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1.
Fumonisins are mycotoxins produced by Fusarium verticillioides, a widespread pathogen of corn. Although the gene cluster for the biosynthesis of fumonisins has been cloned, the majority of the genes have not been biochemically characterized. Here, we report the biochemical characterization of FUM13, a gene that encodes a short-chain dehydrogenase/reductase required for fumonisin biosynthesis. FUM13 has been expressed in E. coli, and the produced protein, Fum13p, has been purified. When the protein was incubated with 3-keto fumonisin B(3) (FB(3)) in the presence of NADPH, FB(3) was produced. The data provide direct evidence for the role of FUM13 in the 3-ketoreduction of fumonisins. In a functional complementation experiment, FUM13 gene was introduced into tsc10 mutants of the yeast Saccharomyces cerevisiae, which carry a mutation in the 3-ketosphinganine reductase gene in the sphingolipid pathway. The tsc10 mutants were not able to grow on the selection medium, but the same mutants transformed with FUM13 were able to grow. The results further confirm the function of FUM13 in 3-ketoreduction in vivo.  相似文献   

2.
Fumonisins are carcinogenic mycotoxins produced by the maize ear rot pathogen Gibberella moniliformis (anamorph Fusarium verticillioides). These toxins consist of a linear polyketide-derived backbone substituted at various positions with an amine, one to four hydroxyl, two methyl, and two tricarballylic ester functions. In this study, we generated and characterized deletion mutants of G. moniliformis for five genes, FUM7, FUM10, FUM11, FUM14, and FUM16 in the fumonisin biosynthetic gene cluster. Functional analysis of mutants in four genes, predicted to encode unrelated proteins, affected formation of the tricarballylic esters. FUM7 deletion mutants produced a previously undescribed homologue of fumonisin B1 with an alkene function in both tricarballylic esters, FUM10 and FUM14 deletion mutants produced homologues of fumonisin B3 and fumonisin B4 that lack tricarballylic ester functions, and FUM11 deletion mutants produced fumonisins that lack one of the tricarballylic ester functions. These phenotypes indicated specific roles for FUM7, FUM10, FUM11, and FUM14 in fumonisin biosynthesis that are consistent with the predicted proteins encoded by each gene. Deletion of FUM16 had no apparent effect on fumonisin production. The phenotypes of the deletion mutants provide further insight into the order of steps in fumonisin biosynthesis.  相似文献   

3.
Fumonisins are polyketide-derived mycotoxins produced by the filamentous fungus Gibberella moniliformis (anamorph Fusarium verticillioides). Wild-type strains of the fungus produce predominantly four B-series fumonisins, designated FB(1), FB(2), FB(3), and FB(4). Recently, a cluster of 15 putative fumonisin biosynthetic genes (FUM) was described in G. moniliformis. We have now conducted a functional analysis of FUM13, a gene in the cluster that is predicted by amino acid sequence similarity to encode a short chain dehydrogenase/reductase (SDR). Mass spectrometric analysis of metabolites from FUM13 deletion mutants revealed that they produce approximately 10% of wild-type levels of B-series fumonisins as well as two previously uncharacterized compounds. NMR analysis revealed that the new compounds are similar in structure to FB(3) and FB(4) but that they have a carbonyl function rather than a hydroxyl function at carbon atom 3 (C-3). These results indicate that the FUM13 protein catalyzes the reduction of the C-3 carbonyl to a hydroxyl group and are the first biochemical evidence directly linking a FUM gene to a specific reaction during fumonisin biosynthesis. The production of low levels of FB(1), FB(2), FB(3), and FB(4), which have a C-3 hydroxyl, by the FUM13 mutants suggests that G. moniliformis has an additional C-3 carbonyl reductase activity but that this enzyme functions less efficiently than the FUM13 protein.  相似文献   

4.
Fumonisins, a family of mycotoxins produced by Fusarium moniliforme and Fusarium proliferatum, are found in maize worldwide and have been associated with animal diseases. There is concern that high dietary intake of a maize-based diet may expose people in Mexico and Central America to fumonisins. Nixtamalized maize products from Mexico and the United States were examined to evaluate methods for quantitation of the different forms of fumonisins. The chelating reagent EDTA (exceeding the calcium concentration by a factor of 1. 36) was added to enhance extraction of fumonisins because calcium remained in the samples as a result of processing. It was expected that the majority of the fumonisin detected would be in the hydrolyzed form, yet the highest level of hydrolyzed fumonisin B(1) detected was 0.1 ppm. The amount of fumonisin B(1) was significantly higher in Mexican samples (mean = 0.79 ppm) than in samples purchased in the United States (mean = 0.16 ppm).  相似文献   

