Heritability of fumonisin B1 production in Gibberella fujikuroi mating population A.

Fumonisins are mycotoxins produced by strains belonging to several different mating populations of Gibberella fujikuroi (anamorphs, Fusarium section Liseola), a major pathogen of maize and sorghum worldwide. We studied the heritability of fumonisin production in mating population A by crossing fumonisin-producing strains collected from maize and sorghum in the United States with fumonisin-nonproducing strains collected from maize in Nepal. Random ascospore and tetrad progeny from three of these crosses were analyzed by gas chromatography-mass spectrometry and high-performance liquid chromatography for their ability to produce fumonisins on autoclaved cracked maize. In all three crosses, the ability to produce fumonisins, predominately fumonisin B1, segregated as a single gene or group of closely linked genes. Intercrosses between appropriate progeny and parents were poorly fertile, so we could not determine if the apparent single genes that were segregating in each of these crosses were allelic with one another. Mating type and spore-killer traits were scored in some crosses, and each segregated, as expected, as a single gene that was unlinked to the ability to produce fumonisins. We conclude that G. fujikuroi mating population A provides a powerful genetic system for the study of this important fungal toxin.     

Linkage among genes responsible for fumonisin biosynthesis in Gibberella fujikuroi mating population A.

Most naturally occurring strains of the fungus Gibberella fujikuroi mating population A produce high levels of the mycotoxin fumonisin B1 (FB1), which is oxygenated at both carbons C-5 and C-10. Some strains, however, produce only FB2 or FB3, suggesting that they lack the ability to hydroxylate position C-10 or C-5, respectively. Genetic analysis indicates that these different phenotypes are due to single gene defects at closely linked loci designated fum2 and fum3. Further allellism tests indicate that both fum2 and fum3 are closely linked to fum1, a previously identified gene that regulates fumonisin production. The recovery frequency of FB1-producing progency from cross 510 between fum1 and fum2 mutations suggests a map distance of approximately 6.2 cM between these two loci. Amplified fragment length polymorphism analysis of parents and progeny of cross 510 was employed to confirm that the FB1-producing strains are recombinant progeny. We conclude that fum1, fum2, and fum3 constitute a fumonisin biosynthetic gene cluster on chromosome 1 of the restriction fragment length-map of G. fujikuroi.     

A polyketide synthase gene required for biosynthesis of fumonisin mycotoxins in Gibberella fujikuroi mating population A.

Fumonisins are toxins associated with several mycotoxicoses and are produced by the maize pathogen Gibberella fujikuroi mating population A (MP-A). Biochemical analyses indicate that fumonisins are a product of either polyketide or fatty acid biosynthesis. To isolate a putative polyketide synthase (PKS) gene involved in fumonisin biosynthesis, we employed PCR with degenerate PKS primers and a cDNA template prepared from a fumonisin-producing culture of G. fujikuroi. Sequence analysis of the single PCR product and its flanking DNA revealed a gene (FUM5) with a 7.8-kb coding region. The predicted FUM5 translation product was highly similar to bacterial and fungal Type I PKSs. Transformation of a cosmid clone carrying FUM5 into G. fujikuroi enhanced production in three strains and restored wild-type production in a fumonisin nonproducing mutant. Disruption of FUM5 reduced fumonisin production by over 99% in G. fujikuroi MP-A. Together, these results indicate that FUM5 is a PKS gene required for fumonisin biosynthesis.     

Nitrogen repression of fumonisin B1 biosynthesis in Gibberella fujikuroi

Fumonisins are a group of structurally related mycotoxins produced by Gibberella fujikuroi. The fungus produced fumonisin B1 (FB1) as early as 18 hour in a defined medium containing 1.25 mM or 2.5 mM ammonium phosphate, whereas fumonisin B1 production was repressed for 75 hour and 125 hour when mycelia were resuspended in media containing ammonium phosphate at 10 mM or 20 mM, respectively. Although total fumonisin B1 production was greater in resuspension cultures grown in higher concentrations of ammonium phosphate, the accumulation was independent of the inoculum size and carbon/nitrogen ratio. The addition of ammonium phosphate to cracked corn cultures also repressed fumonisin B1 production by 97%, and persisted for at least three weeks. Thus, biosynthesis of fumonisin B1 is regulated by a mechanism involving nitrogen metabolite repression, suggesting that control strategies that target the regulatory elements of nitrogen metabolism may be effective at reducing the risk of fumonisin contamination in food.     

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.     

