Influence of a Fusarium culmorum inoculation of wheat on the progression of mycotoxin accumulation, ingredient concentrations and ruminal in sacco dry matter degradation of wheat residues

The Fusarium head blight (FHB)-susceptible winter wheat cv. Ritmo was inoculated with spores of Fusarium culmorum at the beginning of full blossom. Samples of whole wheat plants were taken once weekly from anthesis until harvest and subsequently fractionated into straw, glumes and spindles, which were examined for deoxynivalenol (DON) and zearalenone (ZON). Additionally, the content of crude protein (CP) and non-starch polysaccharides (NSP) was scrutinized. Synthesis of the Fusarium toxins DON and ZON generally differed in terms of date of formation and concentration. Final mean DON concentrations of 37.5, 28.1 and 5.0 mg/kg DM were measured in glumes, spindles and straw, respectively, at the time of harvest. At this time, maximal mean ZON concentrations of 587, 396 and 275 microg/kg DM in spindles, glumes and straw, respectively, were determined. Moreover, Fusarium infected wheat residues contained higher CP but lower NSP contents at the last three sampling dates. In addition, collective samples of wheat straw and chaff were taken to investigate the effect of the Fusarium contamination on their in sacco DM degradation in dairy cows. Samples were analysed for mycotoxins and selected quality parameters. The dried and milled collective samples of straw and chaff were weighed into nylon bags and subjected to ruminal incubation for 4, 8, 16, 24, 48, 72, 96 and 120 h in two dairy cows equipped with a permanent rumen cannula. Marked differences in level of mycotoxin contamination as well as in ingredient composition between the variants of straw and chaff were detected. Moreover, after 120 h rumen incubation the in sacco DM degradation of inoculated straw and chaff were lower compared to the accordant controls. The soluble fraction was increased in inoculated samples, whereas a diminishment in the potentially degradable but insoluble fraction was more pronounced. Thereby, a decrease in the potential degradability was obtained for inoculated straw and even if less pronounced for chaff compared to the non inoculated corresponding controls. In conclusion, infection with F. culmorum of wheat involves an increased risk of mycotoxin contamination in straw. Also, a Fusarium infection may have an impact on chemical composition and may result in Fusarium growth-related modifications of host cell wall components.     

Mycotoxins produced by <Emphasis Type="Italic">Fusarium</Emphasis> spp. associated with Fusarium head blight of wheat in Western Australia

An isolated occurrence of Fusarium head blight (FHB) of wheat was detected in the south-west region of Western Australia during the 2003 harvest season. The molecular identity of 23 isolates of Fusarium spp. collected from this region during the FHB outbreak confirmed the associated pathogens to be F. graminearum, F. acuminatum or F. tricinctum. Moreover, the toxicity of their crude extracts from Czapek-Dox liquid broth and millet seed cultures to brine shrimp (Artemia franciscana) was associated with high mortality levels. The main mycotoxins detected were type B trichothecenes (deoxynivalenol and 3-acetyldeoxynivalenol), enniatins, chlamydosporol and zearalenone. This study is the first report on the mycotoxin profiles of Fusarium spp. associated with FHB of wheat in Western Australia. This study highlights the need for monitoring not just for the presence of the specific Fusarium spp. present in any affected grain but also for their potential mycotoxin and other toxic secondary metabolites.     

Fusarium toxins in bread cereals of the land brandenburg — Comparison of integrated/ecological cultivation of 2000 to 2002

Over a period of three years (2000–2002), wheat and rye samples of integrated and ecological cultivation in the land Brandenburg were analyzed by HPLC for theFusarium toxins deoxynivalenol and zearalenone. In the years 2000 and 2001, the contamination generally was lower than in the year 2002. In the 3 years of testing, the contamination byFusarium toxins was significantly less frequent and lower in cereals of ecological cultivation than in cereals of integrated cultivation. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

On the effects ofFusarium-contaminated wheat and the feed intake level on ruminal fermentation and toxin-turnover of cows

