Biosynthesis of labeled fumonisins in liquid cultures of Fusarium moniliforme

Fumonisins were readily produced in cultures of Fusarium moniliforme using a defined liquid medium. Addition of 200 mg of d₃-methyl L-methionine to 100-ml cultures of F. moniliforme gave increased overall yields and high levels of deuterium (²H) incorporation into fumonisin B₁. Approximately 90% of the resulting fumonisin B₁ contained 6 deuterium atoms, while 9% of the product contained 3 deuterium atoms. Deuterium was shown to be incorporated exclusively in the methyl groups of the fumonisin backbone. The addition of as little as 5 mg of labeled methionine stimulated fumonisin production, but only about 5% of the fumonisin produced contained 3 deuterium atoms.Abbreviations ELEM equine leukoencephalomalacia Mention of companies or products by name does not imply their endorsement by the US Department of Agriculture over others not cited.     

Comparative studies of hepatotoxicity and fumonisin B1 and B2 content of water and chloroform/methanol extracts of Fusarium moniliforme strain MRC 826 culture material

Fusarium moniliforme has been associated with several diseases including equine leukoencephalomalacia, human esophageal cancer and hepatotoxicity/hepatocarcinogenicity in laboratory animals. The potential health risks to animals and humans posed by F. moniliforme contaminated grains cannot be assessed until the toxins are identified and toxicologically evaluated. As part of a systematic approach to identifying the hepatotoxins produced by F. moniliforme, diets containing aqueous and chloroform/methanol (11) extracts of F. moniliforme strain MRC 826 culture material (CM) and/or the extracted CM residues were fed to male Sprague-Dawley rats for four weeks. Serum alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase activities were increased after two and four weeks and microscopic liver lesions were found in those animals fed aqueous CM extract and the CM residue after chloroform/ methanol extraction. Fumonisins B₁ and B₂ were extracted from the CM by water, but not chloroform/ methanol, and were present in the toxic diets at concentrations of 93–139 and 82–147 ppm, respectively. Nontoxic diets contained 22 ppm fumonisin B₁ and 65 ppm fumonisin B₂.Abbreviations CM culture material - ELEM equine leukoencephalomalacia Mention of a trademark, proprietory name or vendor does not imply its approval by the US Department of Agriculture to the exclusion of others that may also be suitable.     

Fertility ofFusarium moniliforme from maize and sorghum related to fumonisin production in Italy

Forty-three strains ofFusarium moniliforme isolated from infected maize and sorghum plants in Italy were assayed for their ability to produce fertile crosses with A and F mating population tester strains, in relation to their ability to produce fumonisins on maize substrate. Most of the strains isolated from maize (ear and stalk rot and maize-based feed), producing fumonisin B₁ (FB₁) and B₂ (FB₂) (up to 4,100 and 855 mg/kg, respectively), belonged to the A mating population. All of the strains isolated from sorghum belonged to the F mating population and produced little or no FB₁ and FB₂. This is the first report of the occurrence of mating population F in Europe. Our data on strains from Italy are consistent with previous studies from the United States that found significant differences in sexual fertility and fumonisin production between strains from maize and sorghum.     

Mycoflora and fumonisin-producing strains ofFusarium moniliforme in mixed poultry feeds and component raw material

In a survey of the mycoflora and mycotoxins in foods and feeds, 66 samples of mixed poultry feeds and some component raw materials were investigated. Fungal counts ranged from < 10² to 1.3 × 10⁶ CFU/g.Fusarium spp. counts ranged from 10² to 1.0 × 10⁶ CFU/g. TheFusarium spp. strains isolated were screened for their potential to produce fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) in maize cultures. Samples and maize cultures were analysed for FB₁ and FB₂ using TLC and fluorescamine-derivative HPLC. No fumonisins were detected in the samples (<6 ppm).Fusarium moniliforme was isolated in 59.1% of samples, and 97.4% of the strains produced FB₁ and 79.4% of strains produced FB₂ in maize cultures. Some isolates produced higher FB₁ and FB₂ levels than the reference strainF. moniliforme MRC 826.     

