A fumonisin biosynthetic gene cluster in Fusarium oxysporum strain O-1890 and the genetic basis for B versus C fumonisin production

Most species of Fusarium that produce fumonisin mycotoxins produce predominantly B fumonisins (FBs). However, Fusarium oxysporum strain O-1890 produces predominantly C fumonisins (FCs). In this study, the nucleotide sequence of the fumonisin biosynthetic gene (FUM) cluster in strain O-1890 was determined. The order and orientation of FUM genes were the same as in the previously described clusters in Fusarium verticillioides and Fusarium proliferatum. Coding regions of F. oxysporum and F. verticillioides FUM genes were 88-92% identical, but regions flanking the clusters did not share significant identity. The FUM cluster gene FUM8 encodes an alpha-oxoamine synthase, and fum8 mutants of F. verticillioides do not produce fumonisins. Complementation of a fum8 mutant with the F. verticillioidesFUM8 restored FB production. Complementation with F. oxysporumFUM8 also restored production, but the fumonisins produced were predominantly FCs. These data indicate that different orthologues of FUM8 determine whether Fusarium produces predominantly FBs or FCs.     

Production of fumonisins by Fusarium moniliforme strains isolated from corn grain

Fusarium moniliforme is the predominant fusarium species in the grain mycoflora of corn grown in Northern Caucasus, accounting for 95% of fusarium isolates. Eighty-five Fusarium moniliforme strains were grown on grain substrate and checked for the presence of fumonisins (B1 + B2 + B3) by indirect solid-phase enzyme immunoassay (EIA). All strains were capable of producing fumonisins (0.95 to 32,000 mg/kg). Strains sampled in the Krasnodar krai produced the highest fumonisin levels (averaging 5490 mg/kg).     

Genetic analysis of fumonisin production and virulence of Gibberella fujikuroi mating population A (Fusarium moniliforme) on maize (Zea mays) seedlings.

The phytopathogenic fungus Gibberella fujikuroi mating population A (anamorph, Fusarium moniliforme) produces fumonisins, which are toxic to a wide range of plant and animal species. Previous studies of field strains have identified a genetic locus, designated fum1, that can determine whether fumonisins are produced. To test the relationship between fumonisin production and virulence on maize seedlings, a cross between a fum1+ field strain that had a high degree of virulence and a fum1- field strain that had a low degree of virulence was made, and ascospore progeny were scored for these traits. Although a range of virulence levels was recovered among the progeny, high levels of virulence were associated with production of fumonisins, and highly virulent, fumonisin-nonproducing progeny were not obtained. A survey of field strains did identify a rare fumonisin-nonproducing strain that was quite high in virulence. Also, the addition of purified fumonisin B1 to virulence assays did not replicate all of the seedling blight symptoms obtained with autoclaved culture material containing fumonisin. These results support the hypothesis that fumonisin plays a role in virulence but also indicate that fumonisin production is not necessary or sufficient for virulence on maize seedlings.     

Ochratoxin A and fumonisins (B1 and B2) in maize from Balkan nephropathy endemic and non endemic areas of Croatia

The occurrence of ochratoxin A, fumonisin B1 and B2 has been investigated in maize samples collected in 1996 (105 samples) and 1997 (104 samples) in 14 counties of Croatia, including Brodsko-Posavska county, the main area of Balkan endemic nephropathy in Croatia. Ochratoxin A and fumonisins co-occurred in 21% of the examined samples. In particular, ochratoxin A (OTA) was found in 10 samples (10%) of the 1996 and 36 samples (35%) of the 1997 crops with mean concentrations of positive samples of 37.9 ng/g and 57.1 ng/g, and highest concentrations at 223.6 ng/g and 613.7 ng/g, respectively. Similar incidence of OTA contamination was observed in 1996 samples from both endemic and non endemic areas of Balkan nephropathy, whereas a significant difference (P<0.01) was found between the two areas in 1997, with 50% and 20% incidence of contamination in the endemic and non endemic area, respectively, and relevant OTA mean concentration of positive samples of 73.4 ng/g and 20.2 ng/g. High incidence of infection byPenicillium spp. (potential OTA producers) was found in all tested samples, with mean values of 88% and 93% in samples of 1996 and 1997, respectively. With respect to fumonisin B1 (FB1) and B2 (FB2) all but one of the 1996 samples were contaminated, with highest and mean concentrations of positive samples (FB1+FB2) at 11661 ng/g and 645 ng/g, respectively. Similar incidence of positive samples (93%), but lower contamination levels (mean 134 ng/g, maximum 2524 ng/g) were found in 1997 samples. The results of fumonisin analysis were in agreement with the mycological analysis showing higher incidence of Fusarium infection in samples of 1996 with respect to those of 1997. These data provide additional information on the occurrence of ochratoxin A in Balkan endemic nephropathy areas and, for the first time, its co-occurrence with other nephrotoxic compounds, such as fumonisins, that may contribute to the disease development. However the finding of these mycotoxins in the non-endemic areas, also at high levels, do not allow to draw a conclusion about their role in the etiology of the disease.     

