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.

Toxigenic fungal species and natural occurrence of mycotoxins in crops harvested in Argentina / Especies fúngicas toxigénicas y ocurrencia natural de micotoxinas en cultivos y productos cosechados en Argentina

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.

Effect of interacting conditions of water activity, temperature and incubation time on Fusarium thapsinum and Fusarium andiyazi growth and toxin production on sorghum grains.

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.

Effect of water activity and temperature on growth and trichothecene production by Fusarium meridionale.

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.

Tolerance of triazole-based fungicides by biocontrol agents used to control Fusarium head blight in wheat in Argentina.

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.

Genetic variability and fumonisin production by Fusarium proliferatum isolated from durum wheat grains in Argentina.

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.

Biological control as a strategy to reduce the impact of mycotoxins in peanuts, grapes and cereals in Argentina.

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.

Development of amplified fragment length polymorphism (AFLP)-derived specific primer for the detection of Fusarium solani aetiological agent of peanut brown root rot.

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.

Natural occurrence of alternariol and alternariol monomethyl ether in soya beans.

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.

Phylogenetic characterization and ochratoxin A--fumonisin profile of black Aspergillus isolated from grapes in Argentina.

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.

Molecular characterization and toxigenic profile of Aspergillus section Nigri populations isolated from the main grape-growing regions in Argentina.

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.

Influence of Planococcus ficus on Aspergillus section Nigri and ochratoxin A incidence in vineyards from Argentina.

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.

Strategies to reduce mycotoxin levels in maize during storage: a review.

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.

Impact of water activity and temperature on growth and alternariol and alternariol monomethyl ether production of Alternaria alternata isolated from soybean.

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.

Fate of ochratoxin A content in Argentinean red wine during a pilot scale vinification / Evolución del contenido de ocratoxina A en vinos tintos argentinos durante el proceso de vinificación a escala piloto

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 l 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 μg/l of OTA while that from the Bonarda variety with 0.3 μg/l 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.

El objetivo del presente trabajo fue evaluar la evolución del contenido de ocratoxina A (OTA) en mostos durante un proceso de vinificación a escala piloto. Se utilizaron mostos de dos variedades de uvas tintas (Bonarda y Tempranillo) contaminados artificialmente con dos niveles distintos de OTA. El ensayo fue llevado a cabo por duplicado en tanques de fermentación de 100 l cada uno. La fermentación se inició mediante la inoculación de dos cepas de Saccharomyces spp. con diferentes características fermentativas. El mosto de la variedad Tempranillo fue contaminado con 6 μg/l de OTA y el mosto de la variedad Bonarda con 0,3 μg/l de la toxina. Se colectaron muestras durante los diferentes estadios del proceso de vinificación. Se estableció el avance de dicho proceso sobre la base de la evolución de las fermentaciones alcohólica y maloláctica. Se determinó la acidez total y volátil, el pH y el contenido de etanol, de azúcar y de SO2 siguiendo los protocolos estándares propuestos por la Oficina Internacional de la Vid y el Vino (OIV). El contenido de OTA se evaluó por HPLC. Los límites de detección y cuantificación fueron 0,01 y 0,1 ng/ml, respectivamente. Los niveles de OTA disminuyeron alrededor del 86,5% al final del proceso de vinificación. El tipo de cepa de Saccharomyces spp. utilizada no tuvo efecto sobre la reducción de OTA.

Aspergillus section Nigri species isolated from different wine-grape growing regions in Argentina.

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.

Effect of environmental factors on tenuazonic acid production by Alternaria alternata on soybean-based media.

AIMS: To determine the effects of water activity (a(W); 0.995-0.90), temperature (5, 18, 25 and 30 degrees C), time of incubation (7-35 days) and their interactions on tenuazonic acid (TA) production on 2% soybean-based agar by two Alternaria alternata strains isolated from soybean in Argentina. METHODS AND RESULTS: TA production by two isolates of A. alternata was examined under interacting conditions of a(W), temperature and time of incubation on 2% soybean-based agar. Maximum TA production was obtained for both strains at 0.98 a(W), but at 30 and 25 degrees C for the strains for RC 21 and RC 39, respectively. The toxin concentration varied considerably depending on a(W), temperature, incubation time and strain interactions. TA was produced over the temperature range from 5 to 30 degrees C and a(W) range from 0.92 to 0.995, however at 5 and 18 degrees C little TA was produced at a(W) below 0.94. Contour maps were developed from these data to identify areas where conditions indicate a significant risk for TA accumulation. CONCLUSIONS: The optimum and marginal conditions for TA production by A. alternata on soybean-based agar were identified. The results indicated that TA production by A. alternata is favoured by different temperatures in different strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Data obtained provide very useful information for predicting the possible risk factors for TA contamination of soybean as the a(W) and temperature range used in this study simulate those occurring during grain ripening. The knowledge of TA production under marginal or sub-optimal temperature and a(W) conditions for growth are relevant as 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.

Fate of ochratoxin A content in Argentinian red wine during a pilot scale vinification.

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.

Population genetic structure of Gibberella zeae isolated from wheat in Argentina.

Gibberella zeae (anamorph Fusarium graminearum) causes Fusarium head blight of wheat. The authors used amplified fragment length polymorphisms (AFLPs) to characterize the genetic structure of two G. zeae populations from commercial wheat fields. The working hypothesis was that sufficient genetic exchange occurs between local populations to prevent significant partitioning of allelic variation. We analysed 216 AFLP loci for 113 isolates collected during the 2002 harvest season. All strains had AFLP profiles typical of G. zeae lineage 7. Both populations were genotypically diverse but genetically similar and potentially part of a larger, randomly mating population, with significant genetic exchange probably occurring between the two subpopulations. Linkage disequilibrium was low, but higher than reported for many other populations of G. zeae, and about 20% of the alleles detected were specific to one of the two subpopulations - results consistent with limited gene exchange between the two subpopulations. This study extends previous work with populations of G. zeae to include those found in Argentina, one of the world's largest wheat growing countries.