Formation of fusarenone X, nivalenol, zearalenone, alpha-trans-zearalenol, beta-trans-zearalenol, and fusarin C by Fusarium crookwellense.

Fusarium crookwellense KF748 (NRRL A-28100) (isolated from dry rotted potato tubers in Central Poland) produced six mycotoxins on both rice and corn substrates at 25 degrees C. The metabolites detected were zearalenone, alpha-trans-zearalenol, beta-trans-zearalenol, fusarin C, and the trichothecenes fusarenone X and nivalenol. This is the first report of formation of alpha-trans-zearalenol, beta-trans-zearalenol, fusarenone X, and nivalenol by F. crookwellense.     

Effect of incubation temperature on zearalenone production by strains of Fusarium crookwellense

After 6 weeks incubation on rice 2 strains of Fusarium crookwellense produced more zearalenone (6060–5010 mg/kg dry wt of culture) at ambient temperature (16–29°C) in daylight than at ambient temperature (18–23 °C) in darkness or at controlled temperatures of 11 °C, 20 °C or 25 °C in darkness. Yields at 25 °C were low. Incubation at 11 °C during the second 3 weeks incubation increased yields only when preliminary incubation had been at 25 °C. After 6 weeks incubation at controlled temperatures in darkness, 4 strains produced most zearalenone at 20 °C (2460-21 360 mg/kg), 1 strain at 11 °C (6570 mg/kg). Yields at a temperature oscillating daily from 10–20 °C were less than at 15 °C. One of the 5 strains produced appreciable amounts of a-zearalenol (1645 mg/kg at 20°C) and 2 of nivalenol (340 and 499 mg/kg at 20 °C).     

Effect of incubation temperature on zearalenone production by strains of Fusarium crookwellense.

After 6 weeks incubation on rice 2 strains of Fusarium crookwellense produced more zearalenone (6060-5010 mg/kg dry wt of culture) at ambient temperature (16-29 degrees C) in daylight than at ambient temperature (18-23 degrees C) in darkness or at controlled temperatures of 11 degrees C, 20 degrees C or 25 degrees C in darkness. Yields at 25 degrees C were low. Incubation at 11 degrees C during the second 3 weeks incubation increased yields only when preliminary incubation had been at 25 degrees C. After 6 weeks incubation at controlled temperatures in darkness, 4 strains produced most zearalenone at 20 degrees C (2460-21 360 mg/kg), 1 strain at 11 degrees C (6570 mg/kg). Yields at a temperature oscillating daily from 10-20 degrees C were less than at 15 degrees C. One of the 5 strains produced appreciable amounts of a-zearlaenol (1645 mg/kg at 20 degrees C) and 2 of nivalenol (340 and 499 mg/kg at 20 degrees C).     

Production of fusarin C on cereal and soybean by Fusarium moniliforme

Two isolates of Fusarium moniliforme were compared with respect to production of a mutagenic compound, fusarin C, on seven corn varieties as well as on soybean, wheat, rye, barley, and a liquid culture medium. The isolates were originally obtained from corn and barley. Both isolates produced fusarin C on seed of all five crops within a 21-day period, and one isolate produced the largest amount on oats. Soybean was the poorest substrate for both isolates. Although the quantity of fusarin C produced on grain was isolate dependent, specific substrate requirements for each strain were suggested. The isolates differed in their ability to grow and produce fusarin C on corn with different moisture contents (16, 20, 24, and 28%). One isolate was more xerotolerant and grew at 16% moisture but did not produce the mutagen.     

Production of fusarin C on cereal and soybean by Fusarium moniliforme.

Two isolates of Fusarium moniliforme were compared with respect to production of a mutagenic compound, fusarin C, on seven corn varieties as well as on soybean, wheat, rye, barley, and a liquid culture medium. The isolates were originally obtained from corn and barley. Both isolates produced fusarin C on seed of all five crops within a 21-day period, and one isolate produced the largest amount on oats. Soybean was the poorest substrate for both isolates. Although the quantity of fusarin C produced on grain was isolate dependent, specific substrate requirements for each strain were suggested. The isolates differed in their ability to grow and produce fusarin C on corn with different moisture contents (16, 20, 24, and 28%). One isolate was more xerotolerant and grew at 16% moisture but did not produce the mutagen.     