Absence of detectable fumonisins in the milk of cows fed Fusarium proliferatun (Matsushima) Nirenberg culture material

Fumonisins, a group of mycotoxins produced by the ubiquitous fungi Fusarium moniliforme and F. proliferatum, were first identified about eight years ago. They have been shown to cause a variety of health effects in animals, including epidemiological evidence of esophageal cancer in humans. Cattle are less sensitive to ill effects than horses and swine. Fumonisins are common contaminants of low quality grain fed to cattle. Culture material containing fumonisins (FB1, FB2, and FB3) was mixed into the total diet and fed for 14 days to two midlactation Jersey cows to determine if fumonisins are excreted in milk. The dietary equivalent of fumonisin was approximately 75 ppm and the two cows consumed an average of 3 mg fumonisin Bl /kg body weight (b wt)/day. Fumonisins were not detected in any of the milk samples by two analytical laboratories using methods with a sensitivity of 5 ng/ml. Except for transient diarrhea at the beginning of the contaminant feeding period and an increase in serum cholesterol, clinical and hematologie changes were not observed in the animals. The appearance or carry over of fumonisins from feed to milk in dairy cows does not appear to be significant and likely not a hazard or food safety concern for humans.     

Occurrence of fumonisins in asparagus (asparagus officinalis L.) and garlic (allium sativum L.) from Germany

Fusarium proliferatum is able to produce fumonisins and is considered a pathogen of many economically important plants (e.g. corn, rice, asparagus) [1]. The occurrence of fumonisin FB₁ inF. proliferatum infected asparagus spears from Germany was investigated using a liquid chromatography/electrospray ionization-mass spectrometry (LC-ESI-MS) method with isotopically labeled fumonisin FB₁-d₆ as internal standard. Asparagus samples were harvested in July 2000 and screened forFusarium species. AltogetherF. oxysporum, F. proliferatum and F. sambucinum were isolated from the spears. The samples infected with F.proliferatum were subsequently analyzed for fumonisins. FB₁ was detected in 9 of the 10 samples in amounts ranging from 36.4 ng/g to 4513.7 ng/g (based on dry weight). Fumonisins FB₂ and FB₃ were found in six samples in lower concentrations. In asparagus spears of June 2002 we could findF. proliferatum in 6% of the samples, however no fumonisins were detectable. Furthermore the capability of producing FB₁ by the fungus in garlic bulbs was investigated. Therefore garlic was cultured inF. 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 FB₁ (26.0 ng/g to 94.6 ng/g). This is the first report of the natural occurrence of FB₁ in German asparagus spears and furthermore our findings suggest a potential for natural contamination of garlic bulbs with fumonisins. For detailed results and methods see Ref. [2].     

Natural occurrence of the C series of fumonisins in moldy corn

We analyzed 44 moldy corn samples for the B and C series of fumonisins by high-performance liquid chromatography. Of the 44 samples, 32 (73%) were contaminated with both the B and C series of fumonisins and 6 were contaminated with only the B series of fumonisins. The incidence of fumonisin C1 in moldy corn was 71%; the incidence was 11% for fumonisin C3 and 43% for fumonisin C4. Their mean levels ranged from 500 to 1,900 ng/g. This is the first report on the natural occurrence of the C series of fumonisins and fumonisin B4 in moldy corn.     

