CHARACTERIZING OF TOXIGENIC STRAINS OF Fusarium BY MOLECULAR BIOLOGY TECHNIQUES.

Mycotoxins are metabolites produced by a wide group of fungi, including the genus Fusarium. Mycotoxins have been identified as natural pollutants to a greater or lesser degree in many agricultural and manufactured products and can cause several toxic effects in humans and animals if they become eaten. 

Maize is one of the most vulnerable cereals to colonization by Fusarium species and hence the contamination by mycotoxins produced by such genus. In this work we have isolated Fusarium strains from maize grains for human and animal feed. We have carried out a comparative study between Potato Dextrose Agar (PDA) and Agar malachite green (AVM) to isolate Fusarium strains, highlighting the latter for its selectivity for isolation Fusarium genus.

We focus on the identification by PCR of Fusarium isolates from maize grains and detection of trichothecenes producing strains (deoxynivalenol (DON) and nivalenol (NIV)). As controls, were used strains reference provided by the Spanish Type Culture Collection (CECT). 

We have selected 377 isolates belonging to Fusarium genus and subjected to PCR analysis using different primers specific. With this method we have identified the species F. graminearum, F. proliferatum, F. oxysporum and F. verticillioides. The PCR technique also allowed the detection of the gene that codifies the production of trichothecenes, as well as isolated producers of DON. 

To check the specificity of the identification of Fusarium strains by PCR, sequencing of the fragment ITS1-5.8S-ITS2 DNA was carried out. With this technique we have identified 43 isolates that could not be identified by PCR.  We have also analyzed the region IGS through technical PCR-RFLP of 96 isolated, founding a high intra and interspecific variability. 

The production of deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), fusarenon X, 15-acetyldeoxynivalenol (15-AcDON) and nivalenol from isolated strains in three different culture media at different periods of incubation, was determined by gas chromatography with mass detector (GS-MS). We studied 19 isolated F. graminearum containing the genes involved in the production of trichothecenes and DON. By GC-MS was found that all strains produced one of the mycotoxins, DON, 3-AcDON and 15-AcDON. The culture medium in which was obtained a higher production of mycotoxin PDA was at 21 days.
    

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