The aim of the study was to examine the effects of dry matter intake level and the feeding ofFusarium-contaminated wheat on the toxin-turnover and ruminal fermentation of dairy cows. Fourteen dairy cows equipped with ruminal and duodenal cannulae were used. All animals were fed the same diet, only the daily feed amounts were adjusted to the current performance stage of the cow. On a dry matter basis, the diet consisted of 60% concentrate including 55% wheat (Fusarium-contaminated wheat [Mycotoxin period] or control wheat [Control period]). Each cow was fed with both the contaminated and the control wheat. TheFusarium-contamination of the wheat significantly decreased the flow of undegraded protein at the duodenum with increased intakes of organic matter. The duodenal flow of microbial protein and the activities of aspartate aminotransferase (ASAT), glutamate dehydrogenase (GLDH) and gamma glutamyl transferase (γ-GT) in the serum were not affected by dietary treatment, but increased with feed intake. The duodenal flow of deoxynivalenol (DON) and de-epoxy DON related to DON intake ranged between 12 and 77% when theFusarium-contaminated wheat was fed. DON was almost completely metabolized to de-epoxy DON independent of the feed intake level. The zearalenone (ZON) flow at the duodenum increased moderately with increasing ZON/feed intake.     

Progression of mycotoxin and nutrient concentrations in wheat after inoculation with Fusarium culmorum

The objective of this study was to follow the mycotoxin formation and changes in nutrient composition of wheat (cv. Ritmo) artificially inoculated with Fusarium culmorum. From anthesis until harvest, samples were taken once a week from the inoculated and control plots. The investigations were focused on monitoring the progression of the contamination of the wheat kernels with deoxynivalenol (DON) and zearalenone (ZON). Both the uncontaminated control kernels and the contaminated kernels were examined also for the presence of zearalenone-4-beta-D-glucopyranoside and several trichothecenes at harvest. Furthermore, the impact of the Fusarium inoculation on some nutrients as starch, crude protein, amino acid composition, crude ash, non starch polysaccharides (NSP) as well as viscosity and thousand seed weight (TSW) was examined. Also proteolytic and amylolytic activity as well as the NSP-degrading enzyme activities of inoculated and control samples were analysed at the time of harvest. DON was detected in higher concentrations and in earlier stages, while ZON was found later and in smaller amounts. On average 7.79?mg/kg DM of DON and 100?μg/kg DM of ZON were found in the inoculated kernels at the time of harvest. Neither in the contaminated nor in the control samples glucose conjugates of ZON (Zearalenone-4-beta-D-glucopyranoside) were detected. Moreover, the infection with Fusarium culmorum had pronounced effects on some quality parameters. The crude protein content of the inoculated kernels showed significantly higher values over the whole period compared to the control kernels. The protein content of the inoculated kernels amounted 13.9% DM at harvest, while only a concentration of 12.5% DM was detected in the control samples. Similarly, in almost all stages of development the crude ash content of inoculated samples was higher than in control samples. These distinct differences in kernel composition resulted possibly from the changes of the thousand seed weight. In the present work the grain harvested from the control plots showed a significantly higher TSW (24.2?g) as compared to their inoculated counterparts (15.5?g). Despite lower extract viscosity of inoculated samples at time of harvest, the content of soluble NSP of inoculated plots was higher than in control samples at the same time. Moreover, inoculation resulted in markedly increased activities of protease, amylase and several NSP-degrading enzyme activities. This would suggest that the cell wall penetrating properties of the fungus itself and/or that the fungus induced alterations of the metabolic activity of the embryo or other constituents of the wheat kernel could be responsible.     

