Occurrence of peptide and clavine ergot alkaloids in tall fescue grass.

Evidence is presented that ergot alkaloids are ubiquitous in tall fescue pastures infected with the clavicipitaceous fungal endophyte Sphacelia typhina (or Acremonium coenophialum). Ergopeptide alkaloids, predominantly ergovaline, constituted 10 to 50 percent of the total ergot alkaloid concentration, which was as high as 14 milligrams per kilogram in sheaths and 1.5 milligrams per kilogram in blades. Ergot alkaloid concentrations were substantially increased by application of large amounts (10 millimoles per liter) of potassium nitrate or ammonium chloride to infected plants in the greenhouse. The results indicate that ergot alkaloids are probably responsible for the toxicity to cattle of this common pasture and lawn grass and that ergotism-like toxicoses may be caused by clavicipitaceous fungi other than Claviceps.

New conjugated hydroxydienoic fatty acids and acetotriacylglycerols from Securidaca longipedunculata seed oil.

Like other members of the plant family Polygalaceae, Securidaca longipedunculata Fres., is a source of fatty acids and triacylglycerols with unusual structures. Its seed oil contains at least seven chromatographically distinct groups of triacylglycerols divided into two series: One series represents monoacetotriacylglycerols, and the other 'normal' triacylglycerols having only long-chain fatty acids. Each series has groups containing zero, one or two conjugated hydroxydienoic acids. In addition, there is a small amount of triacylglycerol incorporating three hydroxy acids. In addition to coriolic (13-hydroxyoctadeca-cis-9,trans-11-dienoic) acid (27%), two of its previously unknown homologs are present: 11-hydroxyhexadeca-cis-7,trans-9-dienoic acid (15%) and 9-hydroxytetradeca-cis-5,trans-7-dienoic acid (2%).

A genetic map of Gibberella zeae (Fusarium graminearum).

We constructed a genetic linkage map of Gibberella zeae (Fusarium graminearum) by crossing complementary nitrate-nonutilizing (nit) mutants of G. zeae strains R-5470 (from Japan) and Z-3639 (from Kansas). We selected 99 nitrate-utilizing (recombinant) progeny and analyzed them for amplified fragment length polymorphisms (AFLPs). We used 34 pairs of two-base selective AFLP primers and identified 1048 polymorphic markers that mapped to 468 unique loci on nine linkage groups. The total map length is approximately 1300 cM with an average interval of 2.8 map units between loci. Three of the nine linkage groups contain regions in which there are high levels of segregation distortion. Selection for the nitrate-utilizing recombinant progeny can explain two of the three skewed regions. Two linkage groups have recombination patterns that are consistent with the presence of intercalary inversions. Loci governing trichothecene toxin amount and type (deoxynivalenol or nivalenol) map on linkage groups IV and I, respectively. The locus governing the type of trichothecene produced (nivalenol or deoxynivalenol) cosegregated with the TRI5 gene (which encodes trichodiene synthase) and probably maps in the trichothecene gene cluster. This linkage map will be useful in population genetic studies, in map-based cloning, for QTL (quantitative trait loci) analysis, for ordering genomic libraries, and for genomic comparisons of related species.

FUM1--a gene required for fumonisin biosynthesis but not for maize ear rot and ear infection by Gibberella moniliformis in field tests.

We have analyzed the role of fumonisins in infection of maize (Zea mays) by Gibberella moniliformis (anamorph Fusarium verticillioides) in field tests in Illinois and Iowa, United States. Fumonisin-nonproducing mutants were obtained by disrupting FUM1 (previously FUM5), the gene encoding a polyketide synthase required for fumonisin biosynthesis. Maize ear rot, ear infection, and fumonisin contamination were assessed by silk-channel injection in 1999 and 2000 and also by spray application onto maize silks, injection into maize stalks, and application with maize seeds at planting in 1999. Ear rot was evaluated by visual assessment of whole ears and by calculating percentage of symptomatic kernels by weight. Fumonisin levels in kernels were determined by high-performance liquid chromatography. The presence of applied strains in kernels was determined by analysis of recovered isolates for genetic markers and fumonisin production. Two independent fumonisin-nonproducing (fum1-3 and fum1-4) mutants were similar to their respective fumonisin-producing (FUM1-1) progenitor strains in ability to cause ear rot following silk-channel injection and also were similar in ability to infect maize ears following application by all four methods tested. This evidence confirms that fumonisins are not required for G. moniliformis to cause maize ear rot and ear infection.

