Production of fumonisin B and C analogues by several fusarium species

Six strains of Fusarium verticillioides, two of F. oxysporum, one strain of F. proliferatum, and a strain of an unidentified species were cultured on maize patties and rice and evaluated for their ability to simultaneously produce fumonisin B (FB) and C (FC) series analogues. Fumonisins were quantified by LC-MS-MS using positive ion electrospray ionization. FC1 provided characteristic fragment ions at m/z 690, 672, 654, 532, 514, and 338 corresponding to sequential loss of H2O and tricarboxylic acid moieties from the alkyl backbone, while FC3 and FC4 provided equivalent product ions 16 and 32 amu lower than the corresponding FC1 fragments, respectively. All isolates cultured on maize produced FC4. All isolates except for that of F. proliferatum also produced FC1, and three of the six strains of F. verticillioides produced FC3. All isolates except those of F. oxysporum produced detectable amounts of FB1, FB2, and FB3. Isolates that produced fumonisin B analogues produced at least 10 fold more of the B series analogues than they did of the C series analogues. The results confirm that at least some strains of F. oxysporum produce FC, but not FB, fumonisin analogues and also suggest that the genetics and physiological regulation of fumonisin production may be more complicated than previously envisaged since some strains of F. verticillioides and F. proliferatum as well as the strain of the unidentified species can simultaneously produce both FB and FC analogues.     

Rapid detection of kernel rots and mycotoxins in maize by near-infrared reflectance spectroscopy

Near-infrared (NIR) spectroscopy is a practical spectroscopic procedure for the detection of organic compounds in matter. It is particularly useful because of its nondestructiveness, accuracy, rapid response, and easy operation. This work assesses the applicability of NIR for the rapid identification of micotoxigenic fungi and their toxic metabolites produced in naturally and artificially contaminated products. Two hundred and eighty maize samples were collected both from naturally contaminated maize crops grown in 16 areas in north-central Italy and from ears artificially inoculated with Fusarium verticillioides. All samples were analyzed for fungi infection, ergosterol, and fumonisin B1 content. The results obtained indicated that NIR could accurately predict the incidence of kernels infected by fungi, and by F. verticillioides in particular, as well as the quantity of ergosterol and fumonisin B1 in the meal. The statistics of the calibration and of the cross-validation for mold infection and for ergosterol and fumonisin B1 contents were significant. The best predictive ability for the percentage of global fungal infection and F. verticillioides was obtained using a calibration model utilizing maize kernels (r2 = 0.75 and SECV = 7.43) and maize meals (r2 = 0.79 and SECV = 10.95), respectively. This predictive performance was confirmed by the scatter plot of measured F. verticillioides infection versus NIR-predicted values in maize kernel samples (r2 = 0.80). The NIR methodology can be applied for monitoring mold contamination in postharvest maize, in particular F. verticilliodes and fumonisin presence, to distinguish contaminated lots from clean ones, and to avoid cross-contamination with other material during storage and may become a powerful tool for monitoring the safety of the food supply.     

Fumonisin production by Fusarium verticillioides strains isolated from maize in Mexico and development of a polymerase chain reaction to detect potential toxigenic strains in grains

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.     

Incidence of Fusarium verticillioides and levels of fumonisins in corn from main production areas in Iran

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.     

