Identification by PCR of Fusarium culmorum strains producing large and small amounts of deoxynivalenol.

Thirty deoxynivalenol-producing F. culmorum strains, isolated from wheat grains, were incubated in vitro and analyzed for trichothecene production. Seventeen strains produced more than 1 ppm of deoxynivalenol and acetyldeoxynivalenol and were considered high-deoxynivalenol-producing strains, whereas 13 F. culmorum strains produced less than 0.07 ppm of trichothecenes and were considered low-deoxynivalenol-producing strains. For all strains, a 550-base portion of the trichodiene synthase gene (tri5) was amplified and sequenced. According to the tri5 data, the F. culmorum strains tested clustered into two groups that correlated with in vitro deoxynivalenol production. For three high-producing and three low-producing F. culmorum strains, the tri5-tri6 intergenic region was then sequenced, which confirmed the two separate clusters within the F. culmorum strains. According to the tri5-tri6 sequence data, specific PCR primers were designed to allow differentiation of high-producing from low-producing F. culmorum strains.

Fungal growth and fusarium mycotoxin content in isogenic traditional maize and genetically modified maize grown in France and Spain.

Fungi of the genus Fusarium are common fungal contaminants of maize and are also known to produce mycotoxins. Maize that has been genetically modified to express a Bt endotoxin has been used to study the effect of insect resistance on fungal infection of maize grains by Fusarium species and their related mycotoxins. Maize grain from Bt hybrids and near-isogenic traditional hybrids was collected in France and Spain from the 1999 crop, which was grown under natural conditions. According to the ergosterol level, the fungal biomass formed on Bt maize grain was 4-18 times lower than that on isogenic maize. Fumonisin B(1) grain concentrations ranged from 0.05 to 0.3 ppm for Bt maize and from 0.4 to 9 ppm for isogenic maize. Moderate to low concentrations of trichothecenes and zearalenone were measured on transgenic as well as on non-transgenic maize. Nevertheless, significant differences were obtained in certain regions. The protection of maize plants against insect damage (European corn borer and pink stem borer) through the use of Bt technology seems to be a way to reduce the contamination of maize by Fusarium species and the resultant fumonisins in maize grain grown in France and Spain.

Toxigenic potential of Fusarium culmorum strains isolated from French wheat.

Sixty F. culmorum strains were isolated from wheat grains collected from different wheat-growing areas in France and from different cultivars. The isolates were grown on autoclaved wheat grain to assess their ability to produce trichothecenes and zearalenone. Fungal biomass was evaluated through the ergosterol grain content. All the isolates produced zearalenone (0.39-1660 mg kg(-1)). Thirty-five of the 60 F. culmorum produced nivalenol (0.11-11.7 mg kg(-1)), 12 of 60 produced fusarenone X (0.05-8.42 mg kg(-1)), five of 60 produced 15-acetyldeoxynivalenol (0.48-27.7 mg kg(-1)), 13 of 60 produced 3-acetyldeoxynivalenol (0.07-21.0 mg kg(-1) and 24 of 60 produced deoxynivalenol (0.92-51.9 mg kg(-1)). According to the results, the distribution of the different chemotypes as well as the high and the low mycotoxin-producing Fusarium strains could not be associated to geographical origin.

Study of the biosynthesis of fumonisins B1, B2 and B3 by different strains of Fusarium moniliforme.

The relationship between fungal growth and the production of fumonisin on maize grain by 25 strains of Fusarium moniliforme of different origins has been investigated. Although sporulation was essentially the same for all the strains (about 10(8) propagules g-1 dry matter), ergosterol assays revealed marked variations in fungal biomass. All strains studied produced highly variable amounts of fumonisin B1, the highest levels being observed in strains of ergosterol content above 400 micrograms g-1. However, no correlation could be established between the synthesized biomass and the quantity of fumonisins produced. We verified that ergosterol is an indicator of mycelial growth, and therefore of the potential toxicity of the analysed grain.

Growth of Fusarium moniliforme and its biosynthesis of fumonisin B1 on maize grain as a function of different water activities.

The production of fumonisin by Fusarium moniliforme during its growth on maize depends on extrinsic factors. In particular, experiments on maize grain at different water activities (aw) (1, 0.95, 0.90, 0.85) have demonstrated the influence of aw on fumonisin biosynthesis, and on fungal growth defined by measurement of ergosterol levels. Fumonisin levels dropped threefold when aw was lowered by 5%, but growth rate was unchanged. A 10% reduction in aw from 1 to 0.90 resulted in a 20-fold drop in fungal growth, and fumonisin production was reduced 300-fold. At a threshold aw of 0.85-0.86, F. moniliforme exhibited virtually no measurable metabolic activity, and hence no fumonisin production.

Labeled proteinase inhibitors: versatile tools for the characterization of serine proteinases in solid-phase assays.

Peptidyl chloromethyl ketones were used for the specific labeling of proteinases by attaching a biotin group to the N-terminal end of the peptide. Such labeled peptide inhibitors allowed the detection and quantitation of proteolytic enzymes immobilized on the plastic surface of a microtiter plate, as well as on nitrocellulose. The validity of these solid-phase assays was demonstrated using subtilisin Carlsberg as a model enzyme and biotinyl-epsilon-aminocaproyl-L-alanyl-L-alanyl-L-propyl-L-phenylal++ + anyl- chloromethyl ketone as a specific reagent. In addition to being usable for the screening of a particular proteinase in a large number of samples, these assays can be adapted for the analysis of specific proteolytic enzyme present in complex mixtures.

Determination of cytotoxic trichothecenes in corn by cell culture toxicity assay.

The great sensitivity of some cell species to toxins has been adapted to a direct biological determination of trichothecene contamination of food and feeds. The murine spleen lymphocyte stimulated by PHA (Phaseolus vulgaris phytohaemagglutinin) appeared to be the most convenient cells because of their particular sensitivity to cytotoxic trichothecenes and the opportunity to translate this cytotoxicity to immunosuppressive hazard, one of the most important concerns for trichothecenes. In this paper, the use of cell cultures was adapted for a survey of corn. The toxins were extracted by aqueous methanol, and the extract was defatted with hexane and purified on a silica gel/Florisil column. This extract was then used for a gas chromatographic (GC) determination and the biological test. The growth of cells was measured by the incorporation of tritiated thymidine (3H Tdr), and the inhibition was expressed by the IC50: concentration of corn extract inhibiting by half the 3H Tdr incorporation. We have tested pure toxins, control corn, corn spiked with T-2 toxin, corn experimentally inoculated with toxigenic Fusarium strains, and naturally contaminated corn. A good correlation exists between IC50 and the T-2 toxin concentration as determined by GC analysis. The response is not affected by the presence of zearalenone or by small amounts of deoxynivalenol. A quantitative evaluation of cytotoxic trichothecenes in corn is valuable in the range of 100 ppb to 10 ppm, expressed as T-2 toxin equivalents. The result is obtained in 48 h.