An overview of rodent toxicities: liver and kidney effects of fumonisins and Fusarium moniliforme.

Fumonisins are produced by Fusarium moniliforme F. verticillioides) and other Fusarium that grow on corn worldwide. They cause fatal toxicoses of horses and swine. Their effects in humans are unclear, but epidemiologic evidence suggests that consumption of fumonisin-contaminated corn contributes to human esophageal cancer in southern Africa and China. Much has been learned from rodent studies about fumonisin B1(FB1), the most common homologue. FB1 is poorly absorbed and rapidly eliminated in feces. Minor amounts are retained in liver and kidneys. Unlike other mycotoxins, fumonisins cause the same liver cancer promotion and subchronic (studies (3/4) 90 days) liver and kidney effects as (italic)F. moniliforme. FB 1 induces apoptosis of hepatocytes and of proximal tubule epithelial cells. More advanced lesions in both organs are characterized by simultaneous cell loss (apoptosis and necrosis) and proliferation (mitosis). Microscopic and other findings suggest that an imbalance between cell loss and replacement develops, a condition favorable for carcinogenesis. On the molecular level, fumonisins inhibit ceramide synthase, and disrupt sphingolipid metabolism and, theoretically, sphingolipid-mediated regulatory processes that influence apoptosis and mitosis. Liver sphingolipid effects and toxicity are correlated, and ceramide synthase inhibition occurs in liver and kidney at doses below their respective no-observed-effect levels. FB1 does not cross the placenta and is not teratogenic in vivoin rats, mice, or rabbits, but is embryotoxic at high, maternally toxic doses. These data have contributed to preliminary risk evaluation and to protocol development for carcinogenicity and chronic toxicity studies of FB1 in rats and mice.

Methylation and acetolysis of extracellular D-mannans from yeast.

Methylation-fragmentation analyses were conducted on a series of extra-cellular, yeast alpha-D-linked mannans representing six different structural types. D-Mannans of low degree of branching were produced by Hansenula capsulata strains and by species related to H. holstii, The former consisted primarily of (1 leads to 2)- and (1 leads to 6)-linked D-mannosyl residues; the latter, of (1 leads to 2)- and (1 leads to 3)-linked D-mannosyl residues. Although the remaining structural types were highly branched, each gave distinct methylation-patterns indicative of (1 leads to 6)-linked backbones to which are appended non-(1 leads to 6)-linked side-chains. Acetolysis studies were correlated with the methylation analyses, and the correlation demonstrated that each branched polymer possesses side chains of heterogeneous length.

Rapid purification of fumonisins and their hydrolysis products with solid-phase extraction columns.

Fumonisins B(3) and B(4) (FB(3) and FB(4)) were recovered from the 50:50 acetonitrile/water extract of corn cultures of a strain of Fusarium moniliforme that does not make FB(1) or FB(2) by stirring the extract with IRA-68, a weak anion-exchange resin. The fumonisins were desorbed with 5% acetic acid in the same solvent. After dilution with water, the desorbed fumonisins were separated into FB(3) (FB(3) and FA(3)) and FB(4) (FB(4), FC(4), and FA(4)) fractions with a tC(18) solid-phase extraction (SPE) cartridge. The FB(3) fraction was then separated into FB(3) and FA(3) by using an NH(2) SPE cartridge and eluting with 5% acetic acid and increasing amounts of acetonitrile in water. Finally, FB(1) and FA(3) were hydrolyzed with calcium hydroxide. After recovery from the reaction mixture using a tC(18) cartridge, the hydrolyzed and partially hydrolyzed analogues were separated and the unreacted fumonisins recovered by using an NH(2) cartridge, initially in the normal-phase mode with increasing amounts of water in acetonitrile and then in the reversed-phase mode after the addition of 5% acetic acid to the solvent and eluting in the reverse order.