Mycoflora and fumonisin contamination in Brazilian corn from sowing to harvest

The present study aimed to analyze the mycoflora and potential mycotoxin contamination of soil and corn samples collected at different plant maturity stages in Cap?o Bonito and Ribeir?o Preto, two regions of the State of S?o Paulo, Brazil. In addition, the data obtained were correlated with the occurrence of wind-dispersed fungi and the predominant climatic conditions of the two regions studied. Corn mycoflora profiles showed that Fusarium verticillioides prevailed in 35% of the samples from Cap?o Bonito and in 49% of the samples from Ribeir?o Preto. Examination of wind-dispersed fungi also revealed a high incidence of F. verticillioides. Soil mycoflora analyses showed that Penicilliumwas the most prevalent genus, although F. verticillioides was present in 55.5% of Cap?o Bonito's samples and in 26.7% of Ribeir?o Preto's samples. With respect to water activity, the corn kernels most contaminated with F. verticillioides had water activity levels of 0.70-0.80. HPLC analysis of fumonisins revealed that 88.5% of Cap?o Bonito's kernels were contaminated with fumonisin B(1) (FB(1)) (0.09-10.87 microg/g) and 53.8% with fumonisin B(2) (FB(2)) (0.05-0.52 microg/g); Ribeir?o Preto's kernels presented contamination levels of 93.5% for FB(1) (0.11-17.69 microg/g) and 61.3% for FB(2) (0.05-5.24 microg/g). No aflatoxins were detected by thin-layer chromatography in corn grains of either region. The concomitant occurrence of F. verticillioides and fumonisins in most of the field corn assayed demonstrates the importance of an effective control of cultivation throughout the plant maturity stages.     

Gibberella ear rot of maize (Zea mays) in Nepal: distribution of the mycotoxins nivalenol and deoxynivalenol in naturally and experimentally infected maize

The fungus Fusarium graminearum (sexual stage Gibberella zeae) causes ear rot of maize (Zea mays) and contamination with the 8-ketotrichothecenes nivalenol (1) or 4-deoxynivalenol (2), depending on diversity of the fungal population for the 4-oxygenase gene (TRI13). To determine the importance of 1 and 2 in maize ear rot, a survey of naturally contaminated maize in Nepal was combined with experiments in the field and in a plant growth room. In the survey, 1 contamination was 4-fold more frequent than 2 contamination and 1-producers (TRI13) were isolated more than twice as frequently as 2-producers (Psi TRI13). In maize ear rot experiments, genetically diverse 1-producers and 2-producers caused ear rot and trichothecene contamination. Among strains with the same genetic background, however, 1-producers caused less ear rot and trichothecene contamination than did 2-producers. The high frequency of 1 contamination and the high virulence of many 1-producers are of concern because maize is a staple food of rural populations in Nepal and because 1 has proven to be more toxic than 2 to animals.     

Volatiles from Fusarium verticillioides (Sacc.) nirenb. and their attractiveness to nitidulid beetles

It is known that sap beetles (Coleoptera: Nitidulidae) can vector the fungus Fusarium verticillioides (Sacc.) Nirenb. (= F. moniliforme Sheldon), which causes an important ear-rot disease in corn and also produces fumonisin mycotoxins. The volatiles produced by this fungus were studied to establish whether they could attract sap beetles. Such an association would suggest more than just an incidental role in transmission of the fungus by the beetles. F. verticillioides consistently produces a blend of five alcohols (ethanol, 1-propanol, 2-methyl-1-propanol, 3-methyl-1-butanol, and 2-methyl-1-butanol), acetaldehyde, and ethyl acetate. Ethanol is the most abundant alcohol. The fungus also produces four phenolic compounds (the most abundant of which is ethylguaiacol), a series of presently unidentified sesquiterpene hydrocarbons, and an unidentified compound that is probably a 10-carbon ketone. Solid-phase microextraction was the key technique used in volatile analysis. The volatile profiles change over time and differ somewhat among fungal strains: The alcohols, aldehyde, and ester always appeared first and were present for each strain. Production of the phenolics lagged by several days, and in some strains these compounds were barely detectable. Volatile production eventually diminished in all strains. All strains were attractive to the sap beetle, Carpophilus humeralis (F.), in wind-tunnel bioassays. Attraction was correlated primarily to the presence of the alcohols, acetaldehyde, and ethyl acetate, rather than to the phenolics. To verify that the identified culture volatiles were responsible for beetle attraction, cultures were quantitatively simulated with synthetic chemicals, and the cultures and corresponding synthetic mixtures were then compared by bioassay. The comparisons were favorable. Volatile emission patterns from cultures were fairly robust with respect to inoculum level or incubation temperature, but some manipulation was possible. For example, after freeze-drying and rehydrating (a rapid simulation of winter/spring conditions), F. verticillioides produced ethyl acetate and other esters at unusually high levels. The fungus produced attractive volatiles following ear inoculation of milk-stage field corn as well as on sterile, mature kernels in the laboratory.     