5.
Fumonisins are polyketide-derived mycotoxins produced by the maize pathogen Fusarium verticillioides. Previous analyses identified naturally occurring variants of the fungus that are deficient in fumonisin C-10 hydroxylation or that do not produce any fumonisins. In the current study, gene deletion and genetic complementation analyses localized the C-10 hydroxylation deficiency to a cytochrome P450 monooxygenase gene in the fumonisin biosynthetic gene (FUM) cluster. Sequence analysis indicated that the hydroxylation deficiency resulted from a single nucleotide insertion that caused a frame shift in the coding region of the gene. Genetic complementation localized the fumonisin-nonproduction phenotype to the polyketide synthase gene in the FUM cluster, and sequence analysis indicated that the nonproduction phenotype resulted from a nucleotide substitution, which introduced a premature stop codon in the coding region. These results provide the first direct evidence that altered fumonisin production phenotypes of naturally occurring F. verticillioides variants can result from single point mutations in the FUM cluster.  相似文献   

6.
The fungus Fusarium verticillioides infects maize and produces fumonisins. The purpose of this study was to determine the ability of F. verticillioides to produce fumonisins in synthetic and natural soils and their biological availability to maize roots. Maize seeds were inoculated with a pathogenic strain of F. verticillioides (MRC826) and planted in synthetic and three different natural soils. There were statistically significant reductions in stalk weight and root mass and increased leaf lesions in the MRC826-treated seedlings in all soil types. Fumonisins were detected in all of the soils of seedlings grown from MRC826-inoculated seeds. The fumonisin produced in the soils was biologically available to seedlings as demonstrated by the statistically significant elevation of free sphingoid bases and sphingoid base 1-phosphates in their roots. These results indicate that F. verticillioides produced fumonisins in the autoclaved synthetic and natural soils and that the fumonisin produced is biologically available on the basis of evidence of inhibition of ceramide synthase.  相似文献   

7.
To extract fumonisin B1 (FB1) and fumonisin B2 (FB2) from Thai white rice flour, different solvent mixtures, temperatures, pH values, and addition of enzymes or ethylenediaminetetraacetic acid disodium salt (Na2EDTA) were examined. Three extractions with 0.1 M Na2EDTA achieved the highest recoveries. Initial recoveries of fumonisins added to white rice flour, cornstarch, cornmeal, and glucose varied with commodity. Fumonisins disappeared in Thai white rice flour after 12 h, but 55% remained in another white rice flour. With cornstarch 20-30% fumonisins remained after 24 h; only 43% of 14C-labeled FB1 materials extracted from cornstarch was eluted with methanol from an immunoaffinity column. Fumonisins were stable in cornmeal for 24 h but only approximately 50% remained after 30 days. With glucose, 25% of FB1 and FB2 remained 24 h after addition; N-(1-deoxy-D-fructos-1-yl)FB(1) andN-(carboxymethyl)FB(1) were detected in lower amounts than residual FB(1) after 3 months.  相似文献   

8.
Fumonisins are mycotoxins produced by Fusarium verticillioides (=F. moniliforme) and other Fusarium species. They are found in corn and corn-based foods. Cooking decreases fumonisin concentrations in food products under some conditions; however, little is known about how cooking effects biological activity. Baked cornbread, pan-fried corncakes, and deep-fried fritters were made from cornmeal that was spiked with 5% w/w F. verticillioides culture material (CM). The cooked materials and the uncooked CM-spiked cornmeal were fed to male rats (n = 5/group) for 2 weeks at high (20% w/w spiked cornmeal equivalents) or low (2% w/w spiked cornmeal equivalents) doses. A control group was fed a diet containing 20% w/w unspiked cornmeal. Toxic response to the uncooked CM-spiked cornmeal and the cooked products included decreased body weight gain (high-dose only), decreased kidney weight, and microscopic kidney and liver lesions of the type caused by fumonisins. Fumonisin concentration, as determined by HPLC analysis, in the 20% w/w pan-fried corncake diet [92.2 ppm of fumonisin B(1) (FB(1))] was slightly, but not statistically significantly, lower than those of the 20% w/w baked cornbread (132.2 ppm of FB(1)), deep-fried fritter (120.2 ppm of FB(1)) and CM-spiked cornmeal (130.5 of ppm FB(1)) diets. Therefore, baking and frying had no significant effect on the biological activity or concentration of fumonisins in these corn-based products, and the results provided no evidence for the formation of novel toxins or "hidden" fumonisins during cooking.  相似文献   