Fumonisin B(1)-nonproducing strains of Fusarium verticillioides cause maize (Zea mays) ear infection and ear rot.

Fumonisins are polyketide mycotoxins produced by Fusarium verticillioides (synonym F. moniliforme), a major pathogen of maize (Zea mays) worldwide. Most field strains produce high levels of fumonisin B(1) (FB(1)) and low levels of the less-oxygenated homologues FB(2) and FB(3), but fumonisin B(1)-nonproducing field strains have been obtained by natural variation. To test the role of various fumonisins in pathogenesis on maize under field conditions, one strain producing FB(1), FB(2), and FB(3), one strain producing only FB(2), one strain producing only FB(3), and one fumonisin-nonproducing strain were applied to ears via the silk channel and on seeds at planting. Disease severity on the harvested ears was evaluated by visible symptoms and by weight percent symptomatic kernels. Fumonisin levels in kernels were determined by high-performance liquid chromatography. The presence of the applied FB(1)-nonproducing strains in kernels was determined by analysis of recovered strains for fumonisin production and other traits. All three FB(1)-nonproducing strains were able to infect ears following either silk-channel application or seed application at planting and were as effective as the FB(1)-producing strain in causing ear rot following silk-channel application. These results indicate that production of FB(1), FB(2), or FB(3) is not required for F. verticillioides to cause maize ear infection and ear rot.

Occurrence of Fusarium species and mycotoxins in nepalese maize and wheat and the effect of traditional processing methods on mycotoxin levels.

Maize (Zea mays) and wheat (Triticum aestivum) collected in the foothills of the Nepal Himalaya Mountains were analyzed for Fusarium species and mycotoxins: fumonisins, nivalenol (NIV), and deoxynivalenol (DON). Predominant species were Gibberella fujikuroi mating population A (F. moniliforme) in maize and F. graminearum in maize and wheat; G. fujikuroi mating population D (F. proliferatum), F. acuminatum, F. avenaceum, F. chlamydosporum, F. equiseti, F. oxysporum, F. semitectum, and F. torulosum were also present. Strains of G. fujikuroi mating population A produced fumonisins, and strains of F. graminearum produced NIV or DON. By immunoassay or high-performance liquid chromatography, fumonisins were >1000 ng/g in 22% of 74 maize samples. By immunoassay or fluorometry, NIV and DON were >1000 ng/g in 16% of maize samples but were not detected in wheat. Fumonisins and DON were not eliminated by traditional fermentation for producing maize beer, but Nepalese rural and urban women were able to detoxify contaminated maize by hand-sorting visibly diseased kernels.

Fluorescence polarization as a means for determination of fumonisins in maize.

Fumonisins, mycotoxins produced by certain species of Fusaria, are commonly found worldwide as contaminants in maize. This paper reports the development of a rapid, portable fluorescence polarization-based assay for fumonisins in maize. The assay was based on the competition of unlabeled fumonisin, from a sample, with a fluorescently tagged fumonisin (FB(1)-FL) for a fumonisin-specific monoclonal antibody in solution. The fluorescence polarization (FP) of the tagged fumonisin was increased upon binding with the antibody. In the presence of free toxin, less of the FB(1)-FL was bound and the polarization signal was decreased. The assays were very simple to perform, requiring only mixing of an aqueous extract of maize with the tagged fumonisin and antibody, and required <2 min per sample, excluding extraction time. Two permutations of the assay were tested, one with each sample matrix serving as its own blank, and the other with all of the samples compared relative to a PBS blank with normalization of the data similar to an ELISA. The limit of detection, defined as the toxin content associated with a fluorescence polarization signal 5 standard deviations from that of a fumonisin-free control, was 0.5 microg of FB(1)/g in spiked maize. Recoveries from spiked maize over the range of 0.5-20 ppm averaged 94.3 +/- 13.8%. Forty-eight samples of field-contaminated maize were tested by the FP and an established HPLC method, with a good correlation between the two (r(2) = 0.85-0.88). For these samples, the two variations of the FP assay also compared well to one another (r(2) = 0.97), suggesting the assay principle is very robust. The results, combined with the speed and ease of use for the assay, suggest that this technology has substantial potential as a screening tool for mycotoxins in foods.