Maize ear rot and moniliformin contamination by cryptic species of Fusarium subglutinans

Fusarium subglutinans causes maize ear rot and contaminates grain with the mycotoxin moniliformin. Previous DNA sequence analysis divided F. subglutinans from maize into two cryptic species, designated groups 1 and 2. Here, it was determined whether the two groups differ in the agriculturally important traits of virulence on maize and moniliformin production in planta. Thirty-seven strains from U.S. maize were assigned to groups 1 and 2 by DNA sequence analysis. In field tests, all strains were highly virulent on maize inbred B73 and four maize hybrids. In planta, 82% of group 1 strains and 25% of group 2 strains produced high levels (100-1500 microg/g) of moniliformin. All group 2 strains from more northern states produced little or no moniliformin (0-5 microg/g). These data indicate that moniliformin production is highly variable in F. subglutinans from U.S. maize and that production may not be required for the fungus to cause maize ear rot.     

Natural occurrence of fumonisins B(1) and B(2) in domestic maize of Taiwan.

Samples of maize grown in various districts of Taiwan were collected and analyzed for the presence of fumonisin B(1) (FB(1)) and fumonisin B(2) (FB(2)) using high-performance liquid chromatography. Forty-nine (44.5%) and 2 (1.8%) of 110 samples were found to contain FB(1) (109-1148 ng/g) and FB(2) (222-255 ng/g), respectively. The frequency of detection and also the maximum FB(1) concentration were found in samples from Penton (2/2, 262 ng/g), followed by Chiayi (18/26, 264 ng/g), Tainan (8/16, 160 ng/g), Hualinen (5/14, 1148 ng/g), Taitung (7/20, 109 ng/g), and Yunlin (9/26, 361 ng/g). Of the 110 samples examined, only 2 samples from Hualinen had been detected containing FB(2). During an analysis of the distribution pattern of FB(1), it became apparent that >79% of tested samples had FB(1) concentrations <100 ng/g, whereas 2.7% (or 3 samples) contained FB(1) >300 ng/g. These results clearly illustrated that domestically produced maize for human consumption is frequently contaminated with FB(1).     

Deletion analysis of FUM genes involved in tricarballylic ester formation during fumonisin biosynthesis

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.     

FUM13 encodes a short chain dehydrogenase/reductase required for C-3 carbonyl reduction during fumonisin biosynthesis in Gibberella moniliformis

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.     

Effect of nixtamalization (Alkaline cooking) on fumonisin-contaminated corn for production of masa and tortillas

Studies were undertaken to determine the fate of the mycotoxins, fumonisins, during the process of alkaline cooking (nixtamalization), using normal-appearing corn that was naturally contaminated with fumonisin B(1) (FB(1)) at 8.79 ppm. Corn was processed into tortillas, starting with raw corn that was cooked with lime and allowed to steep overnight; the steeped corn (nixtamal) was washed and ground into masa, which was used to make tortillas. Calculations to determine how much of the original fumonisin remained in the finished products took into consideration that FB(1) will be converted to hydrolyzed fumonisin B(1) (HFB(1)) by the process of alkaline cooking. All fractions, including steeping and washing water, were weighed, and percent moisture and fumonisin content were determined. Tortillas contained approximately 0.50 ppm of FB(1), plus 0.36 ppm of HFB(1), which represented 18.5% of the initial FB(1) concentration. Three-fourths of the original amount of fumonisin was present in the liquid fractions, primarily as HFB(1). Nixtamalization significantly reduced the amount of fumonisin in maize.     

