Ronald D. Plattner

Co-expression of 15 contiguous genes delineates a fumonisin biosynthetic gene cluster in Gibberella moniliformis.

Fumonisins are mycotoxins produced by the maize pathogen Gibberella moniliformis and are associated with cancer in rodents. In this study, we determined the nucleotide sequence of a 75-kb region of G. moniliformis DNA and identified 18 heretofore undescribed genes flanking a cluster of five previously identified fumonisin biosynthetic (FUM) genes. Ten of the newly identified genes downstream of the cluster were coregulated with FUM genes and exhibited patterns of expression that were correlated with fumonisin production. BLASTX analyses indicated that the predicted functions of proteins encoded by the 10 genes were consistent with activities expected for fumonisin biosynthesis or self-protection. These data indicate that the 10 newly identified genes and the previously identified FUM genes constitute a fumonisin biosynthetic gene cluster. Disruption of two of the new genes, encoding longevity assurance factors, had no apparent effect on fumonisin production, but disruption of a third, encoding an ABC transporter, had a subtle effect on ratios of fumonisins produced.

Evidence for disruption of sphingolipid metabolism as a contributing factor in the toxicity and carcinogenicity of fumonisins

Fumonisins are inhibitors of the biosynthesis of sphingosine and more complex sphingolipids. In eucaryotic cells, fumonisin inhibition of sphingolipid biosynthesis is a result of inhibition of the enzyme ceramide synthase. Large increase in free sphinganine concentration in plant and animal cells are observed within a few hours after exposure to fumonisins and/or Alternaria toxins (AAL-toxins). Some of the sphinganine is metabolized to other bioactive intermediates, and some is released from cells. In animals, free sphinganine accumulates in tissues and quickly appears in blood and urine. Free sphingoid bases are toxic to most cells, and complex sphingolipids are essential for normal cell growth. Fumonisin B1 stimulates sphinganine-dependent DNA synthesis in Swiss 3T3 cells, but is mitoinhibitory in other cell types. In cultured cells the accumulation of bioactive long-chain sphingoid bases and depletion of complex sphingolipids are clearly contributing factors in growth inhibition, increased cell death, and (in Swiss 3T3 cells) mitogenicity of fumonisins. While disruption of sphingolipid metabolism directly affects cells, it may indirectly affect some tissues. For example, fumonisin B1 impairs the barrier function of endothelial cells in vitro. Adverse effects on endothelial cells could indirectly contribute to the neurotoxicity and pulmonary edema caused by fumonisins. It is hypothesized that fumonisin-induced changes in the sphingolipid composition of target tissues could directly or indirectly contribute to all Fusarium moniliforme-associated diseases.

Discontinuous distribution of fumonisin biosynthetic genes in the Gibberella fujikuroi species complex.

Production of the carcinogenic mycotoxins fumonisins has been reported in several Fusarium species, most of which are members of the Gibberella fujikuroi (Gf) complex. In this study, we examined 15 Fusarium species in the Gf complex and 12 other species for fumonisin production and the presence of fumonisin biosynthetic genes (FUM). Among the species within the Gf complex, fumonisin production was detected only in F. fujikuroi, F. globosum, F. proliferatum, F. nygamai, F. oxysporum and F. verticillioides. These five species include members of two of the three major clades delineated in the Gf complex. The FUM genes were detected in these same five species and in F. anthophilum, a member of the third clade. Among the species outside the Gf complex, fumonisin production and FUM genes were detected only in F. oxysporum. Phylogenetic analyses of nucleotide sequences from two FUM gene fragments inferred relationships similar but not identical to those inferred from previous analyses of other genes. The results indicate the FUM genes are discontinuously distributed in the Gf complex and that this distribution gives rise to the differences in the abilities of closely related Fusarium species to produce fumonisins.

The occurrence of ochratoxin A in dust collected from a problem household

Accumulated dust samples were collected from the heating ducts in a household where signs resembling ochratoxin poisoning in animals occurred. Several Penicillium spp. and Aspergillus ochraceous had been identified previously from air samples taken from this house. A composite sample from six collected samples was examined by HPLC, and it was determined that 58 ppb of ochratoxin A was present in this sample. A second set of six samples was collected and determinations were made by HPLC of the ochratoxin content in each sample. All samples, including one sample of dirt from a crawl space, yielded at least a trace of ochratoxin A; however, one sample of dust collected from the heating ducts yielded over 1500 ppb of ochratoxin A, and another sample of dust from a different heating duct yielded 306 ppb of ochratoxin A. Ochratoxin A was confirmed in all samples by LC-MS, and ochratoxin was evident in the samples by TLC analysis. This is believed to be the first report of finding ochratoxin inhouse dust.

Genetic analysis of the role of trichothecene and fumonisin mycotoxins in the virulence of Fusarium

The phytotoxicity of the Fusarium trichothecene and fumonisin mycotoxins has led to speculation that both toxins are involved in plant pathogenesis. This subject has been addressed by examining virulence of trichothecene and fumonisin-nonproducing mutants of Fusarium in field tests. Mutants were generated by transformation-mediated disruption of genes encoding enzymes that catalyze early steps in the biosynthesis of each toxin. Two economically important species of Fusarium were selected for these studies: the trichothecene-producing species Fusarium graminearum, which causes wheat head blight and maize ear rot, and the fumonisin-producing species F. verticillioides, which causes maize ear rot. Trichothecene-non-producing mutants of F. graminearum caused less disease than the wild-type strain from which they were derived on both wheat and maize, although differences in virulence on maize were not observed under hot and dry environmental conditions. Genetic analyses of the mutants demonstrated that the reduced virulence on wheat was caused by the loss of trichothecene production rather than by a non-target mutation induced by the gene disruption procedure. Although the analyses of virulence of fumonisin-non-producing mutants of F. verticillioides are not complete, to date, the mutants have been as virulent on maize ears as their wild-type progenitor strains. The finding that trichothecene production contributes to the virulence of F. graminearum suggests that it may be possible to generate plants that are resistant to this fungus by increasing their resistance to trichothecenes. As a result, several researchers are trying to identify trichothecene resistance genes and transfer them to crop species.