9.
Corn silage was dried, ground, and then extracted with 0.1 M ethylenediaminetetraacetic acid. The filtrate was applied to a FumoniTest immunoaffinity column. Fumonisins were derivatized with naphthalene-2,3-dicarboxaldehyde, separated on a C(18) liquid chromatographic column, and detected by fluorescence. The detection limits for fumonisin B(1), fumonisin B(2), and fumonisin B(3) were 50, 25, and 25 ng/g of dried silage, respectively. Recoveries of fumonisin B(1), fumonisin B(2), and fumonisin B(3) from wet and dried corn silage spiked over the range of 100-5000 ng/g averaged 91-106%. The method was applied to corn silage samples collected from the midwestern area of the United States during 2001-2002. Of 89 corn silage samples, fumonisin B(1), fumonisin B(2), and fumonisin B(3) were found in 86 (97%), 64 (72%), and 51 (57%) of the samples. The mean positive levels of fumonisin B(1), fumonisin B(2), and fumonisin B(3) were 615, 93, and 51 ng/g, respectively, in dried silage. This suggests that fumonisins may be frequent low level contaminants in corn silage.  相似文献   

10.
Fumonisins are mycotoxins produced by Fusarium verticillioides (Sacc. Nirenberg) in maize (Zea mays L.), a staple crop in Mexico. In this study, we report the isolation and identification of 67 Fusarium strains isolated from maize kernels collected in Northwest and Central Mexico. The strains were characterized regarding fumonisin B(1) production and the presence of the FUM1 gene. F. verticillioides was the predominant species isolated in both geographic regions, but the isolates from Northwest Mexico produced higher levels of fumonisin. A polymerase chain reaction (PCR)-based method, to detect a region of the FUM1 gene involved in fumonisin biosynthesis, was developed and employed to detect mycotoxigenic fungi in pure culture and in contaminated maize. The presence of the FUM1 gene was associated with fumonisin production in most isolates, except seven that did not synthesize fumonisin but contained the gene in their genome. The PCR method allowed the direct detection of fungal contamination in ground corn and could be employed to screen for the presence of potential mycotoxigenic fusaria.  相似文献   

11.
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.  相似文献   

12.
Fumonisin B2 production by Aspergillus niger   总被引：1，自引：0，他引：1  
The carcinogenic mycotoxin fumonisin B2 was detected for the first time in the industrially important Aspergillus niger. Fumonisin B2, known from Fusarium verticillioides and other Fusaria, was detected in cultures of three full genome sequenced strains of A. niger, in the ex type culture and in a culture of F. verticillioides by electrospray LC-MS analysis of methanolic extracts from agar plugs of cultures grown on several substrates. Whereas F. verticillioides produced fumonisins B1, B2, and B3 on agar media based on plant extracts, such as barley malt, oat, rice, potatoes, and carrots, A. niger produced fumonisin B2 best on agar media with a low water activity, including Czapek yeast autolysate agar with 5% NaCl. Of the media tested, only rice corn steep agar supported fumonisin production by both F. verticillioides and A. niger. However, A. niger had a different regulation of fumonisin production and a different quantitative profile of fumonisins, producing only B2 as compared to F. verticillioides. Fumonisin production by A. niger, which is a widely occurring species and an extremely important industrial organism, will have very important implications for biotechnology and especially food safety. A. niger is used for the production of citric acid and as producer of extracellular enzymes, and also as a transformation host for the expression of heterologous proteins. Certain strains of A. niger produce both ochratoxin A and fumonisins, so some foods and feeds may potentially contain two types of carcinogenic mycotoxins from this species.  相似文献   

13.
Concerns that raisins may be contaminated by fumonisins stem from the persistent occurrence of Aspergillus niger spores on raisins and the recent discovery of fumonisin production by A. niger on grapes, which leads to the widespread occurrence of fumonisin B(2) in wine. This study presents an LC-MS/MS survey of fumonisins in retail raisins. In 10 of 21 brands collected in Denmark, Germany, and The Netherlands, fumonisins B(2) and B(4) were detected at levels up to 13 and 1.3 μg/kg, respectively. Only fumonisin B(2) has been detected in wine, so the presence of fumonisin B(4) in raisins suggests that the fumonisins are produced mainly during the drying process concomitant with the decreasing water activity. Analysis of multiple packages from one manufacturer showed a 3-fold package-to-package variation, suggesting that a few raisins per package are contaminated.  相似文献   

14.
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.  相似文献   

15.
A total of 52 corn samples collected in 2000 from four main corn production provinces of Iran (Fars, Kermanshah, Khuzestan, and Mazandaran) were analyzed for contamination with Fusarium verticillioides and fumonisins (FB(1), FB(2), FB(3), and 3-epi-FB(3)). The mean incidence of F. verticillioides (percent of kernels infected) for these four areas was 26.7, 21.4, 24.9, and 59.0%, respectively. The incidence in Mazandaran was significantly (p < 0.05) above that of the other areas. All samples from Mazandaran were contaminated with fumonisins with a mean level of total fumonisins of 10674 microg/kg. In contrast, the incidence of fumonisin contamination above 10 microg/kg was 53 (8/15), 42 (5/12), and 57% (8/14) in the samples from Fars, Kermanshah, and Khuzestan, respectively, and the corresponding mean total fumonisin levels were 215, 71, and 174 microg/kg, respectively. No statistical differences (p > 0.05) were observed in the fumonisin levels of the corn samples from these three provinces, which were significantly (p < 0.05) lower than the fumonisin contamination in samples from Mazandaran.  相似文献   