Fusarium toxins in bread cereals of the land Brandenburg in the years 2000 to 2004 — Comparison of integrated and ecological cultivation

Over a period of five years (2000–2004), wheat and rye samples of integrated and ecological cultivation in the land Brandenburg were analyzed by HPLC for theFusarium toxins deoxynivalenol and zearalenone. In the years 2000, 2001, 2003 and 2004 the contamination generally was lower than in the year 2002. In the five years of testing, the contamination byFusarium toxins was significantly less frequent and lower in cereals of ecological cultivation than in cereals of integrated cultivation. Presented at the 27^th Mykotoxin-Workshop, Dortmund, Germmany, June 13–15, 2005 Financial support: The Ministry of rural development, environment and consumer protection of the land Brandenburg     

Short review: Metabolism of theFusarium mycotoxins deoxynivalenol and zearalenone in plants

Plants have a high capacity to transform and thereby detoxify deleterious or poisonous compounds, like mycotoxins. The formation of glucose conjugates has a central role in this process. Mammals, however, are able to (partly) release the precursor substances during digestion, reactivating the mycotoxins. This short review provides a brief summary about the metabolism of theFusarium mycotoxins deoxynivalenol and zearalenone in plants. Two examples are discussed in greater detail. First, the formation of deoxynivalenol-3-glucoside in wheat is linked to a quantitative trait locus that is often used forFusarium head blight resistance breeding. Secondly, the metabolism of zearalenone inArabidopsis thaliana results in at least 17 different metabolites, all of which are potentially hazardous for humans and animals. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006 Financial support: Christian Doppler Society, the Austrian Genome Research Initiative GEN-AU, the Lower Austrian government, the Austrian Science Fund FWF     

Toxigenic Fusarium species infecting wheat heads in Poland

Toxigenic Fusarium species are common pathogens of wheat and other cereals worldwide. In total, 449 wheat heads from six localities in Poland, heavily infected with Fusarium during 2009 season, were examined for Fusarium species identification. F. culmorum was the most common species (72.1% on average) with F. graminearum and F. avenaceum the next most commonly observed, but much less frequent (13.4 and 12.5% respectively). F. cerealis was found in 1.8% of all samples, and F. tricinctum was found only in one sample (0.2%). Subsequent quantification of the three major mycotoxins (deoxynivalenol, zearalenone and moniliformin) in grain and chaff fractions with respect to associated prevailing pathogen species uncovered the following patterns. Moniliformin (MON) was found in low amounts in all samples with F. avenaceum present. In contrast, deoxynivalenol (DON) and zearalenone (ZEA) were the contaminants of F. culmorum- and F. graminearum-infected heads. The highest concentration of DON was recorded in grain sample collected in Radzików (77 µg g^?1). High temperatures in Central Poland during July and August accompanied with high rainfall in July were responsible for this high DON accumulation. Trichothecene, zearalenone, enniatin and beauvericin chemotypes were identified among 21 purified isolates using gene-specific PCR markers.     

Effect of delayed harvest on contamination of pearl millet grain with mycotoxin-producing fungi and mycotoxins

The response to delayed harvest of fungal and mycotoxin contamination of grain of the pearl millet hybrid HGM 100 was examined in 1992 and 1993. Samples of grain were assayed from seven plantings at locations near Tifton, Georgia, USA. Grain was harvested at 30, 40, and 50 days after anthesis and evaluated for infection byFusarium species andAspergillus flavus, and mycotoxin contamination. Mean isolation frequencies ofF. semitectum (35.6%) andF. chlamydosporum (17.2%) increased linearly with delayed harvest.Fusarium moniliforme andF. equisiti were infrequently isolated (<0.5%) and did not increase in the grain when harvest was delayed. Low mean concentrations of zearalenone (0.17 ppm), nivalenol (0.42 ppm), and deoxynivalenol (0.01 ppm) were detected but were not affected by delayed harvest. Isolation frequencies ofF. chlamydosporum andF. equiseti were correlated (P=0.07) with levels of nivalenol.Aspergillus flavus was not isolated from the grain, and aflatoxin concentrations averaged 1.9 ppb.     

Influence of aFusarium culmorum inoculation of wheat onin sacco dry matter degradation of wheat straw and wheat chaff

The aim of this study was to investigate the effect of aFusarium culmorum inoculation of wheat on thein sacco dry matter degradation (DG) of wheat straw and wheat chaff in dairy cows. The ruminal disappearance of dry matter was measured with thein situ nylon bag technique. Samples of wheat straw and wheat chaff from non-inoculated andFusarium-inoculated wheat were used to examine the ruminal dry matter degradability. Samples were subjected to ruminal incubation in two dairy cous fitted with a permanent rumen fistula and incubated for 4, 8, 16, 24, 48, 72, 96 and 120 h. To describe the degradation kinetics, the equation by Ørskov and McDonald (1979) was used. DG rates obtained for contaminates straw and chaff were higher compared to the corresponding rates of the non-contaminated samples, which is assumed to be due to the activity of fungal enzymes. It can be concluded that an infection of wheat withF. culmorum may have an influence on the dry matter degradation of straw and chaff.     

