OCHRATOXIN A AND OCHRATOXIN - PRODUCING ISOLATES OF ASPERGILLUS SECTION NIGRI IN RAISIN IN IRAN

Ochratoxin A (OTA) is a mycotoxin which contaminates different plant products, including cereals, coffee, and dried grape. Aspergillus and penicillium species were identified as the main producer of OTA. Ochratoxin A is a potent nephrotoxin. The presence of OTA in raisin is an important barrier to raisin export. The maximum tolerable level of OTA in raisin is 10 µg /kg. During 2010- 2012 a research was carried out on the monitoring of ochratoxin A and ochratoxin A - producing fungi in raisin of Khorasan, Azarbaijan, and Qazvin provinces. The samples were collected from different processing stages of raisin, storages and factories. To study fungal contamination two methods of plating surface disinfected raisin and washing of raisin surface (suspension) on artificial media were used. Aspergillus section Nigri species were isolated and the growth of Aspergillus section Nigri species which were assumed to produce ochratoxin A were recorded after incubation period. To confirm morphological identification of A. carbonarius isolates, DNA of three A. carbonarius isolates were extracted and their ITS regions of ribomal DNA and β- tubulin genes were amplified by PCR and sequenced. Ochratoxin A contaminations of the raisin samples were estimated using HPLC- IAC method. To study ochratoxin A producing ability of Aspergillus section Nigri species isolated from raisin, 14 isolates selected as representative were cultured on PDB medium and OTA produced by each isolate was evaluated using HPLC- IAC method. All raisin samples were contaminated by Aspergillus section Nigri species. The mean of the assumed ochratoxin A producing -fungi contamination of raisin samples was 56.31 (8-100%) but varied depending on the processing method, raisin variety and location. The highest contamination was found in injured not directly edible raisin. Twenty percent of the isolates belonged to A. carbonarus species and the rest to A. nigra. Ochratoxin A was found in five samples out of 44 (11%) raisin samples at the contamination  levels of 0.4 to 100 ng/g. 71% of tested A. carbonarius isolates produced ochratoxin between 0.78- 108.8 ppb. DNA sequencing data were generated for a part of 18S-ITS1-5.8S-ITS2 and 28S region of the rDNA, and β- tubulin gene region. These sequences were compared to other sequences of A. carbonarus in GenBank and showed 100 % and 99% similarity, respectively.  These data confirmed the morphological examinations. Sequences were submitted to GenBank under the accession numbers KF434631, KF434632, and KF434633 for ITS, and KF434634 ، KF434635 for β- tubulin gene. Since the highest contamination was found in injured raisins, they can be easily removed to reduce contamination by fungi and ochratoxin.