Characterization of gibberellin producing strains of Fusarium moniliforme based on DNA polymorphism

The gibberellins are one of the major groups of growth promoting hormones and are secondary metabolites of the fungus Fusarium moniliforme (Perfect stage: Gibberella fujikuroi). Sixteen strains of Fusarium from different geographical regions and different hosts were analysed for their ability to produce gibberellins (GA) and for genetic relatedness by random amplified polymorphic DNA (RAPD). Range of gibberellin production varied between 28.9 to 600.0 mg g^-1 dry weight of mycelium in different strains of Fusarium. RAPD analysis showed completely different pattern between high, moderate and low producing strains. High producers formed nearly identical RAPD patterns, whereas the low and moderate producers gave heterologous amplification patterns. Since Fusarium pallidoroseum was in another group, it was possible to distinguish between different species of the genus Fusarium by RAPD. These investigations may find an application in the diagnosis of unknown Fusarium species and in distinguishing isolates of Gibberella fujikuroi within the section of Liseola. This revised version was published online in June 2006 with corrections to the Cover Date.     

Isolation of beauvericin as an insect toxin from Fusarium semitectum and Fusarium moniliforme var. subglutinans

Nonpolar methylene chloride-soluble extracts from the mycelia of Fusarium semitectum and Fusarium moniliforme var. subglutinans were toxic to Colorado potato beetles. The major toxic metabolite was isolated and found to be the cyclodepsipeptide, beauvericin. This is the first report of the isolation of beauvericin from the genus Fusarium.     

Developmental effects of fumonisin B1-containingFusarium moniliforme culture extract in CD1 mice

Pregnant Charles River CD1 mice were treated with a semipurified extract ofFusarium moniliforme culture containing 0, 12.5, 25, 50 or 100 mg FB₁/kg each day orally (diluted in distilled water) between gestational days (GD) 7 and 15 to evaluate the developmental toxicity of FB₁. Following sacrifice of dams on GD 18, litters were examined for gross abnormalities and divided equally for skeletal or visceral examination by routine techniques. Significant maternal mortality was observed at doses of 50 and 100 mg FB₁/kg. Dose-dependant decreases in maternal body weight gains, number of live offsprings per litter, and mean body weight of the offspring were produced at FB₁ doses of 25 mg/kg or higher. The percentage of implants resorbed increased at all doses in a dose-dependant manner. A dose-dependant increase, except at the lowest dose tested, in the incidence of ossification deficits involving digits and sternum, short and wavy ribs, and hydrocephalus of lateral and third ventricles was also evident. Cleft palate was seen only at the highest FB₁ dose. Maternal intoxication manifested as a dose-dependant increase in the severity of ascites associated mainly with increased histopathologic scores reflecting hepatocellular damage at day 18. Concommittant increases in serum alanine amino transferase (ALT) on GD 12, reflecting parenchymal liver cell damage, was also observed at all doses above 12.5 mg of FB₁/kg. These results suggest that FB₁-containingF. moniliforme culture extract is developmentally toxic in mice, and that this toxicity may be mediated by maternal hepatotoxicity.     

Fumonisin B1 alters sphingolipid metabolism and immune function in BALB/c mice: Immunological responses to fumonisin B1

Fumonisins have been reported to have diverse effects on animals, including immunosuppression in chickens and feeder calves; therefore, the effects of fumonisin B₁ (FB₁) on immune function in BALB/c mice was investigated. When administered i.p. with sheep red blood cells (SRBC), 5 to 100 µg of FB₁ reduced the number of plaque-forming cells (PFC) produced against SRBC; however, when administered daily, 1 to 50 µg of FB₁ caused a 4 to 12-fold increase in the number of PFC after SRBC injection. Therefore, FB₁ is not only immunosuppressive; but also, immunostimulatory. To test the possibility that there may have been an immune response to FB₁ as an antigen, FB₁ was injected into mice and the number of splenic cells forming rosettes on FB₁-treated SRBC was determined. There were dose-dependent increases in the antigen-binding cells, with up to 4.9- and 4.6-fold increases, respectively, upon primary and secondary immunization. FB₁-binding immunoglobulins could be detected in sera from some treated mice, but this response was not obtained in every experiment. In summary, these results show that FB₁ has diverse effects on the immune system, causing both stimulation and suppression of the response to foreign antigens, and apparently inducing an antigenic response to FB₁.Abbreviations DMEM Dulbecco's Modified Eagle's Medium - FB₁ fumonisin B₁ - FCS fetal calf serum - PBS phosphate buffered saline - PFC plaque forming cells - RFC rosette forming cells - SRBC sheep red blood cells     