Fate of fumonisin B(1) in corn kernel steeping water containing SO(2)

Six 100 ppm fumonisin B(1) (FB(1)) solutions were prepared by dissolving pure standard in six different solvents containing SO(2). Two of the solvents contained 0.2 or 0.4% SO(2) in distilled water. The other four solvents were obtained by steeping corn kernels at 60 degrees C in a 0.2% SO(2) aqueous solution for 6, 12, 24, or 48 h. After the addition of FB(1), all solutions were maintained at 60 degrees C for 7 days. Fumonisin B(1) content in each solution was determined in triplicate by HPLC. Steeping corn kernels in 0.2% solution at 60 degrees C for 6 h seems to be the most effective treatment to decrease the amount of FB(1).     

Fumonisins: probable role as effectors in the complex interaction of susceptible and resistant maize hybrids and Fusarium verticillioides

Fusarium verticillioides is best known for its worldwide occurrence on maize resulting in highly variable disease symptoms, ranging from asymptomatic to severe rotting and wilting and fumonisin production. The aim of this study was to investigate the influence of hybrid genotypes in the early stages of F. verticillioides infection, and the role of fumonisins as effectors in the outcome of this complex interaction. Disease symptoms, growth parameters, root morphology, and fungal colonization were evaluated at 7, 14, and 21 days after planting in seedlings from maize seeds of resistant (RH) and susceptible (SH) hybrids inoculated with F. verticillioides or watered with solutions of fumonisins. F. verticillioides induced growth enhancement or retardation depending on the plant genetic background and the fungal colonization rate, while fumonisins caused severe reduction in biomass and fitness. Seedlings watered with high fumonisin concentrations displayed lesions similar to those seen in F. verticillioides maize seedling disease, and also elicited inhibitory effects on root growth and morphology and on functional properties. In summary, these data strongly suggest a dual role for fumonisins in the F. verticillioides-maize interaction, acting as pathogenic factors at high concentrations, or triggering the plant detoxification mechanisms at low levels.     

Development of a sensitive ELISA for the determination of fumonisin B(1) in cereals

Monoclonal fumonisin B(1) antibodies with high titer were raised by using FB(1)-glutaraldehyde-keyhole limpet hemocyanin immunogen prepared by a short cross-linker reagent (glutaraldehyde). Mean cross-reactivities of the selected monoclonal antibody for FB(1), FB(2), and FB(3) were 100, 91.8, and 209%, respectively; no reactivity was found with hydrolyzed fumonisin. A direct, competitive enzyme-linked immunosorbent assay for the quantitative determination of FB(1) in cereals has been developed with this antibody. Fifty percent acetonitrile-based solvent with some additives was used for extraction of cereals, and the diluted extracts were used without cleanup in the test. The mean within-assay and interassay coefficients of variation for the standard curve were <10%. The measuring range of this test is 10-500 ng/g, with a detection limit of 7.6 ng/g FB(1). The toxin recovery from cereals infected with 50-200 ng/g of FB(1) varied between 61 and 84%. According to the comparable results of naturally infected maize samples, this test proved to be suitable for the rapid screening of food and feed samples for the presence of FBs.     

Fumonisin disruption of ceramide biosynthesis in maize roots and the effects on plant development and Fusarium verticillioides-induced seedling disease

The fungus Fusarium verticillioides infects maize and produces fumonisins, inhibitors of ceramide synthase. Seeds of the cultivar Silver Queen were inoculated with fumonisin-producing or non-fumonisin-producing strains of F. verticillioides. Leaf lesion incidence and severity of effects on root and stalk growth were significantly correlated with fumonisin in roots and disruption of sphingolipid metabolism in roots. Uninoculated seeds grown in soil watered with solutions of fumonisin B1 exhibited above-ground symptoms indicative of F. verticillioides-induced seedling disease and dose-dependent reduction in root mass that was inversely correlated with fumonisin B1, sphingoid bases, and sphingoid base 1-phosphates in roots. There was also evidence of an adaptive response to disrupted sphingolipid metabolism in both the virulence and watering assays, suggesting induction of pathways responsible for metabolism of sphingoid base 1-phosphates after prolonged exposure. The results suggest that fumonisin, and its effects on sphingolipids, could contribute to all aspects of F. verticillioides maize seedling disease.     

Fumonisin production and bioavailability to maize seedlings grown from seeds inoculated with Fusarium verticillioides and grown in natural soils

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.     

