María M. Reynoso

Trichothecene genotypes and chemotypes in Fusarium graminearum strains isolated from wheat in Argentina

Argentina is the fourth largest exporter of wheat in the world. The main pathogen associated with Fusarium Head Blight (FHB) of wheat in Argentina is Fusarium graminearum lineage 7 also termed F. graminearum sensu stricto in the F. graminearum species complex, which can produce the Type B trichothecenes, usually deoxynivalenol (DON) and its acetylated forms (3-ADON and 15-ADON) or nivalenol (NIV). We used a multiplex PCR assay of Tri3, Tri7, and Tri13 to determine the trichothecene genotype of 116 strains F. graminearum collected from three locations in Argentina and then verified the chemotype by chemical analysis. PCR assays and chemical analyses gave the same results for all strains that produced trichothecenes. Most strains (> 92%) had the 15-ADON genotype, with the remaining strains having the DON/NIV genotype. We observed neither the NIV nor the 3-ADON genotypes amongst the strains evaluated. The nine strains with the DON/NIV genotype produced DON when analyzed chemically. Thus, the Argentinean populations of F. graminearum are similar to those from wheat elsewhere in the world, in that all the strains produced DON/15-ADON and belong to lineage 7. However approximately 8% of the strains tested were incorrectly diagnosed as DON/NIV producers with the current multiplex PCR and were only DON producers by chemical analysis.

Population genetic structure of Gibberella zeae isolated from wheat in Argentina

Gibberella zeae (anamorph Fusarium graminearum) causes Fusarium head blight of wheat. The authors used amplified fragment length polymorphisms (AFLPs) to characterize the genetic structure of two G. zeae populations from commercial wheat fields. The working hypothesis was that sufficient genetic exchange occurs between local populations to prevent significant partitioning of allelic variation. We analysed 216 AFLP loci for 113 isolates collected during the 2002 harvest season. All strains had AFLP profiles typical of G. zeae lineage 7. Both populations were genotypically diverse but genetically similar and potentially part of a larger, randomly mating population, with significant genetic exchange probably occurring between the two subpopulations. Linkage disequilibrium was low, but higher than reported for many other populations of G. zeae, and about 20% of the alleles detected were specific to one of the two subpopulations—results consistent with limited gene exchange between the two subpopulations. This study extends previous work with populations of G. zeae to include those found in Argentina, one of the worlds largest wheat growing countries.

Vegetative compatibility and mycotoxin chemotypes among Fusarium graminearum (Gibberella zeae) isolates from wheat in Argentina

Gibberella zeae (anamorph Fusarium graminearum) is the main pathogen causing Fusarium head blight of wheat in Argentina. The objective of this study was to determine the vegetative compatibility groups (VCGs) and mycotoxin production (deoxynivalenol, nivalenol and 3-acetyl deoxynivalenol) by F. graminearum populations isolated from wheat in Argentina. VCGs were determined among 70 strains of F. graminearum isolated from three localities in Argentina, using nitrate non-utilizing (nit) mutants. Out of 367 nit mutants generated, 41% utilized both nitrite and hypoxanthine (nit1), 45% utilized hypoxanthine but not nitrite (nit3), 9% utilized nitrite but not hypoxanthine (NitM) and 5% utilized all the nitrogen sources (crn). The complementations were done by pairing the mutants on nitrate medium. Fifty-five different VCGs were identified and the overall VCG diversity (number of VCGs/number of isolates) averaged over the three locations was 0.78. Forty-eight strains were incompatible with all others, thus each of these strains constituted a unique VCG. Twenty-two strains were compatible with other isolates and were grouped in seven multimembers VCGs. Considering each population separately, the VCG diversity was 0.84, 0.81 and 1.0 for San Antonio de Areco, Alberti and Marcos Juarez, respectively. Toxin analysis revealed that of the 70 strains of F. graminearum tested, only 90% produced deoxynivalenol, 10% were able to produce deoxynivalenol and very low amounts of 3-acetyldeoxynivalenol. No isolate produced nivalenol. The results indicate a high degree of VCG diversity in the F. graminearum populations from wheat in Argentina. This diversity should be considered when screening wheat germplasm for Fusarium head blight resistance.