Interactions of Fusarium moniliforme,its metabolites and bacteria with corn

Fusarium moniliforme Sheldon is an economically important pathogen of corn (Zea mays L.) which causes stalk, root and ear rot. Several mycotoxins have also been isolated, identified and implicated in both animal and human toxicoses. The fungus can be disseminated in symptomless corn seed and can also survive in crop residues in the soil. Asymptomatic infection may be related to different corn cultivars, fungal strains, and environmental factors. Symptomatic expression of pathogenicity may vary, but usually the result of such infections is death of the plant. The greatest concern is the asymptomatic infection, since it is in this form that fungal toxins may surreptitiously enter animal and human food chains. F. moniliforme produces both fusaric acid, which is phytotoxic to corn and interferes with seed germination, and plant growth regulators that may affect pathogenicity of the fungus or be associated with the production of mycotoxins. Other metabolites, including fusarin C, moniliformin, and the fumonisins, may or may not be phytotoxic, but are associated with animal and human toxicoses. The control of F. moniliforme in corn is therefore quite important. One potential means to accomplish this reduction is biocontrol by the application of antagonistic rhizobacteria to corn kernels at planting. To be effective the bacteria must be able to colonize the corn root system and be able to prevent root infection by successful competing with F. moniliforme which may be accomplished by siderophore and or antibiotic activity.     

Factors affecting gibberellic acid production by Fusarium moniliforme in solid-state cultivation on starch

The production of gibberellic acid (GA₃) by Fusarium moniliforme M-7121 in solid-state culture was evaluated in flask cultures as well as in 3-I horizontal rotary reactors. The highest production rate of GA₃ was with 80% (w/v) maize flour mixed with wheat bran. The optimum initial moisture content was inversely dependent on the ambient relative humidity. The initial water activity range for optimal growth and GA₃ accumulation was about 0.98 to 0.99, which is unusually high for a filamentous fungus. A low O₂ concentration resulted in a much decreased GA₃ yield and the appearance of a yellow to reddish pigmentation in the mycelium. The lag phase was short and rapid growth continued for up to 2 days in the rotary reactor, with a maximum specific growth rate of 0.12 h^–1. The maximum rate of GA₃ production occurred during the subsequent 3 to 10 days of incubation and the final GA₃ concentration reached was 18.7 mg to 19.3 mg/g dry culture. The point of maximum GA₃ accumulation after 10 to 12 days of incubation was usually marked by a sharp increase in pH.The authors are with the Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, 9300 Bioemfontein, Republic of South Africa     

Molecular characterization of endophytic strains of Fusarium verticillioides (= Fusarium moniliforme) from maize (Zea mays. L)

The species Fusarium verticillioides (= F. moniliforme) is often found in maize seeds, constituting an important source of inoculum in the field. Fusarium spp., associated with symptomatic and asymptomatic plants, may be a primary causal agent of disease, a secondary invader or an endophyte. In the present work, endophytic fungi were isolated from two populations of Zea mays (BR-105 and BR-106) and their respective inbred lines. Within different inbred lines of maize, Fusarium was found at a frequency of 0 to 100% relative to the number of total isolated fungi. The frequency with which the genus occurred was practically the same in the two field sites (around 60%). Twenty-one F. verticillioides strains were analysed using the random amplified polymorphic DNA (RAPD) technique, employing 10 random primers. Variability analysis of endophytic isolates via RAPD showed genome polymorphism taxa of species around 60%. Endophytic isolates were clustered by their sites of origin. RAPD analysis clustered the endophytic isolates by their maize inbred lines hosts (Mil-01 to Mil-06), whereas at site A they clustered into two major groups related to the maize gene pool (BR-105 or BR-106 population). All strains isolated from seeds collected in Site A, except strains L9 and L10, were sub-grouped according to maize inbred lines. The analysis showed a discrete sub-grouping at site B. Results obtained here could be explained by a co-evolution process involving endophytic isolates of F. verticillioides and maize inbred lines.     