Production of beauvericin,moniliformin, fusaproliferin,and fumonisins b(1), b(2), and b(3) by fifteen ex-type strains of fusarium species

Fifteen Fusarium species were analyzed by high-performance liquid chromatography for the production of six mycotoxins in corn grits cultures. Production of mycotoxins ranged from 66 to 2,500 micro g/kg for fumonisin B(1), 0.6 to 1,500 micro g/g for moniliformin, 2.2 to 720 micro g/g for beauvericin, and 12 to 130 micro g/g for fusaproliferin. Fumonisin B(2) (360 micro g/kg) was produced by two species, fumonisin B(3) was not detected in any of the 15 species examined, and Fusarium bulbicola produced none of the six mycotoxins that we analyzed.     

Binding of Fusarium mycotoxins by fermentative bacteria in vitro

AIMS: Fusarium toxins can occur in conserved forages impairing farm animal performances and health. On-farm biological decontamination methods could be an alternative to traditional physico-chemical methods. In this work, the ability to remove Fusarium toxins by fermentative bacteria was evaluated in vitro. METHODS AND RESULTS: Twenty-nine strains of lactic (LAB) and propionic acid bacteria (PAB) were tested for their ability to remove deoxynivalenol (DON) and fumonisins B1 and B2 (FB1, FB2) from an acid, pH 4, medium. Mycotoxin removal was widespread for LAB, but differences among strains were large. Removal was up to 55% for DON, 82% for FB1 and 100% for FB2. Selected strains were also capable of removing up to 88% zearalenone. The PAB strains were less efficient than the LAB. Binding, not biodegradation appeared to be the mode of action, as no toxin derivatives were observed and removal was not impaired in nonviable bacteria. Binding was not affected by pH, except for fumonisins that decreased to nearly 0% at neutral pH. CONCLUSIONS: Selected fermentative bacteria are able to bind main Fusarium mycotoxins. SIGNIFICANCE AND IMPACT OF THE STUDY: The binding ability of selected strains could be used to decrease the bioavailability of toxins in contaminated silages.     

Survey of fumonisin production by Fusarium species

Fumonisins B1 (FB1) and B2 (FB2), two structurally related mycotoxins with cancer-promoting activity, were recently isolated from corn cultures of Fusarium moniliforme MRC 826. These toxins have been reported to be produced also by isolates of F. proliferatum. Contamination of foods and feeds by F. moniliforme has been associated with human esophageal cancer risk, and FB1 has been shown to be the causative agent of the neurotoxic disease leukoencephalomalacia in horses. Because of the toxicological importance of the fumonisins, the potential to produce FB1 and FB2 was determined in a study of 40 toxic Fusarium isolates representing 27 taxa in 9 of the 12 sections of Fusarium, as well as two recently described species not yet classified into sections. With the exception of one isolate of F. nygamai, fumonisin production was restricted to isolates of F. moniliforme and F. proliferatum, in the section Liseola. The F. nygamai isolate produced 605 micrograms of FB1 g-1 and 530 micrograms of FB2 g-1, and the identity of the toxins was confirmed by capillary gas chromatography-mass spectrometry. This is the first report of the production of the fumonisins by F. nygamai.     

Duckling toxicity and the production of fumonisin and moniliformin by isolates in the A and E mating populations of Gibberella fujikuroi (Fusarium moniliforme).

Two biological species of Gibberella fujikuroi (A and F mating populations) share the Fusarium moniliforme anamorph. Twenty strains of each of these biological species were tested for the ability to produce fumonisins B1, B2, and B3 and moniliformin and for toxicity to 1-day-old ducklings. Most of the members of the A mating population (19 of 20 strains) produced more than 60 micrograms of total fumonisins per g, whereas only 3 of 20 members of the F mating population produced more than trace levels of these toxins and none produced more than 40 micrograms of total fumonisins per g. In addition, only 3 of 20 members of the A mating population produced more than 1 microgram of moniliformin per g (and none produced more than 175 micrograms/g), while all 20 strains of the F mating population produced more than 85 micrograms of this toxin per g and 1 strain produced 10,345 micrograms/g. The duckling toxicity profiles of the strains of the two mating populations were similar, however, and the level of either toxin by itself was not strongly correlated with duckling toxicity. On the basis of our data we think that it is likely that the members of both of these mating populations produce additional toxins that have yet to be chemically identified. These toxins may act singly or synergistically with other compounds to induce the observed duckling toxicity.     

