Use of ISSR-PCR analysis to discriminate ochratoxigenic species in the Aspergillus section Nigri.

GIAJ-MERLERA, G.; TORRES, A.M.; REYNOSO M.M.

Rotterdam

Simposio; 7th Conference of The World Mycotoxin Forum and XIII IUPAC International Symposium on Mycotoxins and Phycotoxins; 2012

IUPAC

Aspergillus Section Nigri are worldwide distributed, they can contaminate agricultural products at different stages including pre-harvest, harvest, processing and handling. Some members of this group are able to produce two mycotoxins, ochratoxin A and the B2 fumonisin. These black aspergilli are one of the more difficult groups concerning classification and identification. In the present study, we evaluated the discriminatory power of ISSR-PCR analysis, derived from seven ISSR primers, to distinguish morphotypic species. For this purpose, 25 morphotyped black-spore Aspergillus species isolated from different commodities were identified based on phenotypic criteria and species-specific primers. Additionally, all the strains were tested for OTA production. Species identity was confirmed by partial calmodulin gene sequence analyses. A pair-wise similarity matrix was calculated using the Dice coefficient and the dendrogram was generated by UPGMA. Dendogram showed three different clusters: the uniseriate cluster (I), the A. carbonarius cluster (with two sub-clusters II.A and II.B), and the normal A. niger aggregate cluster (with three sub-clusters named III.A, III.B and III.C). The sub-cluster II.A grouped five strains with a style similarity of 82% with A. carbonarius reference strain (bootstrap value 100%). All of these isolates were OTA producers and were amplified by the A. carbonarius specific primers. One isolate (sub-cluster II.B) showed a similarity of 36% with A. carbonarius. A. carbonarius specific primers and, was not OTA producer. This isolated was confirmed as A. ibericus by sequencing. of 17 A. niger aggregatewere clearly identified as "A. niger similarity with A. nigerFRR5695 reference strain, bootstrap value=100%) confirmed by specific primers (sub cluster III.A); five isolates showed a similarity of 70% with A. tubingensis ITEM4845 (bootstrap value=96%). All these five strains were amplified by the A. tubingensis specific primers and, were not OTA producers (sub cluster III.B). Two strains closely related at this cluster, but not identical, were finally confirmed as A. acidus by sequencing. Based on these results, ISSR-PCR procedure is a reliable tool for identifying the black-spored aspergilli. Besides, species-specific bands were found that represent target sites for the possible development of specific primers for the species.