Formation of [13C]- and [14C]zearalenone by Fusarium graminearum DSM 4529

Summary To raise the yields for the production of ¹⁴C-labelled zearalenone in Fusarium cultures the influence of growth conditions and known effectors or precursors of toxin biosynthesis was studied. Benzoic acid and 2,4-dihydroxybenzoic acid used as precursors decreased toxin formation; in the presence of different pesticides such as 2,4-dichlorophenoxyacetic acid, however, toxin production increased up to 140%. The known pathway of zearalenone biosynthesis could be confirmed from the relative extents of ¹³C-incorporation into the zearalenone molecule by incubating Fusarium graminearum DSM 4529 with d-(+)-[1-¹³C]glucose as carbon source. When grown in the presence of d-[U-¹⁴C]glucose or [2-¹⁴C]malonic acid the strain produced [¹⁴C]zearalenone with specific activities of 0.07 and 0.09 Ci/mg, the ¹⁴C-incorporation rates being 0.34% and 0.48%, respectively.     

Mycotoxin formation by different geographic isolates of Fusarium crookwellense

Eighteen Fusarium crookwellense isolates from the continents of Australia, Europe, and North America were compared for their ability to produce mycotoxins on corn at 25 °C after 2 weeks. Extracts from corn fermented with each Fusarium isolate were analyzed by thin-layer chromatography (TLC) and gas chromatography/mass spectroscopy (GS/MS) for mycotoxins. Toxins detected were zearalenone (13 isolates), fusarin C (11 isolates), nivalenol (4 isolates), and diacetoxyscirpenol (2 isolates). Zearalenone and fusarin C were produced by isolates from each continent, while nivalenol was detected in the Fusarium isolates originating from Australia and one isolate from the United States.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned     

DNA adduct formation by Fusarium culture extracts: lack of role of fusarin C

Fusarium fungi have been shown to infect corn and other crops worldwide, and have a significant impact on human health through loss of crops or contamination of food with mycotoxins. Isolates of Fusarium fungi from an area of South Africa with high incidence of esophageal cancer have been shown to induce esophageal and liver cancer in rats. Several isolates of Fusarium fungi were grown on corn to determine if genotoxic products were produced. We report the incubation of methanol extracts of Fusarium verticillioides cultures with DNA in the presence of rat liver fractions (S9) resulted in the formation of a unique DNA adduct that was detected by (32)P-postlabeling. Fusarin C was purified from cultures of Fusarium verticillioides RRC 415, and was not responsible for the formation of the DNA adduct. Treatment of the methanolic extracts with ultraviolet B radiation reduced the fusarin C content in the extract; however, this had no effect on the formation of the DNA adduct following incubation of the extract with DNA and S9. The unique DNA adduct was formed following the incubation of several Fusarium verticillioides isolates from the US and South Africa, while extracts of cultures of Fusarium graminearium and Fusarium sacchari isolates formed very little of the DNA adduct when incubated with DNA and S9. These data suggest that neither fusarin C nor any of its metabolites are responsible for formation of the DNA adduct, and that an unidentified compound is present in F. verticillioides cultures that forms a DNA adduct, and may be important in the etiology of human esophageal cancer.     

Decontamination ofFusarium mycotoxins,nivalenol, deoxynivalenol,and zearalenone,in barley by the polishing process

Korean dehusked and unhusked barley naturally contaminated withFusarium mycotoxins were polished using a Satake Grain Testing Mill. The pearled barley and bran fractions with different degrees of polishing were analyzed for nivalenol (NIV) and deoxynivalenol (DON) by gas chromatography with an electron capture detector, and for zearalenone (ZEN) by high-performance liquid chromatography with a fluorescence detector. NIV was detected in all the pearled barley fractions, but DON and ZEN were not detected in ≥27 % pearled barley fractions from dehusked barley and ≥36% pearled barley fractions from unhusked barley. However, for all degrees of polishing, NIV, DON, and ZEN were detected in bran fractions. The levels of NIV, DON, and ZEN in the bran fractions increased several fold over the original barley. Polishing was effective in removing DON and ZEN from the naturally contaminated barley, but not NIV.     

