Molecular strategy to discriminate between two ochratoxin A producing Aspergillus niger aggregate species isolated from fresh and dried grapes

Aspergillus genus is an ubiquitous fungal group that colonizes a wide range of substrates. A total of 100 Tunisian fungal strains isolated at harvest time from fresh and dried grapes were identified within the section Nigri and tested for their ochratoxin A (OTA) producing abilities. Of the isolates, 45% were identified as Aspergillus tubingensis, 34% as Aspergillus niger, 12% as Aspergillus japonicus and 9% as Aspergillus carbonarius. The OTA production was assessed using Czapeck yeast extract agar which revealed that 25% of the isolates belonging to the Aspergillus niger aggregate were OTA producers, while 97% were identified as belonging to the A. carbonarius species. To distinguish between A. niger and A. tubingensis, PCR was used to amplify the ITS-5.8S rDNA for these two strains. The sequence analysis of the PCR products revealed a full similarity between the two species. Thus, a new molecular strategy has been developed based on the targeting of the gene from nitrate reductase (niiA) and nitrite reductase gene (niaD), as well as the intergenic region of the gene (niiA-niaD). The subsequent screening revealed that two new specific primer pairs can be used to identify specifically A. niger and A. tubingensis.