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 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.

Evaluation of potential biocontrol agent for aflatoxin in Argentinean peanuts.

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.

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.

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.

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.

Molecular characterization of Aspergillus section Flavi isolates collected from peanut fields in Argentina using AFLPs.

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.

Genetic diversity in Aspergillus parasiticus population from the peanut agroecosystem 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.