Molecular characterization of the black Aspergillus isolates responsible for ochratoxin A contamination in grapes and wine in relation to taxonomy of Aspergillus section Nigri.

This work examines ochratoxigenic mycobiota in grapes by ap-PCR analysis sequence analysis of the ITS and IGS regions and ability to produce OTA. A comparison was also made with many reference strains of Aspergillus section Nigri. Based on ap-PCR profiles, derived from two microsatellite primers, three main groups were obtained by UPGMA cluster analysis corresponding to A. carbonarius, A. niger and A. tubingensis. The cophenetic correlation values corresponding to ap-PCR UPGMA analysis revealed a higher genetic variability in A. niger and A. tubingensis than in A. carbonarius. In addition, no genotypical differences could be established between OTA producers and nonproducers in all species analysed. Phylogenetic relationships inferred from ITS and IGS sequences are, mostly, congruent with earlier works. A. niger and A. tubingensis strains were closely related, but not identical, and they clustered into two distinct groups within the A. niger aggregate. Sequence analysis also showed genetic divergences between strains of A. foetidus and the rest of the Aspergillus section Nigri. Additionally, the phylogenetic analysis was consistent in separating a new group of ochratoxigenic strains, frequently isolated from grapes, named A. tubingensis-like. All strains of A. carbonarius analysed by sequence analysis had identical ITS and IGS sequences confirming the lack of significant genetic variability within this important ochratoxigenic species.

Mycobiota and mycotoxin producing fungi from cocoa beans.

The present study reports on the natural mycobiota occurring in cocoa beans, paying special attention to the incidence of fungal species that are potential producers of mycotoxins. The results show that predominant fungi were different species of the genus Aspergillus belonging to section Flavi and Nigri. Of the 214 strains of Aspergillus section Flavi collected from cocoa beans, 120 were identified as A. flavus and 94 as A. tamarii. Of Aspergillus section Nigri 138 strains were isolated, with 132 belonging to A. niger aggregate and 6 to A. carbonarius species. Potential ability to produce aflatoxins (AFs) B1, B2, G1 and G2, cyclopiazonic acid (CPA) and ochratoxin A (OTA) was studied by isolate culture followed by HPLC analysis of these mycotoxins in the culture extracts. Results indicated that 64.1% and 34.2% of the A. flavus strains produced AFs and CPA, respectively. Most of the A. flavus strains presented moderate toxigenicity with mean levels of AFs ranging from 100 ng g(-1) to 1000 ng g(-1). All the CPA-producing strains of A. flavus were highly toxigenic producing >30 microg g(-1) of CPA. Furthermore, 98% of A. tamarii strains produced CPA and over 50% of them were highly CPA toxigenic. With respect to OTA-producing fungi, a high percentage of black aspergilli strains (49.2%) were able to produce OTA. Additionally, most of the OTA-producing isolates were of moderate toxigenicity, producing amounts of OTA from 10 microg g(-1) to 100 microg g(-1). These results indicate that there is a possible risk factor posed by AFs, CPA and OTA contamination of cocoa beans, and consequently, cocoa products.