Occurrence of moniliformin,deoxynivalenol, and zearalenone in durum wheat (Triticum durum Desf.)

Forty-eight durum wheat samples from 5 locations in Austria were examined forFusarium infection andFusarium toxin content.F.gramlnearum andF.avenaceum were by far the prevailingFusarium species In durum wheat kernels, followed byEpoae, F.culmorum, andF.equlsetl. Ion-paired HPLC analyses of the samples showed moniliformin contents of kernels up to 0.88 mg/kg. All moniliformin contaminated samples also contained high levels of deoxynivalenol (up to 8.2 mg/kg) and lower levels of zearalenone (<0.33 mg/kg). The levels of zearalenone in naturally contaminated durum wheat samples did not correspond to the high yields of zearalenone found in cultures of the fusaria isolated from the durum wheat kernels. These conflicting results as well as some toxicologlcal aspects of the carry over ofFusarium toxins from durum wheat kernels into pasta are discussed.     

Progression of the mycotoxin and nutrient concentration in wheat after inoculation with<Emphasis Type="Italic">Fusarium culmorum</Emphasis>

The aim of the study was to follow the mycotoxin formation and the changes in nutrient composition of wheat artificially inoculated withFusarium cuimorum. From anthesis until harvest, samples were taken once a week from the inoculated and control plots. The investigations were focused on the progression of the contamination of the wheat with deoxyni-valenol (DON) and zearalenone (ZON). Furthermore, the impact of the Fusarium inoculation on the quality parameters starch, crude protein, crude ash, viscosity, non starch polysaccharides (NSP) and thousand seed weight was examined. Deoxynivalenol was detected in higher concentrations and in earlier stages, while zearalenone was found later and in smaller amounts. Moreover, the infection withFusarium cuimorum had pronounced effects on some quality parameters.     

Effects of Fusarium Toxin Contaminated Wheat and of a Detoxifying Agent on Performance of Growing Bulls,on Nutrient Digestibility in Wethers and on the Carry Over of Zearalenone

Experiments were carried out to examine the effects of a Fusarium contaminated wheat (10mg deoxynivalenol and 0.76mg zearalenone, ZON, perkg dry matter) and of a detoxifying agent (Mycofix ®Plus, Biomin GmbH, Herzogenburg, Austria) on the growing performance of bulls, carry-over of ZON and its metabolites into body fluids and tissues, and on nutrient digestibility in wethers. The experiments were designed according to a complete two by two factorial approach which meant that both the uncontaminated control wheat and the Fusarium toxin contaminated wheat were tested both in the absence and presence of Mycofix ®Plus. The growing experiment with bulls (n = 14 per treatment) covered the live weight range between 244kg and 460kg. The respective wheat batches were included in the concentrate portion at 65%. Concentrates were fed according to plan whereas maize silage was offered for ad libitum consumption. Daily dry matter intake and live weight gain [kg per animal and day] were 7.40, 7.52, 7.51 and 7.49 and 1.367, 1.296, 1.380 and 1.307 for bulls fed the unsupplemented control wheat, the supplemented control wheat, the unsupplemented and Fusarium toxin contaminated wheat and the supplemented Fusarium toxin contaminated wheat, respectively. ZON and its metabolites were not detected in edible tissues. The most striking effects of feeding the Fusarium toxin contaminated wheat on carcass characteristics were a reduced dressing percentage, an increased weight of the emptied gastro-intestinal tract and a reduced weight of the testicles. No effect of the detoxifying agent was seen for these parameters whereas heart weight increased independently of Fusarium toxin contamination of the concentrates. Nutrient digestibility of the two wheat batches, unsupplemented or supplemented with Mycofix ®Plus was evaluated according to the difference method using wethers. Presence of Fusarium toxins in wheat did not influence its feeding value. The effects of the addition of the detoxifying agent were mycotoxin unspecific and resulted in an increase in apparent digestibility of crude protein and a decrease in crude fiber digestibility. It is concluded that feeding of Fusarium toxin contaminated wheat did not adversely affect performance of growing bulls (approximately 2.2mg DON and 0.1mg ZON perkg complete ration at a reference dry matter content of 88%) or nutrient digestibility in wethers. The effects of the detoxifying agent Mycofix ®Plus on growing performance and on nutrient digestibility were rather Fusarium toxin unspecific. The slightly negative effects on growing performance needs to be examined further.     