Fum3p, a 2-ketoglutarate-dependent dioxygenase required for C-5 hydroxylation of fumonisins in Fusarium verticillioides

Fumonisins are polyketide-derived mycotoxins produced by several agriculturally important Fusarium species. The B series fumonisins, FB(1), FB(2), FB(3), and FB(4), are fumonisins produced by wild-type Fusarium verticillioides strains, differing in the number and location of hydroxyl groups attached to the carbon backbone. We characterized the protein encoded by FUM3, a gene in the fumonisin biosynthetic gene cluster. The 33-kDa FUM3 protein (Fum3p) was heterologously expressed and purified from Saccharomyces cerevisiae. Yeast cells expressing the Fum3p converted FB(3) to FB(1), indicating that Fum3p catalyzes the C-5 hydroxylation of fumonisins. This result was verified by assaying the activity of Fum3p purified from yeast cells. The C-5 hydroxylase activity of purified Fum3p required 2-ketoglutarate, Fe(2+), ascorbic acid, and catalase, all of which are required for 2-ketoglutarate-dependent dioxygenases. The protein also contains two His motifs that are highly conserved in this family of dioxygenases. Thus, Fum3p is a 2-ketoglutarate-dependent dioxygenase required for the addition of the C-5 hydroxyl group of fumonisins.     

Fumonisins — Importance and occurence of a new group of mycotoxins

This paper describes the importance of fumonisins for human beings and animals and shows data for the occurence in food. Corn-based food samples (n = 299) purchased in the area of munich were analyzed for fumonisin content using an enzyme immunoassay. Fumonisins are mycotoxins produced byFusarium species, especially byFusarium moniliforme andFusarium proliferatum. Occurrence of fumonisins in corn and in cornbased foods and feeds has been reported from almost all over the world. In several animal species different diseases are traced back to fumonisin toxicosis. Fumonisin levels of 5–10 ppm inhorse feed induce “Equine Leucoencephalomalacia” and hepatic lesions. Hepatotoxic (10 150 ppm fumonisin in feed) and pneumotoxic (>150 ppm fumonisin in feed) effects have been reported for swine. Cattle and poultry appear to be less susceptible to fumonisins. Fumonisin B₁ Revels of 50 ppm in the diet of rats cause hepatotoxic and nephrotoxic effects, long time exposure results in hepatic cancer. A possible role of fumonisins in the etiology of human esophageal cancer is under discussion, although no direct causal evidence is known so far. The mode of action of the fumonisins is probably based on inhibition of sphingolipidbiosynthesis caused by the blockade of the enzyme sphyngosine (sphinganine)-N-acyltrans-ferase.     

Characterization of Gibberella fujikuroi complex isolates by fumonisin B1 and B2 analysis and by RAPD and restriction analysis of PCR-amplified internal transcribed spacers of ribosomal DNA

Twenty nine isolates of Fusarium spp. (twenty four of them belonging to the Gibberella fujikuroi complex) isolated from banana and corn from different geographical regions were analyzed for their ability to produce fumonisins B1 and B2 and for genetic relatedness using random amplified polymorphic DNA (RAPD) and restriction analysis of PCR amplification products of the 5.8s ribosomal DNA-intervening internal transcribed spacer regions (ITS I-5.8S-ITS II). For RAPD analysis, six of twenty oligonucleotide primers were selected after testing with five Fusarium spp. isolates and used to characterize 24 additional isolates. DNA fragments from the 29 isolates of Fusarium spp., which were approximately 560 bp, were amplified with the universal primers ITS1 and ITS4. The restriction enzymes HaeIII, MboI, HpaII and MspI were useful for distinguishing the isolates. The RAPD analysis permitted to find interspecific differences among the isolates of Fusarium spp., between isolates with low and high capacity of fumonisin production and among isolates from different hosts. The restriction fragment length polymorphism (RFLP-PCR) analysis permitted to distinguish among different species of Fusarium. In combination with morphological analysis, the results of this research may find an application for the diagnosis of unknown Fusarium spp. and, particularly, for the characterization of fumonisin-producing isolates, which may be very useful in the food technology field.     

