Chemotypes and genotypes characterization in F. graminearum species complex isolated from soybean in Argentina

BARROS, G.; ALANIZ ZANON M.S.; OVIEDO, M.S.; RAMIREZ, M.L.; REYNOSO M.M.; TORRES, A.; CHULZE, S.

Mérida

Congreso; IV Congreso Latinoamericano de Micotoxicología; 2010

Sociedad Latinoamericana de Micotoxicología

Aim: to characterized the chemotype and genotype diversity among the Fusarium graminearum species complex isolated from soybean. Materials and Methods: For determination of trichothecene chemotype, Fusarium graminearum complex strains were cultured on Erlenmeyer flasks (250 ml) containing 25 g of rice. Ten ml of distilled water was added before autoclaving for 30 min at 121 ºC, twice. Each flask was inoculated with a 3-mm diameter agar disk taken from the margin of a colony grown on synthetic nutrient agar at 25 °C for seven days. Flasks were shaken by hand for 1 week and incubated for 28 days at 25 ºC in dark.  At the end of the incubation period the content of the flask were dried at 50 °C for 24 h and then stored at –20 °C until analyzed for toxin. Toxin analysis was done by using a modified version of that originally reported by Cooney et al. (2001). For Determination of trichothecene genotype, multiplex PCR experiments were conducted with 10–25 ng of fungal DNA in a total volume of 50 mL of 1x reaction buffer containing 1.5 mM MgCl2, 2 U Taq DNA polymerase (Promega), 200 mM dNTPs, 0.2 mM each of the three Tri3 primers (Tri3F971, Tri3F1325 and Tri3R1679) and 0.1 mM each of the remaining four primers (Tri7F340, Tri7R965, 3551H and 4056H) (Quarta et al. 2005, 2006). A negative control, containing all reagents but no DNA, was included with every set of reactions. PCR was conducted in a PTC-2000 Thermal Cycler (MJ Research Inc., Watertown, MA). The PCR conditions were: 94º C, 3 min then 35 cycles of 94º C, 30 s, 53º C, 30 s, 72º C, 1 min, followed by a final extension step of 10 min, 72º C. PCR products were separated by electrophoresis through 2% agarose gels. Gels were stained with 1 mg/ml ethidium bromide and photographed under UV light. Results and Discussion: A total of 40 isolates belonging to the F. graminearum species complex were evaluated for DON and their acetylated derivatives. Chemical analysis showed that 15-ADON was the most frequent chemotype observed among the population (60% of isolates). A 30% of isolated produced only DON and no acetylated derivatives and 7.5% of the strains had an unusual pattern of mycotoxin production because they simultaneously produce DON and NIV. The NIV and 3-ADON chemotype was not observed. PCR assays showed also that 15-ADON was the most frequent genotype observed among the population followed by DON/NIV genotype (12.5%). Out of 5 strains that showed DON/NIV genotype by PCR analysis, 3 of them were able to produce DON and NIV and 2 isolates produced only DON by chemical analysis. Conclusion: The F. graminearum species complex isolated from soybean consisted mostly of the 15-ADON with the remaining strains exhibiting DON/NIV genotype, based on PCR genotype and chemotype determination. We observed neither the NIV nor the 3-ADON genotypes among the member of the population evaluated.