Effects of selected secondary metabolites of Fusarium moniliforme on unscheduled synthesis of DNA by rat primary hepatocytes.

The Fusarium moniliforme mycotoxins--fusarin C, fumonisin B1, moniliformin and bikaverin--were evaluated for genotoxicity by their ability to induce unscheduled DNA synthesis (UDS) in primary rat hepatocytes. Isolated hepatocytes were exposed to several concentrations of moniliformin (5.0-500 microM), bikaverin (1.0-500 microM), fumonisin B1 (0.5-250 microM), or fusarin C (1.0-100 microM). Aflatoxin B1, a known inducer of UDS, was included as a positive control. UDS was determined by autoradiography of cells after their exposure to [3H]thymidine. The highest doses of fusarin C and bikaverin caused cell death, but no cytotoxicity was observed in cells exposed to moniliformin or fumonisin B1. Fumonisin B1, moniliformin and bikaverin were not genotoxic in the UDS assay. The results of the UDS assay with fusarin C were inconclusive since a marginal effect on UDS was obtained.

Hepatotoxicity and renal toxicity in rats of corn samples associated with field cases of equine leukoencephalomalacia.

Currently there is no convenient bioassay to determine the potential toxicity of corn naturally contaminated with Fusarium moniliforme. A short-term bioassay would be useful for future investigations aimed at isolating as yet unidentified toxins produced by this fungus. Two groups of five male Sprague-Dawley rats were each fed one of two F. moniliforme contaminated corn samples, designated CS-1 and CS-2, that were associated with separate field cases of equine leukoencephalomalacia (ELEM). A control group, also consisting of five male rats, was fed uncontaminated seed corn. All animals survived to the end of the study and there were no apparent differences in appearance or behaviour among groups. Weight loss and irregular food consumption occurred in all groups and probably resulted from nutritional deficiencies inherent in the corn diets. Hepatocellular degeneration, necrosis and hyperplasia as well as biliary hyperplasia were found in the test groups only and were attributed to F. moniliforme. Serum transaminase and alkaline phosphatase activities in animals fed CS-1 and CS-2 for 4 wk were significantly increased compared with the controls, while serum bilirubin concentration was increased only in the CS-1 group. Tubular nephrosis was also present in the renal cortex of all animals fed CS-1 and CS-2. These effects may have been related to fumonisins B1 and B2, recently discovered metabolites of F. moniliforme, that were found in both CS-1 and CS-2. Short-term studies of this type may be useful in screening naturally-contaminated grains and other materials for hepatotoxic metabolites produced by F. moniliforme.

Instability of N-acetylated fumonisin B1 (FA1) and the impact on inhibition of ceramide synthase in rat liver slices.

