María L. Ramirez

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.

In vitro control of growth and fumonisin production by Fusarium verticillioides and F. proliferatum using antioxidants under different water availability and temperature regimes

Aims: To examine the effect of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), trihydroxybutyrophenone and propylparaben (PP) (at concentrations of 1–20 mmol l−1) on growth of and fumonisin production by Argentinian strains of Fusarium verticillioides and F. proliferatum.

Impact of osmotic and matric water stress on germination, growth, mycelial water potentials and endogenous accumulation of sugars and sugar alcohols in Fusarium graminearum

Studies were conducted to determine the effect of osmotic (NaCl, glycerol) and matric (PEG 8000) water stress on temporal germination and growth of two F. graminearum strains over the water potential range of −0.7 to −14.0 MPa at 15 and 25 C. The effect on endogenous water potentials and accumulation of sugars and sugar alcohols also were measured. For both strains, germination occurred rapidly over the same range of osmotic or matric potential of −0.7 to −5.6 MPa after 4–6 h incubation. At lower osmotic and matric potentials (−7.0 to −8.4 MPa), there was a lag of up to 24 h before germination. Optimum germ-tube extension occurred between −0.7 and −1.4 MPa for both strains but varied with the solute used. Growth was optimal at −1.4 MPa and 25 C in response to matric stress, with the minimum being about −8.0 and −11.2 MPa at 15 and 25 C, respectively. In contrast, F. graminearum grew fastest at −0.7 MPa and was more tolerant of solute stress modified with either glycerol or NaCl with a minimum of about −14.0 MPa at 15 and 25 C. A decrease in the osmotic/matric water potential of the media caused a large decrease in the mycelial water potential (Ψc) as measured by thermocouple psychrometry. In general, the concentration of total sugar alcohols in mycelia increased as osmotic and matric potential were reduced to −1.2 MPa. However, this increase was more evident in mycelia from glycerol-amended media. The quality of the major sugar alcohol accumulated depended on the solute used to generate the water stress. The major compounds accumulated were glycerol and arabitol on osmotically modified media and arabitol on matrically modified media. In response to matric stress, the concentration of trehalose in colonies generally was higher in the case of osmotic stress. In each water-stress treatment there was a good correlation between Ψc and total sugar alcohol content.

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

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.

Alternaria mycotoxins in sunflower seeds : incidence and distribution of the toxins in oil and meal

A survey of 150 sunflower-seed samples was carried out to evaluate the contamination from infection with Alternaria alternata with alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA). A high percentage of the samples was contaminated with AOH (85%), AME, (47%), and TA (65%). The average levels detected were 187 μg/kg for AOH, 194 μg/kg for AME, and 6,692, μg/kg for TA. When sunflower seeds fermented by Alternaria alternata were processed under laboratory conditions to obtain the oil and meal, different distributions of Alternaria toxins between the oil and the meal were observed: whereas AOH, AME, and TA were detected in the meal, only AME and TA were detected in the oil, and the latter in a low percentage.

Fungal and mycotoxin contamination in Bt maize and non-Bt maize grown in Argentina

A Bt maize hybrid and its non-transgenic counterpart harvested during 2002/2003 and 2003/2004 harvest seasons from different locations within the maize-growing area in Argentina were compared for fungal and mycotoxin contamination. Fusarium species were the most prevalent on both genotypes with an isolation frequency >60% across all locations. The percentage of infection was lower in Bt maize than in non-Bt maize (P<0.05). There were no statistical differences in infection percentage due to genotype, fungicide treatment and their interactions. Fumonisins were detected in all of the samples from all locations and genotypes. Total fumonisin levels (fumonisins B1, B2 and B3) in the Bt and the non-Bt hybrid were significantly different (P<0.001), with lower toxin levels in the Bt maize in all locations but one evaluated during the two harvest season. There was no significant difference in deoxynivalenol levels between Bt and non-Bt maize. Application of the fungicide tebuconazole did not alter either the infe...

Fumonisin production by, and mating populations of, Fusarium section Liseola isolates from maize in Argentina

The relation between the pattern of fumonisin production and the mating population and mating type of Fusarium isolates from maize in Argentina at different maturity stages has been evaluated. Fifty-one isolates of Fusarium species belonging to the Liseola section were identified to mating population and tested for their ability to produce fumonisins (FB 1 , FB 2 and FB 3 ). Only mating populations associated with maize, A ( Fusarium moniliforme , 23 isolates), D ( F. proliferatum , 24 isolates) and E ( F. subglutinans , 4 isolates) were found. All but two isolates of populations A and D produced, when grown on maize substrate, high levels of fumonisins ranging from 0·01 to 3·99 mg g −1 (mean 2·00 mg g −1 and 1·69 mg g −1 , respectively), whereas isolates of population E yielded less than 0·02 mg g −1 (mean 0·01 mg g −1 ). Five isolates of F. proliferatum , all belonging to mating type D − , produced more FB 2′ than FB 1′ which is consistent with finding a relatively large amount of FB 2 (0·01 mg g −1 ; FB 2 /FB 1 = 2·27) in one of the maize samples from which these isolates were obtained. Amounts of FB 3 were similar to FB 2 in cultures of mating population A (mean FB 3 /FB 2 = 0·89), but much lower than FB 2 in cultures of mating population D (mean FB 3 /FB 2 = 0·21).

Impact of water activity and temperature on growth and alternariol and alternariol monomethyl ether production of Alternaria alternata isolated from soybean.

The objective of this study was to determine the effect of water activity (a(w); 0.995, 0.98, 0.96, 0.94, 0.92, and 0.90), temperature (5, 18, 25, and 30 degrees C), incubation time (7 to 35 days), and their interactions on mycelial growth and alternariol (AOH) and alternariol monomethyl ether (AME) production. Two Alternaria alternata strains isolated from soybeans in Argentina were grown on 2% soybean extract agar. Maximum growth rates were obtained at the highest a(w) (0.995) and 25 degrees C, with growth decreasing as the water availability of the medium was reduced. Maximum amount of AOH was produced at 0.98 a(w) and 25 degrees C for both strains. Maximum AME production was obtained for both strains at 30 degrees C but different a(w) values, 0.92 and 0.94, for the strains RC 21 and RC 39, respectively. The concentrations of both toxins varied considerably depending on the a(w) and temperature interactions assayed. The two metabolites were produced from 5 to 30 degrees C and at a(w) values of 0.92 to 0.995. Although at 5 and 18 degrees C little mycotoxin was produced at a(w) lower than 0.94. Two-dimensional profiles of a(w) by temperature interactions were developed from these data to identify areas where conditions indicate a significant risk from AOH and AME accumulation on soybeans. All the conditions of a(w) and temperature that resulted in maximum production of both toxins are those found during soybean development in the field. Thus, field conditions are likely to be conducive to optimum A. alternata growth and toxin production.