Fusarium poae and Fusarium crookwellense, Fungi Responsible for the Natural Occurrence of Nivalenol in Hokkaido

To determine the reasons for the natural occurrence of nivalenol in the northernmost area of Japan, scabby wheat was harvested from 19 crop fields in Hokkaido. Mycological surveys and analysis for mycotoxin contamination were performed. Among 13 wheat grain samples harvested in seven locations, 9, 2, and 6 samples were positive for deoxynivalenol, nivalenol, and zearalenone, respectively, at levels ranging from 0.03 to 1.28 μg/g, 0.04 to 1.22 μg/g, and 2 to 25 ng/g, respectively. The predominant Fusarium species of the scabby wheat examined were F. sporotrichioides, F. avenaceum, F. poae, and F. crookwellense. Fifteen of 48 F. poae isolates and all four F. crookwellense isolates were screened for the production of seven derivatives of trichothecenes and zearalenone respectively, on rice culture. One isolate of F. poae produced diacetoxyscirpenol alone (4.3 μg/g); seven produced nivalenol (1.3 to 23.8 μg/g), 4-acetylnivalenol (0.1 to 4.6 μg/g), and diacetoxyscirpenol (0.9 to 99.5 μg/g); and five produced nivalenol alone (0.4 to 3.5 μg/g). The remaining two isolates produced no trichothecenes. Zearalenone production was not found in any isolate of F. poae tested. All isolates of F. crookwellense produced nivalenol (0.9 to 22.5 μg/g), 4-acetylnivalenol (0.5 to 25.0 μg/g), and zearalenone (1.4 to 162.5 μg/g). From these results, it is apparent that deoxynivalenol and zearalenone, and occasionally nivalenol, occur naturally throughout Hokkaido, and it is suggested that nivalenol-producing F. poae and F. crookwellense strains are responsible for the natural contamination with nivalenol found in the northernmost area of Japan. Furthermore, it was found for the first time that several isolates of F. poae distributed in Hokkaido possessed the ability to produce both type A and type B trichothecenes.     

Production of fusarenon-X,nivalenol, and zearalenone by Gibberella zeae isolates,and their toxicity in fibroblasts and rats

Three isolates ofGibberella zeae, the perfect stage ofFusarium graminearum, were isolated from ground corn cultures obtained from Taiwan in 1985 and identified asGibberella zeae l-1, G. zeae I-5, andG. zeae l-7. The isolates were grown on a solid rice medium and extracts prepared with 75% aqueous methanol. The extracts were examined for toxicity in the following systems: (1) cytotoxicity to cultured normal human diploid skin fibroblasts and mouse fibroblasts; and (2) toxicity to rats of unextracted cultures. The three extracts were highly cytotoxic as indicated by the ability to cause death and disintegration of 3T3 Swiss mouse fibroblasts and human diploid skin fibroblasts during 3 to 4 days in culture. The unextracted cultures of the isolates were highly toxic to rats, causing hemorrhage of tissues (bladder, stomach, and intestine), uterine enlargement, small thymuses, small spleens, weight loss, and death. The extracts were tested for production of trichothecenes (nivalenol and fusarenon-X) and zearalenone on rice grains. Production of the three mycotoxins was greater at room temperature than in the cold room. Detection of the three mycotoxins from the cultures was variable, ranging from 273 to 817ppm for nivalenol, 268 to 662 ppm for fusarenon-X, and 162 to 1095 ppm for zearalenone at room temperature, and 159 to 413 ppm for nivalenol, 113 to 125 ppm for fusarenon-X and 44 to 202 ppm for zearalenone in the cold room (10°C).     

Effects of zinc, iron, cobalt, and manganese on Fusarium moniliforme NRRL 13616 growth and fusarin C biosynthesis in submerged cultures

The influence of zinc, iron, cobalt, and manganese on submerged cultures of Fusarium moniliforme NRRL 13616 was assessed by measuring dry weight accumulation, fusarin C biosynthesis, and ammonia assimilation. Shake flask cultures were grown in a nitrogen-limited defined medium supplemented with various combinations of metal ions according to partial-factorial experimental designs. Zinc (26 to 3,200 ppb [26 to 3,200 ng/ml]) inhibited fusarin C biosynthesis, increased dry weight accumulation, and increased ammonia assimilation. Carbohydrate was found to be the principal component of the increased dry weight in zinc-supplemented cultures. Zinc-deficient cultures synthesized more lipid and lipidlike compounds, such as fusarin C, than did zinc-supplemented cultures. Microscopic examination showed that zinc-deficient hyphae contained numerous lipid globules which were not present in zinc-supplemented cultures. Addition of zinc (3,200 ppb) to 2- and 4-day-old cultures inhibited further fusarin C biosynthesis but did not stimulate additional dry weight accumulation. Iron (10.0 ppm) and cobalt (9.0 ppm) did not affect fusarin C biosynthesis or dry weight accumulation. Manganese (5.1 ppm) did not affect dry weight accumulation but did increase fusarin C biosynthesis in the absence of zinc. Maximum fusarin C levels, 32.3 micrograms/mg (dry weight), were produced when cultures were supplied manganese, whereas minimum fusarin C levels, 0.07 micrograms/mg (dry weight), were produced when zinc, iron, cobalt, and manganese were supplied. These results suggest a multifunctional role for zinc in affecting F. moniliforme metabolism.     