Gibberella fujikuroi mating population E is associated with maize and teosinte

Isolates of Fusarium subglutinans mating population E are usually found on maize. This fungus forms part of the so-called Gibberella fujikuroi species complex. Previously, F. subglutinans has been associated with two additional mating populations (B and H) and a variety of plant hosts. This was mainly due to a lack of diagnostic morphological characters, but the use of DNA sequence information showed that the strains making up mating populations B, E and H, as well as those associated with the different plant hosts, represent separate species. Recently, another putative mating population has been reported on the wild teosinte relatives of maize. Based on sexual compatibility studies, these isolates were apparently closely related to the pitch canker fungus, F. subglutinans f. sp. pini (= F. circinatum;G. fujikuroi mating population H). The aim of the current study was to determine whether the population of F. subglutinans from teosinte constitutes a new or an existing lineage within the G. fujikuroi complex. For this purpose, portions of the mitochondrial small subunit ribosomal DNA, calmodulin and β-tubulin genes from the fungi were sequenced. Phylogenetic analyses and comparison with sequences from public domain databases indicated that the F. subglutinans isolates from teosinte are most closely related to strains of G. fujikuroi mating population E. These results were confirmed using sexual compatibility studies. The putative mating population from the wild relatives of maize therefore forms part of the existing E-mating population and does not constitute a new lineage in the G. fujikuroi species complex.     

Biosynthetic and genetic relationships of B-series fumonisins produced by Gibberella fujikuroi mating population A

Fumonisins are mycotoxins produced by the maize pathogen Gibberella fujikuroi mating population A and frequently contaminate maize. Wild-type G. fujikuroi produces four B-series fumonisins, FB1, FB2, FR3 and FB4. These toxins are identical in structure except for the number and positions of hydroxyls along their linear carbon backbone. To elucidate the genetic and biosynthetic relationships among these fumonisins, we conducted meiotic and biochemical analyses of G. fujikuroi mutants with altered fumonisin production that resulted from defective alleles at three loci, Fum1, Fum2 and Fum3. These mutants produced either no fumonisins, only FR2 and FB4, or only FR3 and FR4. Genetic analyses revealed the orientation of the Fum loci along linkage group 1 of the fungus. The mutants were grown together in pair-wise combinations to determine if their fumonisin production phenotypes could be complemented. When FR3- and FB2-producing mutants were grown together, complementation occurred. However, when a nonproducing mutant was grown with a FR2- or FB3-producing mutant, complementation did not occur or was incomplete. When purified FR2, FR3, or FB4 was fed to mutant cultures, FR4 was converted primarily to FR2, FR3 was converted to FB1 and FB2 was not converted. The results from these assays suggest a previously unrecognized branch in the fumonisin biosynthetic pathway.     

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.     

Molecular standardization of mating type terminology in the Gibberella fujikuroi species complex.

Mating type in the Gibberella fujikuroi species complex is controlled by a single locus with two alleles and is usually identified following sexual crosses with standard, female-fertile tester isolates. The mating type alleles have been arbitrarily designated "+" and "-" within each biological species, and the nomenclature is tied to the standard tester strains. We developed a pair of PCR primers that can be used to amplify a unique fragment of one of the mating type alleles (MAT-2) from at least seven of the biological species in this species complex. Based on the amplification pattern, we propose a replacement for the existing, arbitrary +/- terminology that is presently in use. The new terminology is based on DNA sequence similarities between the mating type allele fragments from the biological species of the G. fujikuroi species complex and the corresponding fragments from other filamentous ascomycetes.     

Nitrogen availability and production of bikaverin and gibberellins in Gibberella fujikuroi

Production of bikaverin and gibberellins by Gibberella fujikuroi started after depletion of the nitrogen source, but not after depletion of phosphate. Despite this similarity, the regulation of both pathways by nitrogen involved two different mechanisms. This conclusion was supported by the fact that the production of bikaverin, in contrast to the gibberellins, was not inhibited by nitrate in a mutant that could not utilize it. The different regulation of both pathways was clearly demonstrated by a mutant that overproduced bikaverin but lacked gibberellins. An optimal bikaverin production required a low pH, with a sharp drop at about pH 5. The syntheses of fungal secondary metabolites, such as bikaverin and gibberellins, are not subject to common regulation, but respond to various combinations of signals, such as nitrogen availability, nitrate and the pH of the medium.     

