Characterization bu molecular markers (AFLP) of Aspergillus section Nigri isolated from grapes in Argentina

CHIOTTA M L; REYNOSO M.M.; TORRES A.M; COMBINA M; CHULZE , S.N

Merida

Congreso; VI Latin American Congress of Mycotoxicology and II International Symposium on Algal and Fungal Toxins for Industry; 2010

Sociedad Latinoameriana de Micotoxicologia

Background: Ochratoxin A (OTA) is a naturally occurring mycotoxin  in  vineyards and their presence cause important economic losses. Aspergillus section Nigri mainly  A. carbonarius and A. niger aggregate produce this  toxin in grapes and wines ( Battilani et al. 2006; Belli et al. 2005, Cabañes et al. 2002). The correct identification of ochratoxigenic fungi occurring in grapes is important to determine the potential toxicological risk in the grape growing areas.  Molecular markers like AFLP provide useful tool to help in the identification of black aspergilli. Aim:To identify and determine the genetic variability within Aspergillus section Nigri isolated from grapes in Argentina. Materials and Methods: A total of 562 Aspergillus strains, section Nigri isolated from grapes collected in 6 grape growing regions of Argentina during 2 vintage years were evaluated to determine their ability for OTA production. Out of these strains, 94 were selected for identification at species level by amplified fragment length polymorphism (AFLP) technique. Fungal DNA extraction was done using the cetyltrimethylammnonium bromide (CTAB) method of Murray and Thompson (1980) as modified by Kerényi et al. (1999). AFLPs reactions were performed as described by Vos et al. (1995), as modified by Zeller et al. (2000). Results and Discussion: Among the strains isolated during vintage 2006/07, 32% were OTA producers, the  levels ranging between 0.5 and 1,285 ng/gr. The strains were identified by classic morphology as A. niger aggregate (69%) and as A. carbonarius.(31%) In the 2007/08 vintage, A lower percentage of strains isolated during the 2007/08 vintage were  toxigenic  (18.7%) but the levels of ochratoxin A produced was higher ranging between 0.5 and 7,583 ng/gr. The OTA-producer strains were identified as A. niger aggregate (59%)and the remaining 41% as A. carbonarius (41%). Uniseriate aspergilli species isolated in the two periods evaluated were not OTA- producers. The AFLP analysis  produced a complex fingerprint pattern. A total of 107 bands were generated following amplification with two different primer pairs: EcoRI + TG / MseI + CG, EcoRI + AT / MseI + CG. Based on cluster analysis, it was clear that the isolates can be readlly assigned into five main groups and three of them showed an intraespecific homology higher 50%, represented the same species: A. carbonarius, A. tubingensis and A. niger aggregate. Regarding the cluster Aspergillus uniseriate showed low homology (42%) and high internal heterogeneity. In addition, high divergence was found between A. aculeatus and A. japonicas (similarity of 33%). The firth cluster not was assigned readily to any of the type strain of A. section Nigri evaluated. No genotypic differences could be established between the potential of the strains to produce ochratoxin A, the geographical origin and the year of vintage. Conclusion: There was a concordance between morphological identification and separation up to species level using molecular markers (AFLP). The genome diversity of the black aspergilli in the vineyards of Argentina evidence the occurrence of two well defined species A. carbonarius and A. tubingensis, and the other species  included in A. niger aggregate and uniseriate aspergilli.