16.
A survey was carried out to determine Fusarium species and fumonisin contamination in 55 durum wheat (Triticum turgidum L. var. durum) samples collected during two harvest seasons (2007 and 2008) using HPLC and further LC-MS/MS confirmation. All samples showed Fusarium contamination with infection levels ranging from 8 to 66%, F. proliferatum being the species most frequently isolated during 2007 and the second most frequently isolated one during the 2008 harvest season, respectively. Natural contamination with fumonisins was found in both harvest seasons. In 2007, 97% of the samples showed total fumonisin (FB(1) + FB(2)) levels ranging from 10.5 to 1245.7 ng/g, while very low levels of fumonisins were detected in samples collected during 2008. These results could be explained by differences in the amount of rainfall during both periods evaluated. A selected number (n = 48) of F. proliferatum isolates showed fumonisin production capability on autoclaved rice. This is the first report of the presence of natural fumonisins in durum wheat grains.  相似文献   

17.
To study the formation of fumonisin artifacts and the binding of fumonisins to matrix components (e.g., saccharides and proteins) in thermal-treated food, model experiments were performed. Fumonisin B(1) and hydrolyzed fumonisin B(1) were incubated with alpha-d-glucose and sucrose (mono- and disaccharide models), with methyl alpha-d-glucopyranoside (starch model), and with the amino acid derivatives N-alpha-acetyl-l-lysine methyl ester and BOC-l-cysteine methyl ester (protein models). The reaction products formed were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry. The incubation of d-glucose with fumonisin B(1) or hydrolyzed fumonisin B(1) resulted in the formation of Amadori rearrangement products. Whereas conjugates were found following the reaction of sucrose, methyl alpha-d-glucopyranoside, and the amino acid derivatives with fumonisin B(1), the heating with hydrolyzed fumonisin B(1) yielded no artifacts. For structural determination, the stable reaction product formed by heating of methyl alpha-d-glucopyranoside (as starch model) with fumonisin B(1) was purified and identified by nuclear magnetic resonance spectroscopy as the diester of the fumonisin tricarballylic acid side chains with methyl alpha-d-glucopyranoside. These model experiments demonstrate that fumonisins are able to bind to polysaccharides and proteins via their two tricarballylic acid side chains.  相似文献   

18.
Cowpea seed samples from South Africa and Benin were analyzed for seed mycoflora. Fusariumspecies detected were F. equiseti, F. chlamydosporum, F. graminearum, F. proliferatum, F. sambucinum, F. semitectum, and F. subglutinans. Cowpea seed from South Africa and Benin and F. proliferatum isolates from Benin, inoculated onto maize patty medium, were analyzed for fumonisin production. Samples were extracted with methanol/water and cleaned up on strong anion exchange solid phase extraction cartridges. HPLC with precolumn derivatization using o-phthaldialdehyde was used for the detection and quantification of fumonisins. Cowpea cultivars from South Africa showed the presence of fumonisin B(1) at concentrations ranging between 0.12 and 0.61 microg/g, whereas those from Benin showed no fumonisins. This is believed to be the first report of the natural occurrence of FB(1) on cowpea seed. Fumonisin B(1), B(2), and B(3) were produced by all F. proliferatum isolates. Total fumonisin concentrations were between 0.8 and 25.30 microg/g, and the highest level of FB(1) detected was 16.86 microg/g.  相似文献   

19.
Fusarium proliferatum is one of a group of fungal species that produce fumonisins and is considered to be a pathogen of many economically important plants. The occurrence of fumonisin B(1) (FB(1)) in F. proliferatum-infected asparagus spears from Germany was investigated using a liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method with isotopically labeled fumonisin FB(1)-d(6) as internal standard. FB(1) was detected in 9 of the 10 samples in amounts ranging from 36.4 to 4513.7 ng/g (based on dry weight). Furthermore, the capability of producing FB(1) by the fungus in garlic bulbs was investigated. Therefore, garlic was cultured in F. proliferatum-contaminated soil, and the bulbs were screened for infection with F. proliferatum and for the occurrence of fumonisins by LC-MS. F. proliferatum was detectable in the garlic tissue, and all samples contained FB(1) (26.0-94.6 ng/g). This is the first report of the natural occurrence of FB(1) in German asparagus spears, and these findings suggest a potential for natural contamination of garlic bulbs with fumonisins.  相似文献   

20.
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.  相似文献   

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