A Novel Kauranoid Isolated from Gibberella fujikuroi

A novel arseno-sugar was isolated from the brown alga Sargassum thunbergii. Instead of the dimethylarsinoyl group reported for algal arseno-sugars, this has a tri-methylarsonium group, which is borne by arsenobetaine, a ubiquitous organoarsenic compound in marine animals. This may be an intermediate between arseno-sugars and arsenobetaine.     

Morphological mutants of Gibberella fujikuroi for enhanced production of gibberellic acid

AIMS: To examine the production of gibberellic acid by selected morphological mutants of Gibberella fujikuroi in liquid cultures. METHODS AND RESULTS: Mutants of G. fujikuroi having different morphological characteristics were selected after UV irradiation. The production of gibberellic acid by mutants that had different hyphal lengths was examined in shake flasks in media with different concentrations of nutrients as well as different volumes of the medium. Fed-batch fermenter study was performed to evaluate the mutant Mor-25 for growth and production of gibberellic acid. The broth was analysed by high performance liquid chromatography for fusaric acid, the common mycotoxin produced by strains of Fusarium. A variety of morphological mutants having different mycelial and soluble pigmentation as well as colony morphologies were generated from G. fujikuroi upon exposure to UV radiation. A nonpigmented mutant (Car-1) was selected as intermediate parent and later, mutants Mor-1 and Mor-25 were selected based on their distinct morphology. The colonies on regeneration agar plates were small, compact and dry. In liquid medium, mutant Mor-25 grew in a micro-pelleted form and the mycelium had short, highly branched hyphae, curly at tips with thick, swollen cells. Mutant Mor-25 grew rapidly in a low-cost medium containing defatted groundnut flour, sucrose and salts. In media with higher nutrient concentrations as well as larger volumes, it produced twofold more gibberellic acid than the parent. Fusaric acid, the common mycotoxin, was absent in the fermentation broth of mutant Mor-25. The mutants have been deposited in National Collection of Industrial Microorganisms (NCIM), National Chemical Laboratory, Pune, India under following culture collection numbers (Car-1, NCIM 1323; Mor-1, NCIM 1322; and Mor-25, NCIM 1321). CONCLUSIONS: Growth of unpigmented, morphological mutants of G. fujikuroi that led to lower viscosity in fermentation broth resulted in increased production of gibberellic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of morphological mutants that have lower viscosity in liquid cultures for gibberellic acid production is not reported earlier. Similar mutants can be useful for other types of fungal fermentations also.     

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.     

Selective production of bikaverin in a fluidized bioreactor with immobilized Gibberella fujikuroi

The best culture medium composition for the production of bikaverin by Gibberella fujikuroi in shake-flasks, i.e. 100 g glucose l^–1; 1 g NH₄Cl l^–1; 2 g rice flour l^–1; 5 g KH₂PO₄ l^–1 and 2.5 g MgSO₄ l^–1, was obtained through a fractional factorial design and then scaled-up to a fluidized bioreactor. The effects of carbon and nitrogen concentrations, inoculum size, aeration, flow rate and bead sizes on batch bikaverin production using immobilized G. fujikuroi in a fluidized bioreactor were determined by an orthogonal experimental design. Concentrations of up to 6.83 g bikaverin l^–1 were obtained when the medium contained 100 g glucose l^–1 and 1 g NH₄Cl l^–1 with an inoculum ratio of 10% v/v, an aeration rate of 3 volumes of air per volume of medium min^–1, and a bead size of 3 mm. Based on dry weight, the bikaverin production was 30–100 times larger than found in submerged culture and approximately three times larger than reported for solid substrate fermentation.     

Light-Stimulated Gibberellin Biosynthesis in Gibberella fujikuroi

Gibberellins (GAs) are a group of plant growth hormones that were first isolated from the fungus Gibberella fujikuroi. The biosynthesis of GA in liquid cultures of the fungus has been examined using high-performance liquid chromatography and combined gas chromatography-mass spectrometry. GA₃ was the predominant GA in well-aerated cultures. GA₄ and GA₇, intermediates in GA₃ biosynthesis, accumulated in cultures with low levels of dissolved oxygen, but were not detectable in more highly aerated cultures. Light stimulated the production of GA₃ in G. fujikuroi cultures grown from young stock cultures. Cell-free enzyme studies revealed a significant stimulation in the levels of kaurenoic acid oxidation in cultures grown in the light in comparison with those grown in the dark. However, measurements of the relative rates of [¹⁴C]mevalonic acid incorporation into kaurene showed no effect of light on this early part of the pathway. Preliminary experiments indicated that blue light is most effective in enhancing kaurenoic acid oxidation.