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium verticillioides. It inhibits ceramide synthase, which is a proposed underlying mechanism responsible for the myriad of toxic endpoints observed. We previously reported that N-acetylation of FB1 prevents ceramide synthase inhibition, but cautioned that impure preparations of FA1 can contain a contaminant with the ability to inhibit ceramide synthase. We now report that FA1 spontaneously rearranges to O-acetylated analogs. These rearrangement products are putative inhibitors of ceramide synthase. Rat liver slices exposed to impure FA1 containing O-acetylated FB1 had sphinganine/sphingosine (Sa:So) ratios of 1.15-1.64. Control slices had Sa:So ratios of 0.07-0.24. Clean-up to remove the O-acetylated FB1 yielded purified FA1, which produced Sa:So ratios in liver slices of 0.08-0.18. After storage for approximately 1 year as either a dry powder in a desiccator, or as a dried film at 4 degrees C, the purified FA1 again contained O-acetylated FB1, and was capable of ceramide synthase inhibition. FA1 was most stable in neutral solution, but in acidic solution the equilibrium shifted towards the O-acetylated forms. FA1 in solid form also rearranged, but more slowly than in acid solution. As FA1 is considerably less cytotoxic than FB1, these results provide additional support for the conclusion that a primary amino group is necessary for both ceramide synthase inhibition and toxicity.

Oxidative degradation and detoxification of mycotoxins using a novel source of ozone.

Practical methods to degrade mycotoxins using ozone gas (O3) have been limited due to low O3 production capabilities of conventional systems and their associated costs. Recent advances in electrochemistry (i.e. proton-exchange membrane and electrolysis technologies) have made available a novel and continuous source of O3 gas up to 20% by weight. It is possible that the rapid delivery of high concentrations of O3 will result in mycotoxin degradation in contaminated grains--with minimal destruction of nutrients. The major objectives of this study were to investigate the degradation and detoxification of common mycotoxins in the presence of high concentrations of O3. In this study, aqueous equimolar (32 microM) solutions of aflatoxins B1 (AfB1), B2 (AfB2), G1 (AfG1), G2 (AfG2), cyclopiazonic acid (CPA), fumonisin B1 (FB1), ochratoxin A (OA), patulin, secalonic acid D (SAD) and zearalenone (ZEN) were treated with 2, 10 and/or 20 weight% O3 over a period of 5.0 min and analysed by HPLC. Results indicated that AfB1 and AfG1 were rapidly degraded using 2% O3, while AfB2 and AfG2 were more resistant to oxidation and required higher levels of O3 (20%) for rapid degradation. In other studies, patulin, CPA, OA, SAD and ZEN were degraded at 15 sec, with no by-products detectable by HPLC. Additionally, the toxicity of these compounds (measured by a mycotoxin-sensitive bioassay) was significantly decreased following treatment with O3 for 15 sec. In another study, FB1 (following reaction with O3) was rapidly degraded at 15 sec, with the formation of new products. One of these appeared to be a 3-keto derivative of FB1. Importantly, degradation of FB1 did not correlate with detoxification, since FB1 solutions treated with O3 were still positive in two bioassay systems.

Linoleate hydroperoxides are cleaved heterolytically into aldehydes by a Lewis acid in aprotic solvent.

Treatment of isomeric methyl linoleate hydroperoxides with a Lewis acid, BF3, in anhydrous ether led to a carbon-to-oxygen rearrangement that caused cleavage into shorter-chain aldehydes. Methyl (9Z,11E)-13-hydroperoxy-9,11-octadecadienoate afforded mainly hexanal and methyl (E)-12-oxo-10-dodecenoate, whereas methyl (10E,12Z)-9-hydroperoxy-10,12-octadecadienoate cleaved into 2-nonenal and methyl 9-oxononanoate. The 2 aldehydes obtained from each hydroperoxide isomer were uncharacteristic of the complex volatile profile usually obtained by ß-scission of oxy radicals derived from homolysis of the hydroperoxide group. Rather, the reaction resembled the one catalyzed by the plant enzyme, hydroperoxide lyase.

Cis-5-polyenoic acids inLarix leptolepis seed oil.

cis-5,cis-9-Octadecadienoic acid (2.7%) andcis-5,cis-9,cis-12-octadecatrienoic acid (24.9%) are present in the seed oil ofLarix leptolepis. The double bond positions were identified by ozonolysis and by gas chromatography-mass spectrometry of methoxy derivatives. Small quantities of branched chain acids of various chain lengths were indicated by gas chromatography. The presence of C17 and C19 branched methyl esters was confirmed by gas chromatography-mass spectrometry.