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.

Gibberella fujikuroi mating population A and Fusarium subglutinans from teosinte species and maize from Mexico and Central America.

Seed samples of maize ( Zea mays ssp. mays ) from Mexico and of teosintes ( Zea spp.), the nearest wild relatives of maize, from Mexico, Guatemala, and Nicaragua were assessed for infection with Fusarium species. Strains similar in morphology to Fusarium moniliforme and F. subglutinans were the most frequent isolates from maize and from teosinte species including Z. diploperennis, Z. luxurians, Z. mays ssp. mexicana , and Z. mays ssp. parviglumis . Analysis of fertility, vegetative compatibility and mycotoxin production identified 63 % of the 70 F. moniliforme strains from teosinte as genetically diverse members of Gibberella fujikuroi mating population A, a common pathogen of maize. The F. subglutinans strains from maize and teosinte were similarly genetically diverse, but were not fertile with standard testers of G. fujikuroi mating populations B and E, common pathogens of Poaceae , or of mating population H, which causes pitch canker disease of pine. Fifty-four percent of the 80 F. subglutinans strains were fertile when crossed with female tester strains from teosinte and maize collected in a field at Netzahualcoyotyl in the state of Mexico. These strains from Mexico and Central America may comprise a new and distinct G. fujikuroi mating population, but a strain from the Netzahualcoyotyl field site was fertile with a strain of G. fujikuroi mating population H from California. Thus, F. subglutinans from teosinte and maize may have a close relationship to mating population H from pine.

A nematicidal toxin fromPleurotus ostreatus NRRL 3526

A nematicidal toxin was purified fromPleurotus ostreatus NRRL 3526 grown on moistened, autoclaved wheat straw for 30 days at room temperature (21–33°C). The active compound, at a concentration of 300 ppm, immobilized 95% of test nematodes (Panagrellus redivivus) within 1 hr. Immobilized nematodes did not recover, even after being rinsed with deionized water. The toxin was identified astrans-2-decenedioic acid.

Biosynthesis of labeled fumonisins in liquid cultures of Fusarium moniliforme

Fumonisins were readily produced in cultures of Fusarium moniliforme using a defined liquid medium. Addition of 200 mg of d3-methyl L-methionine to 100-ml cultures of F. moniliforme gave increased overall yields and high levels of deuterium (2H) incorporation into fumonisin B1. Approximately 90% of the resulting fumonisin B1 contained 6 deuterium atoms, while 9% of the product contained 3 deuterium atoms. Deuterium was shown to be incorporated exclusively in the methyl groups of the fumonisin backbone. The addition of as little as 5 mg of labeled methionine stimulated fumonisin production, but only about 5% of the fumonisin produced contained 3 deuterium atoms.

In vivo effects of fumonisin B1-producing and fumonisin B1-nonproducing Fusarium moniliforme isolates are similar: Fumonisins B2 and B3 cause hepato- and nephrotoxicity in rats

Fumonisins are mycotoxins produced by Fusarium moniliforme, F. proliferatum, and related Fusarium species found on corn. They occur naturally in corn-based feeds and foods and are suspected human esophageal carcinogens. Fumonisin B1 (FB1), the most common homologue, causes the animal diseases associated with F. moniliforme. Hepato- and nephrotoxicities, disrupted sphingolipid metabolism, and liver cancer have been found in rats fed FB1. To determine the in vivo effects of diets containing fumonisins B2 (FB2) or B3 (FB3), male rats were fed culture materials (CM) of FB1 non-producing F. moniliforme isolates to provide low (4.6–6.7 ppm), mid (32–49 ppm) or high (219–295 ppm) dietary levels of either FB2 (FB2CM) or FB3 (FB3CM). Other groups were fed culture material of an FB1 producing isolate (FB1CM) providing 6.9, 53 or 303~ppm total fumonisins (FB1 : FB2 : FB3 = 1.0 : 0.38 : 0.15) and a tenth group was fed a control diet having no detectable fumonisins. One-half (n = 5/group) the animals were killed after three weeks, at which time the toxicological and histopathological effects of the three culture materials were similar, mimicked the effects of FB1, and included decreased body weight gains, serum chemical indicators of hepatotoxicity, decreased kidney weights, and apoptosis of hepatocytes and kidney tubular epithelium. FB1CM, FB2CM, and FB3CM affected sphingolipids, causing increased sphinganine to sphingosine ratios (Sa/So) in both liver and kidneys. The remaining animals (n = 5/group) were fed a control diet for three additional weeks. All body weight and tissue specific effects, including increased Sa/So, induced by the FB2CM, FB3CM and low level FB1CM diets were absent following the recovery period. Except for mild biliary lesions found in the high dose FB1CM group and a few apoptotic hepatocytes present in one mid- and two high-dose FB1CM rats, no evidence of toxicity remained in these groups following the recovery period.