Determination of N-(carboxymethyl)fumonisin B(1) in corn products by liquid chromatography/electrospray ionization--mass spectrometry

It is well-known that fumonisin B(1) (FB(1)) in corn meal decreases during baking, frying, and cooking, but it is still not exactly clear how heating affects the formation of N-(carboxymethyl)fumonisin B(1) (NCM-FB(1)), the reaction product of FB(1) and reducing sugars. In model experiments corn grits were spiked with FB(1) (2 mg/kg) and D-glucose (50 g/kg) or sucrose (50 g/kg) and manufactured into extrusion products at various temperatures (160--180 degrees C) and moisture levels (16--20%). A liquid chromatography/electrospray ionization-mass spectrometry method using isotopically labeled fumonisin FB(1)-d(6) as an internal standard was developed for the determination of NCM-FB(1). For sample cleanup solid-phase C18 cartridges were used. The detection limit achieved with this method was 10 ng/g (signal-noise ratio = 3:1) using the protonated molecule [M + H](+) signal of NCM-FB(1) (m/z 780) in the selected ion monitoring mode. Low concentrations of NCM-FB(1) (29-97 ng/g) were detected in all samples spiked with D-glucose and FB(1), whereas those spiked with FB(1) and sucrose showed only NCM-FB(1) in samples produced at 180 degrees C (NCM-FB(1) = 27 ng/g). Various corn-containing food samples from the German market were analyzed for the presence of NCM-FB(1), FB(1), and hydrolyzed fumonisin B(1) (HFB(1)). All samples were contaminated with FB(1) (22--194 ng/g) and HFB(1) (5--247 ng/g). Six of nine samples contained NCM-FB(1) in low concentrations ranging from 10 to 76 ng/g. From these data and the low toxicity of NCM-FB(1) it can be concluded that the significance of NCM-FB(1) in food seems to be a minor one.     

Fumonisin mycotoxins in traditional Xhosa maize beer in South Africa

The production and consumption of home-brewed Xhosa maize beer is a widespread traditional practice in the former Transkei region of South Africa. HPLC determination of fumonisins B1 (FB1), B2 (FB2), and B3 (FB3) in maize beer samples collected in two magisterial areas, Centane and Bizana, showed a wide range of levels. All samples were positive for FB(1), with a mean level of 281 +/- 262 ng/mL and a range from 38 to 1066 ng/mL. Total fumonisins (FB1 + FB2 + FB3) ranged from 43 to 1329 ng/mL, with a mean of 369 +/- 345 ng/mL. Data on the consumption of home-brewed beer are not available. On the basis of published data for the consumption of commercial beer in South Africa, the fumonisin exposure in these districts among the consumers of maize beer was found to be well above the provisional maximum tolerable daily intake of 2 mug/kg of body weight/day set by the Joint FAO/WHO Expert Committee on Food Additives.     

Fumonisin in tortillas produced in small-scale facilities and effect of traditional masa production methods on this mycotoxin

Four small tortilla plants were visited in Cameron County, Texas, where observations were made on their production methods. Samples of liquids and solids were collected at each stage of the nixtamalization process, and the pH was recorded. Samples were analyzed for fumonisin B(1) (FB(1)) using an immunoaffinity column/HPLC method chosen for its sensitivity for FB(1). It was found that production methods were highly variable among the producers visited, with major differences particularly in the amount of lime added and boiling times. As reported by others working in Mexico and Central America, FB(1) was found in some tortillas. This led to studies of the effects of the various recipes and across a greater range of initial FB(1) concentration/damaged corn than has typically been reported. Five initial concentrations of FB(1) were tested using irradiated corn kernels inoculated with Fusarium verticillioides MRC 826 as the source of FB(1). The amount of FB(1) detected in the masa and tortillas decreased as the concentration of Ca(OH)(2) increased, and boiling time had no apparent effect. Unexpectedly, as the initial concentrations were increased in the corn prior to nixtamalization, greater percentage reductions in FB(1) were observed.