Natural occurrence of fumonisins and their correlation to Fusarium contamination in commercial corn hybrids growth in Argentina

Fifty commercial corn hybrids with different endosperm characteristics, vegetative cycle length and cross class grown in the same geographical area (Cordoba Province, Argentina) were analysed for fumonisin accumulation. All hybrids analysed showed fumonisin B1 and B2 contamination ranging from 185 to 27,050 ng/g for FB1 and from 40 to 9950 ng/g for FB2. Although most of the hybrids analysed had flint-type endosperm, two hybrids with dent-type endosperm (e.g. Prozea 10 and AX 746) showed the highest level of fumonisin (37,000 ng/g) and more FB2 than FB1 (FB2/FB1 ratio 2.42), respectively. There was no correlation between fumonisin concentration and length of the vegetative cycle. Among 18 hybrids examined for Fusarium species contamination there was also no correlation between fumonisin contamination and the level of infection with Fusarium species (Section Liseola). Eighteen hybrids showed fumonisin levels lower than 1000 ng/g. This result suggests that there is some possibility of selecting hybrids resistant or less susceptible to fumonisin and Fusarium contamination.

Efficacy of antioxidant mixtures on growth, fumonisin production and hydrolytic enzyme production by Fusarium verticillioides and F. proliferatum in vitro on maize-based media

The effect of single or mixtures of antioxidants on the lag phase prior to growth, growth rate, hydrolytic enzyme production ( N -acetyl-β-glucosaminidase, β-D-glucosidase and α-D-galactosidase) and fumonisin production by Fusarium verticillioides and F. proliferatum was evaluated on maize-based media at 25 °C, and under different water activity (a w ) conditions. An increase in the lag phase (h) was observed for both F. verticillioides and F. proliferatum especially with propyl paraben (PP) + butylated hydroxyanisole (BHA) treatments at all a w levels tested. For both species PP alone or in combination with BHA, at concentrations of 0.5 and 1 mM reduced the growth rates by >85% at the three a w levels tested (0.995; 0.98 and 0.95). PP + butylated hydroxytoluene (BHT) or trihydroxybutyrophenone (THBP) were less effective in controlling growth, regardless of a w level. Combinations of PP + BHA reduced the fumonisin concentrations produced by F. verticillioides and F. proliferatum at 0.995 and 0.98 a w significantly. However, at low concentrations of antioxidants (0.5 mM) some stimulation in fumonisin production was observed with some treatments. The efficacy of the treatments was reflected in the impact on enzyme production. In the untreated control the highest total enzyme activity of three hydrolytic enzymes was observed at 0.995 a w after 96 h. All the antioxidant treatments alone or combined resulted in a significant reduction ( P w levels tested. Only 10 mM THBP produced an increase in the total amount of N -acetyl-β-D-glucosidase by both F. verticillioides and F. proliferatum. For the three enzymes single factors: time, a w and antioxidant treatments, most two and all three way interactions were significant ( P

Fusaproliferin, beauvericin and fumonisin production by different mating populations among the Gibberella fujikuroi complex isolated from maize

The production of fumonisins, fusaproliferin and beauvericin by Gibberella fujikuroi different mating populations isolated from maize in Argentina was evaluated. From 203 strains of Fusarium verticillioides (G. fujikuroi mating population A), 193 were fumonisin producers. Among members of mating population A, female fertile strains produced 20% more toxin than female sterile ones. Among 78 Fusarium proliferatum strains (G. fujikuroi mating population D) 65 produced fumonisins. The percentage of strains that were high, intermediate and low level toxin producers varied according to the species evaluated and the area from which the strains were isolated. Fusarium subglutinans (G. fujikuroi mating population E) strains produced low levels or were no fumonisin producers. Strains from both G. fujikuroi mating populations D and E were able to produce fusaproliferin and beauvericin. Among the members of F. subglutinans (G. fujikuroi mating population E) the fusaproliferin production was more constant. Co-production of fumonisin, fusaproliferin and beauvericin among the strains belonging to G. fujikuroi D and E was also observed. The co-production of fumonisin, beauvericin and fusaproliferin in maize need to be considered, since from the toxicological point of view interactions between these toxins could occur. The toxigenic ability of the strains evaluated prompt us that is necessary to determine the natural occurrence of fusaproliferin and beauvericin in Argentinean maize.

Molecular characterization of Aspergillus section Flavi isolates collected from peanut fields in Argentina using AFLPs

Aims:  The objectives of this study were: (i) to evaluate genetic relatedness among Aspergillus section Flavi strains isolated from soil and peanut seeds in Argentina; (ii) to determine if AFLP molecular markers could be useful to identify isolates up to species level, and to correlate these markers with the isolates’ toxigenic potentials and/or vegetative compatibility group (VCG) affiliations.