Screening survey of co-production of fusaric acid,fusarin C,and fumonisins B1, B2 and B3 by Fusarium strains grown in maize grains

Fusarium species isolated from Belgian maize were screened for their ability to produce fusarin C, fusaric acid, fumonisins B₁ (FB₁), FB₂ and FB₃ in maize grains. First, cultivation of Fusarium species in Myro liquid medium allowed overcoming the shortage of the standard of fusarin C on the market. All Fusarium verticillioides produced much higher contents of mycotoxins in Myro compared to Fusarium graminearum or Fusarium venenatum. The optimization of the LC-MS/MS method resulted in low limits of detection and quantification for fusarin C, fusaric acid, FB₁, FB₂ and FB₃ determination in maize grains. Its application for screening the potential toxin production ability evidenced that the concentrations of the analytes were significantly increased at various levels when F. verticillioides strains were cultivated in maize grains and reached 441 mg kg^?1 for fusaric acid, 74 mg kg^?1 for fusarin C, 1,301 mg kg^?1 for FB₁, 367 mg kg^?1 for FB₂ and 753 mg kg^?1 for FB₃.     

Effects of zinc, iron, cobalt, and manganese on Fusarium moniliforme NRRL 13616 growth and fusarin C biosynthesis in submerged cultures.

The influence of zinc, iron, cobalt, and manganese on submerged cultures of Fusarium moniliforme NRRL 13616 was assessed by measuring dry weight accumulation, fusarin C biosynthesis, and ammonia assimilation. Shake flask cultures were grown in a nitrogen-limited defined medium supplemented with various combinations of metal ions according to partial-factorial experimental designs. Zinc (26 to 3,200 ppb [26 to 3,200 ng/ml]) inhibited fusarin C biosynthesis, increased dry weight accumulation, and increased ammonia assimilation. Carbohydrate was found to be the principal component of the increased dry weight in zinc-supplemented cultures. Zinc-deficient cultures synthesized more lipid and lipidlike compounds, such as fusarin C, than did zinc-supplemented cultures. Microscopic examination showed that zinc-deficient hyphae contained numerous lipid globules which were not present in zinc-supplemented cultures. Addition of zinc (3,200 ppb) to 2- and 4-day-old cultures inhibited further fusarin C biosynthesis but did not stimulate additional dry weight accumulation. Iron (10.0 ppm) and cobalt (9.0 ppm) did not affect fusarin C biosynthesis or dry weight accumulation. Manganese (5.1 ppm) did not affect dry weight accumulation but did increase fusarin C biosynthesis in the absence of zinc. Maximum fusarin C levels, 32.3 micrograms/mg (dry weight), were produced when cultures were supplied manganese, whereas minimum fusarin C levels, 0.07 micrograms/mg (dry weight), were produced when zinc, iron, cobalt, and manganese were supplied. These results suggest a multifunctional role for zinc in affecting F. moniliforme metabolism.     

Isolation and characterization of zearalenone sulfate produced by Fusarium spp.

A water-soluble compound related to zearalenone was isolated from a culture of Fusarium graminearum 30 grown in rice. The structure of the novel metabolite was determined to be zearalenone-4-sulfate on the basis of fast-atom-bombardment mass spectrometry, proton nuclear magnetic resonance, UV spectroscopy, and by chemical and enzymatic reactions. Strains representing Fusarium equiseti, Fusarium sambucinum, and Fusarium roseum produced the sulfate conjugate as well. In the rat uterus enlargement bioassay, the metabolite or its hydrolysis product was found to retain the estrogenic activity characteristic of zearalenone. Natural occurrence of this novel metabolite might be significant because analytical methods devised for zearalenone in grain cannot detect the conjugate but the conjugate retains the biological properties of the mycotoxin when ingested by animals.     