Pathogenicity of Fusarium Isolates from Wheat, Rye and Triticale Towards Seedlings and their Ability to Produce Trichothecenes and Zearalenone

37 Fusarium isolates (F. culmorum 20, F. graminearum 4, F. avenaceum 3, F. solani 4, and F. equiseti 6 from wheat, rye and triticale) were examined for pathogenicity and tested for zearalenone (F-2) and trichothecenes production in vitro. Strong pathogens (F. culmorum and F. graminearum) produced deoxynivalenol and 3-acetyl-deoxynivalenol and zearalenone in considerable quantities.     

Influence of nitrogen fertilization on deoxynivalenol contamination of winter wheat — experimental field trials and evaluation of analytical methods

Experimental field trials were carried out to study the influence of N-fertilization on deoxynivalenol (DON) contamination of winter wheat. Within four years of investigation, no definite effect of mineral N-input at dosages varying between 0 and 240 kg N/ha could be observed on DON concentration in wheat grain. The main factors affecting DON contamination of wheat were theFusarium infection pressure, the weather conditions and the susceptibility of the wheat varieties againstFusarium head blight. DON was analyzed with enzyme-linked immunosorbent assay (ELISA) and, for comparison, some of the positive samples were additionally analyzed with high performance liquid chromatography (HPLC). There was a good correlation between the ELISA and the HPLC results for DON concentration in wheat.     

Identification of deoxynivalenol, 3-acetyldeoxynivalenol and zearalenone in the galactose oxidase-producing fungus Dactylium dendroides

The galactose oxidase-producing fungus Dactylium dendroides was re-identified as a Fusarium species. Fungi of this genus are well known for the production of mycotoxins. Verification of growth of this fungus on rice, corn and liquid medium described for the production of galactose oxidase is provided to determine whether the fungus could produce Fusarium toxins, namely, moniliformin, fusaric acid, fumonisin, zearalenone and the trichothecenes, deoxynivalenol, 3-acetyldeoxynivalenol, fusarenone, nivalenol, diacetoxyscirpenol, neosolaniol, and toxin T-2. Under the culture conditions used, deoxynivalenol, 3-acetyldeoxynivalenol and zearalenone were detected in the fungal culture medium. The finding is consistent with the hypothesis that the fungus is in fact a Fusarium species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mycotoxins and fungi in wheat harvested during 1990 in test plots in the state of São Paulo,Brazil

Wheat from two cultivars with contrasting characteristics were harvested in ten experimental plots located in wheat producing areas of the State of São Paulo, Brazil. The samples (10 of each cultivar) were analyzed by a gaschromatographic method for deoxynivalenol (DON), nivalenol (NIV), diacetoxyscirpenol (DAS), toxins T-2 (T-2) and HT-2, T-2 tetraol, T-2 triol, and by a thin-layer chromatographic method for zearalenone (ZEN), aflatoxins B₁, B₂, G₁, G₂, ochratoxin A and sterigmatocystin. No mycotoxins were detected in 13 samples. DON was found in four samples (0.47–0.59 µg/g), NIV in three samples (0.16–0.40 µg/g), T-2 in two samples (0.40, 0.80 µg/g), DAS in one sample (0.60 µg/g), and ZEN in three samples (0.04–0.21 µg/g). The wheat samples were also examined for the incidence of fungi.Alternaria, Drechslera, Epicoccum andCladosporium were the prevailing genera. Among theFusarium spp.,F. semitectum was present in 19 samples andF. moniliforme in 18 samples. NoF. graminearum was isolated in the samples.Abbreviations DAS diacetoxyscirpenol - DON deoxynivalenol - NIV nivalenol - T-2 T-2 toxin - ZEN zearalenone     