Occurrence of Fusarium spp. and Fumonisin in Durum Wheat Grains

A survey was carried out to determine Fusarium species and fumonisin contamination in 55 durum wheat (Triticum turgidum L. var. durum) samples collected during two harvest seasons (2007 and 2008) using HPLC and further LC-MS/MS confirmation. All samples showed Fusarium contamination with infection levels ranging from 8 to 66%, F. proliferatum being the species most frequently isolated during 2007 and the second most frequently isolated one during the 2008 harvest season, respectively. Natural contamination with fumonisins was found in both harvest seasons. In 2007, 97% of the samples showed total fumonisin (FB(1) + FB(2)) levels ranging from 10.5 to 1245.7 ng/g, while very low levels of fumonisins were detected in samples collected during 2008. These results could be explained by differences in the amount of rainfall during both periods evaluated. A selected number (n = 48) of F. proliferatum isolates showed fumonisin production capability on autoclaved rice. This is the first report of the presence of natural fumonisins in durum wheat grains.

Natural deoxynivalenol occurrence and genotype and chemotype determination of a field population of the Fusarium graminearum complex associated with soybean in Argentina

Soybean (Glycine max L.), the main source of protein throughout the world, is used both as a food and a feedstuff. Currently, limited information about the occurrence of Fusarium species and mycotoxins in soybean grain and by-products is available. The aims of the present study were: (1) to identify toxigenic Fusarium species associated with soybean during crop reproductive stages; (2) to determine the occurrence of deoxynivalenol (DON) and nivalenol (NIV) in soybean seeds; (3) to determine the genotype and chemotype of selected Fg complex strains using molecular and chemical analysis, respectively; and (4) to characterize the strains using AFLPs markers. One soybean field located at Córdoba Province, Argentina, was monitored and samples of soybean tissue were harvested at three reproductive stages: flowering (R2), full seed (R6) and full maturity (R8). A total of 389 Fusarium strains F. equiseti (40%) was the most frequently species recovered followed by F. semitectum (27%) and F. graminearum (Fg) (11%). From the 40 soybean samples analysed, only two presented detectable DON levels. Based on DON occurrence on soybean seeds at ripening stages, the toxigenic ability of Fg complex strains isolated from soybean seeds, pods and flowers were analysed. The trichothecene genotype was determined by a multiplex PCR using primers based on Tri3, Tri5 and Tri7 toxin genes and then the chemotype was verified by chemical analysis. Most Fg complex strains showed 15-ADON genotype and five strains presented a DON/NIV; these also produced both toxins under in vitro culture. Neither the NIV nor the 3-ADON genotypes were detected among the members of the population evaluated. All the 15-ADON genotype strains were characterized as F. graminearum sensu stricto (lineage 7), while the strains presented a DON/NIV genotype were characterized as F. meridionale (lineage 2). The present study contributes new information on the occurrence of Fusarium species and trichothecenes toxins on soybean at the pre-harvest stages. Also, this is the first report on the chemotype, genotype and lineages among Fg complex isolated from soybean.

Genetic structure of Fusarium verticillioides populations isolated from maize in Argentina

Fusarium verticillioides (sexual stage Gibberella moniliformis) is a common fungal pathogen of maize worldwide that also produces fumonisin mycotoxins. Populations of this fungus can be diverse with respect to neutral and selectable genetic markers. We used vegetative compatibility groups (VCGs) and amplified fragment length polymorphisms (AFLPs) to evaluate the genetic structure of three F. verticillioides populations from commercial maize fields in Argentina. Based on work with similar populations from outside South America, we expected individuals within the populations to be genetically diverse, that genotypic variation would be distributed in a manner consistent with random mating, and that populations from different locations would be genetically indistinguishable from one another. We analysed 62 AFLP loci for 133 fungal isolates. All three populations were genotypically diverse but genetically similar and potentially part of a larger, randomly mating population, with significant genetic exchange occurring between the three subpopulations. There was no evidence for linkage disequilibrium at P = 0.05. The low values of GST, the lack of frequent private alleles, and the lack of a systemic pattern of linkage disequilibrium all suggest that sexual reproduction is sufficiently common in F. verticillioides and that the dispersal of strains is sufficiently efficient for the population of F. verticillioides in the main maize growing region to be a single randomly mating population with no detectable genetic subdivision. Thus differences in disease and/or toxin production observed in this region are best attributed to differences other than the genetic composition of the population.