Isolation and characterization of zearalenone sulfate produced by Fusarium spp.

A water-soluble compound related to zearalenone was isolated from a culture of Fusarium graminearum 30 grown in rice. The structure of the novel metabolite was determined to be zearalenone-4-sulfate on the basis of fast-atom-bombardment mass spectrometry, proton nuclear magnetic resonance, UV spectroscopy, and by chemical and enzymatic reactions. Strains representing Fusarium equiseti, Fusarium sambucinum, and Fusarium roseum produced the sulfate conjugate as well. In the rat uterus enlargement bioassay, the metabolite or its hydrolysis product was found to retain the estrogenic activity characteristic of zearalenone. Natural occurrence of this novel metabolite might be significant because analytical methods devised for zearalenone in grain cannot detect the conjugate but the conjugate retains the biological properties of the mycotoxin when ingested by animals.     

Two-dimensional environmental profiles of growth,deoxynivalenol and nivalenol production by Fusarium culmorum on a wheat-based substrate

AIMS: To determine the effect of interacting conditions of water activity (aw, 0.99-0.85), temperature (15, 25 degrees C) and time (40 days) on growth and production of the mycotoxins deoxynivalenol (DON) and nivalenol (NIV) by Fusarium culmorum on a wheat-based agar medium. METHODS AND RESULTS: Fusarium culmorum grew optimally at 0.995aw and minimally at 0.90 at both 15 and 25 degrees C. No growth was observed at <0.90aw. Overall, temperature, aw and their interaction had a statistically significant effect on the growth rate of F. culmorum. Production of both DON and NIV were over a much narrower range (0.995-0.95aw) than that for growth. The highest concentrations of DON and NIV levels were produced at 0.995aw and 0.981aw at 25 degrees C, respectively, after 40 days of incubation. Statistically, aw, temperature and incubation time, and aw x temperature and temperature x incubation time had a statistically significant effect on DON/NIV production. CONCLUSIONS: This is the first detailed report on the two-dimensional environmental profiles for DON/NIV production by F. culmorum in the UK. SIGNIFICANCE AND IMPACT OF THE STUDY: As part of a hazard analysis critical control point (HACCP) approach, this type of information is critical in monitoring critical control points for prevention of DON/NIV entering the wheat production chain.     

Formation of moniliformin by Fusarium sporotrichioides and Fusarium culmorum

Two strains of Fusarium sporotrichioides and one strain of F. culmorum were shown to produce the mycotoxin moniliformin in rice culture. Identification was by reverse-phase liquid chromatography, thin-layer chromatography, and mass spectrometry.     

Identification of gene clusters associated with fusaric acid, fusarin, and perithecial pigment production in Fusarium verticillioides

The genus Fusarium is of concern to agricultural production and food/feed safety because of its ability to cause crop disease and to produce mycotoxins. Understanding the genetic basis for production of mycotoxins and other secondary metabolites (SMs) has the potential to limit crop disease and mycotoxin contamination. In fungi, SM biosynthetic genes are typically located adjacent to one another in clusters of co-expressed genes. Such clusters typically include a core gene, responsible for synthesis of an initial chemical, and several genes responsible for chemical modifications, transport, and/or regulation. Fusarium verticillioides is one of the most common pathogens of maize and produces a variety of SMs of concern. Here, we employed whole genome expression analysis and utilized existing knowledge of polyketide synthase (PKS) genes, a common cluster core gene, to identify three novel clusters of co-expressed genes in F. verticillioides. Functional analysis of the PKS genes linked the clusters to production of three known Fusarium SMs, a violet pigment in sexual fruiting bodies (perithecia) and the mycotoxins fusarin C and fusaric acid. The results indicate that microarray analysis of RNA derived from culture conditions that induce differential gene expression can be an effective tool for identifying SM biosynthetic gene clusters.     

Biosynthesis of [14C]zearalenone from [1-14C]acetate by Fusarium roseum 'Gibbosum'.

Addition of [1-14C]acetate or [1,2-14C]acetate to actively growing cultures of Fusarium roseum 'Gibbosum' on rice yielded zearalenone with a specific activity ranging between 1.63 and 46.5 microCi/mmol.