Natural occurrence of deoxynivalenol, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol,nivalenol, 4,7-dideoxynivalenol,and zearalenone in polish wheat

Three wheat samples collected in 1987 in Central Poland and naturally infected withFusarium spp were analyzed for the presence ofFusarium spp andFusarium toxins. Heads were separated into three fractions: kernels with visibleFusarium damage, healthy looking kernels, and chaff + rachis. The samples contained deoxynivalenol (2.0 – 40.0μg/g), nivalenol (O.O1μg/g), 4,7-dideoxynivalenol (0.10 – 0.15μg/g). 15-acetyldeoxynivalenol (0.10–2.00 μg/g), 3-acetyldeoxynivalenol (O/1Oμg/g), and zearalenone (0.01–2.00μg/g). This is the first report about 15 - acetyldeoxynivalenol in European wheat and the co-occurrence of 3 - acetyldeoxynivalenol and 15-acetyldeoxynivalenol in the same sample of contaminated cereals.     

Production of zearalenone,moniliformin and trichothecenes in intact sugar beets under laboratory conditions

Five toxigenic isolates of Fusarium species were tested for the production of zearalenone, moniliformin and trichothecenes (deoxynivalenol, 15-acetyldeoxynivalenol, T-2, HT-2 and neosolaniol) when grown on solid sugar beet slices in the laboratory for thirty days. The isolates were also grown on a solid rice medium for comparison. High zearalenone and trichothecene-producing isolates originally obtained from corn and corn-based feedstuff were compared with isolates obtained from sugar beets. One moniliformin-producing isolate from wheat was included in the study. With the exception of moniliformin, all toxins were produced on both substrates; however, the rice medium yielded the greater concentrations except for HT-2 which was produced on sugar beets in equal or greater concentrations. Zearalenone production on rice reached 729–1943 gmg/g whereas on sugar beet it reached 72–193 gmg/g. The moniliformin-producing isolate grew well on both substrates; however, moniliformin was produced only on the rice substrate. This study demonstrates for the first time that Fusarium species can produce both zearalenone and the trichothecenes on a sugar beet substrate.     

Mycotoxins produced by <Emphasis Type="Italic">Fusarium</Emphasis> species associated with annual legume pastures and ‘sheep feed refusal disorders’ in Western Australia

Sheep grazing in Western Australia can partially or completely refuse to consume annual Medicago pods contaminated with a number of different Fusarium species. Many Fusarium species are known to produce trichothecenes as part of their array of toxigenic secondary metabolites, which are known to cause feed refusal in animals. This study reports the identity of Fusarium species using species-specific PCR primers and a characterization of the toxigenic secondary metabolites produced by 24 Fusarium isolates associated with annual legume-based pastures and particularly those associated with sheep feed refusal disorders in Western Australia. Purification of the fungal extracts was facilitated by a bioassay-guided fractionation using brine shrimp. A number of trichothecenes (3-acetyldeoxynivalenol, deoxynivalenol, fusarenon-X, monoacetoxyscirpenols, diacetoxyscirpenol, scirpentriol, HT-2 toxin and T-2 toxin), enniatins (A, A1, B, and B1), chlamydosporol and zearalenone were identified using GC/MS and/or NMR spectroscopy. Some of the crude extracts and fractions showed significant activity against brine shrimp at concentrations as low as 5 μg ml^-1, and are likely to be involved in the sheep feed refusal disorders. This is the first report of chlamydosporol production by confirmed Fusarium spp.; of the incidence of F. brachygibbosum and F. venenatum in Australia and of F. tricinctum in Western Australia; and of mycotoxin production by Fusarium species from Western Australia.