A review and update of animal toxicoses associated with fumonisin-contaminated feeds and production of fumonisins by Fusarium isolates

During the 1989 corn harvest season, numerous reports of equine leukoencephalomalacia (ELEM) outbreaks and a pulmonary edema (PPE) syndrome in swine from several regions of the United States were received by the National Veterinary Services Laboratories (NVSL), Ames, Iowa. Previous and concurrent research linked Fusarium moniliforme and fumonisin-contaminated feeds to both diseases. Chemical and mycological investigations revealed fumonisin B₁ (FB₁) concentrations of 20 to 360 ppm in suspect swine feeds and 8 to 117 ppm in suspect equine feeds. Nonproblem feeds contained concentrations below 8 ppm. Fusarium moniliforme and Fusarium proliferatum were isolated from both problem and nonproblem equine and swine feeds. When cultured on autoclaved corn, the F. moniliforme and F. proliferatum isolates produced respective FB₁ and fumonisin B₂ (FB₂) that range from less than 5 to more than 2450 ppm and less than 5 to more than 1000 ppm, respectively. Isolates from both problem and nonproblem feeds produced high levels (greater than 500 ppm) in culture. Reported here is a review of chemical and mycological data resulting from the study of several cases of PPE and ELEM.     

Fumonisins: Isolation,chemical characterization and biological effects

The fumonisin B mycotoxins (FB₁ and FB₂) have been purified and characterized from corn cultures of Fusarium moniliforme strain MRC 826. Fumonisin B₁ (FB₁, the major fumonisin produced in culture, has been shown to be responsible for the major toxicological effects of the fungus in rats, horses and pigs. Recent investigations on the purification of compounds with chromatographic characteristics similar to FB₁ have led to the identification of two new fumonisins, FB₃ and FB₄. Fumonisins A₁ and A₂, the N-acetyl derivatives of FB₁ and FB₂ respectively, were also purified and shown to be secondary metabolites of the fungus. Short-term carcinogenesis studies in a rat liver bioassay indicated that over a period of 15 to 20 days, at dietary levels of 0.05–0.1%, FB₂ and FB₃ closely mimic the toxicological and cancer initiating activity of FB₁ and thus could contribute to the toxicological effects of the fungus in animals. In contrast, no biological activity could be detected for FA₁ under identical experimental conditions. These studies and others have indicated that the fumonisin B mycotoxins, although lacking mutagenicity in the Salmonella test or genotoxicity in the DNA repair assays in primary hepatocytes, appear to induce resistant hepatocytes similar to many known hepatocarcinogens.     

Occurrence of fumonisin B1 and B2 in corn-based foodstuffs in Taiwan market

Corn-based human foodstuffs purchased in Taiwan were analyzed for fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) using high-performance liquid chromatography. Fifty-two (33.9%) and 32 (20.9%) of 153 samples were found to contain FB₁ (73–2395 ng/g) and FB2 (120–715 ng/g), respectively. The highest frequency of detection and also the highest FB1 concentrations were found in sweetcorn (50%, 1089 ng/g) and cornflour (50%, 608 ng/g), followed by corn snacks (33.3%, 2395 ng/g), miscellaneous corn products (33.3%, 73 ng/g), popcorn (31.8%, 1003 ng/g) and cornflakes (23.5%, 1281 ng/g). 16 corn snacks (= approximately 20.5% of the samples) had an average FB₁ and FB₂ content of 456 and 145 ng/g, respectively, while six sweetcorn (= 25%) samples were contaminated with an average of 400 ng/g of FB₁ and 65 ng/g of FB₂. Of the 22 pop-corn samples examined, 7 had an average of 347 ng/g and 116 ng/g of FB₁ and FB₂, respectively. During an analysis of the distribution pattern for the combined fumonisin levels of FB₁ and FB₂, it became apparent that more than 69% of tested samples had fumonisin concentrations below 100 ng/g, while 11.1% (or 17 samples) contained in excess of 600 ng toxins per g. These results clearly illustrated that commercially available corn-based foodstuffs for human consumption in Taiwan are frequently contaminated with FB₁ and FB₂.This revised version was published online in October 2005 with corrections to the Cover Date.     

Developmental toxicity of fumonisin in Syrian hamsters

The effects of fumonisin on development of Syrian hamster fetuses were studied using fumonisin B₁ and B₂ extracted fromFusarium moniliforme corn-culture and purified fumonisin B₁. A significant increase in litters with fetal deaths occurred with the high doses of purified (18 mg FB₁/kg) and culture-extracted (18 mg FB₁ plus 4.5 mg FB₂) fumonisin. It is concluded that prenatal exposure to fumonisin on days 8 and 9 of gestation is detrimental to fetal hamster survivability but does not induce clinical maternal intoxication at these doses. Equivalent doses of fumonisin B₁, whether from culture-extract or pure solution produced similar results.     