HPLC determination of fumonisin mycotoxins in maize: a comparative study of naphthalene-2,3-dicarboxaldehyde and o-phthaldialdehyde derivatization reagents for fluorescence and diode array detection

Fumonisins are mycotoxins produced by various species of Fusarium and occur naturally in contaminated maize and maize-based foods. Ingestion of fumonisins has considerable health implications for humans and animals. Since fumonisins lack a useful chromophore or fluorophore, their determination in maize is routinely achieved via HPLC with fluorescence detection (FLD) after precolumn derivatization. This study optimized naphthalene-2,3-dicarboxaldehyde (NDA) derivatization of fumonisins in naturally contaminated maize following strong anion exchange (SAX) solid phase extraction (SPE) clean-up and utilizing diode array detection (DAD) as a practical alternative simultaneously to FLD. The limit of detection (LOD) for fumonisin B(1) (FB(1)), fumonisin B(2) (FB(2)) and fumonisin B(3) (FB(3)) with FLD was 0.11 ng, 0.50 ng and 0.27 ng, respectively, and with DAD it was 13.8 ng, 12.5 ng and 6.6 ng, respectively injected on column. The coefficient of variation (CV, n = 6) for FB(1), FB(2) and FB(3) in a naturally contaminated samples obtained with FLD was 2.6%, 1.8% and 5.3%, respectively, compared to 6.0%, 3.4% and 9.5%, respectively, obtained with DAD. Subsequently the optimized NDA derivatization was compared to the widely used o-phthaldialdehyde (OPA) derivatization agent as well as alternative sample clean-up with immunoaffinity column (IAC) by analyzing naturally contaminated maize samples (n = 15) ranging in total fumonisin (TFB = FB(1)+FB(2)+FB(3)) levels from 106 to 6000 μg/kg. After immunoaffinity column clean-up of extracted samples, the recoveries of spiked maize samples for NDA-FLD of FB(1), FB(2) and FB(3) were 62%, 94% and 64%, respectively. NDA proved to be an effective derivatization reagent of fumonisin in naturally contaminated maize samples following IAC clean-up, except for DAD at TFB levels below 1000 μg/kg. In contrast NDA derivatization following SAX clean-up produced results comparable to OPA only for levels below 1000 μg/kg. Aside from the difference in detection limits, FLD and DAD produced comparable results irrespective of the clean-up method or the derivatization agent.     

Occurrence of fungi and mycotoxins in corn silage, Jalisco State, Mexico

The aim of this study was to evaluate the chemical composition, mold count and mycotoxin contamination of corn silage collected during a six month-period. The results indicated that the chemical composition and the physicochemical parameters evaluated did not show significant variation during the sampling time. Fungal count on RBC ranged from 1.7 x 10(3) to 9 x 10e8 CFU/g. Mucor, Penicillium and Aspergillus spp. were the most frequent fungal species in the corn silage. Fusarium count ranged from 1.6 x 10(3) to 1.6 x 10e8 CFU/g in Nash Snyder culture media. Aflatoxin B, fumonisins, ochratoxin A, ochratoxin B, deoxynivalenol, and zearalenone were detected throughout the period of corn silage maintenance (100% positive samples). However, only deoxynivalenol levels were higher than the maximum limit recommended by the FDA.     