Alchornea cordifolia seed oil: A rich source of a new C20 epoxide, (+)cis-14,15-epoxy-cis-11-eicosenoic acid.

A C20 homolog of vernolic acid has been found at the 50% level inAlchornea cordifolia, Euphorbiaceae, seed oil. This new acid, (+)cis-14,15-epoxy-cis-11-eicosenoic (alchornoic) acid, was isolated by high-pressure liquid chromotography and characterized by mass spectrometry, nuclear magnetic resonance and infrared spectroscopy, optical rotary dispersion, and ozonolysis-gas-chromotography.

Fumonisin B1 concentrations in feeds from 45 confirmed equine leukoencephalomalacia cases.

During the fall of 1989 and winter of 1990, numerous reports of equine leukoencephalomalacia (ELEM) occurred from many regions of the United States. Typically, horses were consuming feed partially or entirely composed of corn and/or corn screenings. From October 1989 through May 1990, samples from 55 confirmed or suspected ELEM cases were received at the National Veterinary Services Laboratories, Ames, Iowa, for fumonisin B1 analysis. Samples from 9 cases in 1984-1985 were also obtained. Fumonisin B1, a mycotoxin produced by Fusarium moniliforme, causes ELEM, but little is known of naturally occurring toxic or safe levels in feeds. To determine what levels of fumonisin B1 in feeds are associated with ELEM, 45 selected cases were studied. The fumonisin B1 concentrations ranged from less than 1 ppm to 126 ppm, with the majority of the samples above 10 ppm. All types of feeds were included: corn, screenings, sweet feeds, and commercially pelleted rations. The length of exposure varied from 7 to greater than 35 days. Horse feed samples not associated with ELEM were also collected and analyzed. None of the nonproblem feed samples contained fumonisin B1 levels greater than 8 ppm.

Fumonisin B1 levels associated with an epizootic of equine leukoencephalomalacia.

During the fall of 1989, an episode of equine leukoencephalomalacia involved 18 of 66 purebred Arabian horses at a breeding/training stable in Arizona. Of the 18 horses affected, the condition was fatal in 14. These horses, as well as 48 unaffected horses, had been fed a diet containing a substantial amount of white corn screenings. Gross pathologic findings included liquefactive necrosis in parts of the cerebral white matter and hemorrhagic foci of various sizes in the brain stem. Histopathologic findings included rarefied white matter with pyknotic nuclei and eosinophilic cytoplasm. Thin-layer chromatography, high-performance liquid chromatography, and gas chromatography/mass spectroscopy were utilized to identify and quantitate fumonisin B1 in 3 samples of corn from the farm. Concentrations of fumonisin B1 range from 37 to 122 ppm. Fumonisin B2 was also detected. Using information on diet, animal weights, and feeding practices, estimates of total fumonisin B1 dosage were determined. This is the first definitive report on equine leukoencephalomalacia and associated fumonisin B1 concentrations.

Analytical determination of fumonisins and other metabolites produced by Fusarium moniliforme and related species on corn.

Fumonisins, secondary metabolites of the fungus Fusarium moniliforme are potent toxins that can be found in fungal contaminated corn. The detection and measurement of these toxins by HPLC with detection by an evaporative light scattering detector and by electrospray MS is reported. The light scattering detector had enough sensitivity to analyze culture materials, however, clean-up was necessary to detect fumonisins at sub-ppm levels in naturally contaminated corn extracts. The detection limit for FB1 with the light scattering detector was in the low ng range (10-50) while the detection limit of less than 1 ng injected was observed for the electrospray detector. Several previously unreported fumonisin isomers were observed in electrospray chromatograms of culture extracts. Two of these compounds, FA3 and FA4 were isolated and their proposed structure confirmed by NMR experiments.