Production of fumonisins byFusarium species of Taiwan

Twenty-nineFusarium species isolated from various sources in different districts of Taiwan were tested for their ability to produce fumonisins in corn cultures. OnlyFusarium moniliforme produced fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂). The finding that the other 28Fusarium species produced neither FB₁ nor FB₂ is preliminary because only one strain per species was studied. The detection of FB₁ and FB₂ in cultures ofF. moniliforme was demonstrated by TLC and HPLC, and FB₁ was further confirmed by mass spectrometry. In a separate experiment, in which 38 strains ofF. moniliforme were tested for fumonisins, approximately 66% (25/38) produced FB₁ and/or FB₂. Of the 25 strains, 14 produced only FB₁ and 11 produced both FB₁ and FB₂, and the amounts of FB₁ and FB₂ produced by different strains varied greatly. This is the first report that fumonisins are found in corn cultures experimentally infected withF. moniliforme strains from Taiwan. It is safe to assume that fumonisin producing strains ofF. moniliforme are widely distributed among the economic crops such as corn, rice, sugarcane, and sorghum throughout the Island.Abbreviations FB₁ Fumonisin B₁ - FB₂ Fumonisin B₂ - OPA o-phthalidialdehyde     

Low molecular mass pectate lyase from Fusarium moniliforme: similar modes of chemical and thermal denaturation

A low molecular mass pectate lyase from Fusarium moniliforme was unfolded reversibly by urea and Gdn-HCl at its optimum pH of 8.5, as monitored by intrinsic fluorescence, circular dichroism, and enzymatic activity measurements. Equilibrium unfolding studies yielded a deltaG(H(2)O) of 1.741 kcal/mol, D1/2 of 2.3M, and m value of 0.755kcal/molM with urea and a deltaG(H(2)O) of 1.927kcal/mol, D1/2 of 1.52M, and m value of 1.27 kcal/molM with Gdn-HCl as the denaturant. Thermal denaturation of the pectate lyase at, pH 8.5, was also reversible even after exposure to 75 degrees C for 10 min. Thermodynamic parameters calculated from thermal denaturation curves at pH values from 5.0 to 8.5 yielded a deltaCp of 0.864kcal/(molK). The deltaG(25 degrees C) at, pH 8.5, was 2.06kcal/mol and was in good agreement with the deltaG(H(2)O) values obtained from chemical denaturation curves. There was no exposure of hydrophobic pockets during chemical or thermal denaturation as indicated by the inability of ANS to bind the pectate lyase.     

Mortality in broiler chicks on feed amended withFusarium proliferatum culture material or with purified fumonisin B1 and moniliformin

Two hundred twenty-eight male chicks (Columbia × New Hampshire) were given feed amended with autoclaved culture material (CM) ofFusarium proliferatum Containing fumonisin B₁ (FB₁), fumonisin B₂ (FB₂) and moniliformin in 3 separate feeding trials. Purified FB₁ and moniliformin were given separately and in combination in a fourth feeding trial. Birds were given amended rations at day 1 (Trial 1 and 4), day 7 (Trial 2), and day 21 (Trial 3) and their respective ration was given for 28 days (Trial 1), 21 days (Trial 2), 7 days (Trial 3), and 14 days (Trial 4). FB₁ concentrations were 546, 193, and 61 ppm; FB₂ were 98, 38 and 14 ppm; and moniliformin were 367, 193, and 66 ppm in the first 3 feeding trial regimens. Chicks in Trial 4 were given dietary concentrations of purified FB₁ at 274 and 125 ppm, and moniliformin at 154 and 27 ppm. FB₁ and moniliformin, both alone and in combination, produced dose-responsive clinical signs, reduced weight gains and mortality in chicks. Age of birds given amended feeds had little difference in the clinical response; however, those given the rations from days 7 or 21 were slightly less susceptible than those given rations beginning at 1 day of age. Additive effects were noted when the toxins were given in combination. When toxins were given separately, adverse effects took longer to occur. A system to monitor pattern and rate of defecation (RD) was developed for assessing the chicks' approach to feed, water and heat source as illness progressed. Our results indicate that chicks fed corn heavily infected withF. proliferatum under field conditions could suffer acute death similar to that described for spiking mortality syndrome during the first 3 weeks of age.