Inhibition of sphingolipid biosynthesis by fumonisins. Implications for diseases associated with Fusarium moniliforme

Culture materials and grains contaminated with certain isolates of Fusarium moniliforme cause equine leucoencephalomalacia, porcine pulmonary edema syndrome, and liver cancer in rats. The causative agents are thought to be a family of compounds called fumonisins, which bear considerable structural similarity to the long-chain (sphingoid) base backbones of sphingolipids. Incubation of rat hepatocytes with fumonisins inhibited incorporation of [14C]serine into the sphingosine moiety of cellular sphingolipids with an IC50 of 0.1 microM for fumonisin B1. In contrast, fumonisin B1 increased the amount of the biosynthetic intermediate sphinganine, which suggests that fumonisins inhibit the conversion of [14C]sphinganine to N-acyl-[14C]sphinganines, a step that is thought to precede introduction of the 4,5-trans double bond of sphingosine (Merrill, A.H., Jr. and Wang, E. (1986) J. Biol. Chem. 261, 3764-3769). In agreement with this mechanism, fumonisin B1 inhibited the activity of sphingosine N-acyltransferase (ceramide synthase) in rat liver microsomes with 50% inhibition at approximately 0.1 microM and reduced the conversion of [3H]sphingosine to [3H]ceramide by intact hepatocytes. As far as we are aware, this is the first discovery of a naturally occurring inhibitor of this step of sphingolipid metabolism. These findings suggest that disruption of the de novo pathway of sphingolipid biosynthesis may be a critical event in the diseases that have been associated with consumption of fumonisins.     

FUM cluster divergence in fumonisins-producing Fusarium species

Fumonisins are polyketide-derived mycotoxins, produced by several Fusarium species, and its biosynthetic pathway is controlled by the FUM cluster--a group of genes exhibiting a common expression pattern during fumonisin biosynthesis. The most common are the B analogues with fumonisin B(1) (FB(1)) being the most prevalent. At least a part of the inter- and intraspecific variation in FBs synthesis level can be explained by the sequence differences inside FUM cluster. The aim of our study was to evaluate the toxin production and sequence variability in FUM genes and intergenic regions among thirty isolates of seven species reported as potential fumonisins producers: Fusarium anthophilum, Fusarium fujikuroi, Fusarium nygamai, Fusarium oxysporum, Fusarium proliferatum, Fusarium subglutinans and Fusarium verticillioides, particularly with respect to FBs synthesis. Fumonisins were produced in high amounts (over 1mg g(-1)) by one isolate of F. subglutinans, three of F. verticillioides and all F. proliferatum isolates except one, regardless of the host organism. The remaining isolates produced low amounts of FBs and two F. verticillioides isolates didn't produce it at all. The lowest variation in amount of toxin produced was found among F. proliferatum isolates. Based on the translation elongation factor 1α (tef-1α) sequence of F. fujikuroi, a species-specific marker was developed. The intergenic region presents similar opportunity for F. nygamai identification. The phylogenetic reconstruction based on FUM1 gene generally reflects the scenario presented by tef-1α sequences. Although the sequence similarities for intergenic regions were lower than in coding regions, there are clearly conserved patterns enabling separation of different subsets of species, including the non-producer species.     

Fumonisins production by Fusarium proliferatum strains isolated from asparagus crown

The present work deals with the capability for producing fumonisin by Fusarium proliferatum strains isolated from asparagus in China. Fifty of F. proliferatum strains were randomly selected and incubated on cultures of maize grain and asparagus spear, respectively. Fumonisin levels (FB₁ and FB₂) were determined by high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The results showed that all 50 strains produced fumonisins in maize culture within a wide range of concentrations, 10–11,499 μg/g and 2–6,598 μg/g for FB₁ and FB₂, respectively. On culture of asparagus spear,48 strains (96%) produced fumonisins in the range 0.2–781.6 μg/g and no detected to 40.3 μg/g for FB₁ and FB₂, respectively. All of F. proliferatum strains produced much higher levels of FB₁, FB₂ and total fumonisins (FB₁ + FB₂) in maize grain culture than in asparagus spear culture. Meanwhile, fumonisin B₃ (FB₃) was identified in all maize culture extracts and most of asparagus spear culture extracts. This is the first study carried out the fumonisin-producing ability of F. proliferatum strains isolated from asparagus in China. The information obtained is useful for assessing the risk of fumonisins contamination in asparagus spear. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.