Differentiation of Fusarium subglutinans f. sp. pini by histone gene sequence data.

Fusarium subglutinans f. sp. pini (= F. circinatum) is a pathogen of pine and is one of eight mating populations (i.e., biological species) in the Gibberella fujikuroi species complex. This species complex includes F. thapsinum, F. moniliforme (= F. verticillioides), F. nygamai, and F. proliferatum, as well as F. subglutinans associated with sugarcane, maize, mango, and pineapple. Differentiating these forms of F. subglutinans usually requires pathogenicity tests, which are often time-consuming and inconclusive. Our objective was to develop a technique to differentiate isolates of F. subglutinans f. sp. pini from other isolates identified as F. subglutinans. We sequenced the histone H3 gene from a representative set of Fusarium isolates. The H3 gene sequence was conserved and contained two introns in all the isolates studied. From both the intron and the exon sequence data, we developed a PCR-restriction fragment length polymorphism technique that reliably distinguishes F. subglutinans f. sp. pini from the other biological species in the G. fujikuroi species complex.     

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.     

Characterization and genetic variability of Fusarium verticillioides strains isolated from corn and sorghum in Brazil based on fumonisins production, microsatellites, mating type locus, and mating crosses

Fusarium verticillioides (Gibberella fujikuroi mating population A) is a producer of fumonisins and one of the main contaminants of corn grains. In Brazil, some studies analyzing strains isolated from corn have demonstrated high levels of fumonisins, whereas the levels for strains isolated from sorghum have been found to be low. In the present study, we investigated the genetic diversity of 22 F. verticillioides strains isolated from corn and 21 strains isolated from sorghum cultivated in the State of S?o Paulo, Brazil. Differences in the genetic profile were observed between the strains isolated from the two substrates using single primer amplification reaction by polymerase chain reaction (SPAR-PCR). Fumonisins levels were higher in strains isolated from corn than in those isolated from sorghum. The MAT-1 and MAT-2 alleles were identified by PCR, and the isolates were subsequently crossed with Fusarium thapsinum (G. fujikuroi mating population F) reference strains because this species is morphologically similar to F. verticillioides and produces low levels of fumonisins. The SPAR haplotypes of some strains isolated from sorghum were similar to the F. thapsinum reference strain haplotypes, but there was no fertile mating between the strains isolated from the two substrates and the F. thapsinum references strains. The MAT-1:MAT-2 proportion was 5:17 and 14:7 for isolates from corn and sorghum, respectively.     

A single amino acid substitution in highly similar endo-PGs from Fusarium verticillioides and related Fusarium species affects PGIP inhibition

Endo-polygalacturonase (PG) may be a critical virulence factor secreted by several fungi upon plant invasion. The single-copy gene encoding PG in Fusarium verticillioides and in eight other species of the Gibberella fujikuroi complex (F. sacchari, F. fujikuroi, F. proliferatum, F. subglutinans, F. thapsinum, F. nygamai, F. circinatum, and F. anthophilum) was functionally analyzed in this paper. Both the nucleotide and amino acid sequences were highly similar among the 12 strains of F. verticillioides analyzed, as well as among those from the G. fujikuroi complex. The PGs were not inhibited by the polygalacturonase-inhibiting proteins (PGIPs) from the monocot asparagus and leek plants, but were inhibited to variable extents by bean PGIP. PGs from F. verticillioides, F. nygamai and one strain of F. proliferatum were barely inhibited. Residue 97 within PG was demonstrated to contribute to the different levels of inhibition. Together these findings provide new insights into the structural and functional relationships between the PG from the species of the G. fujikuroi complex and the plant PGIP.     

A single amino acid substitution in highly similar endo-PGs from Fusarium verticillioides and related Fusarium species affects PGIP inhibition.

Endo-polygalacturonase (PG) may be a critical virulence factor secreted by several fungi upon plant invasion. The single-copy gene encoding PG in Fusarium verticillioides and in eight other species of the Gibberella fujikuroi complex (F. sacchari, F. fujikuroi, F. proliferatum, F. subglutinans, F. thapsinum, F. nygamai, F. circinatum, and F. anthophilum) was functionally analyzed in this paper. Both the nucleotide and amino acid sequences were highly similar among the 12 strains of F. verticillioides analyzed, as well as among those from the G. fujikuroi complex. The PGs were not inhibited by the polygalacturonase-inhibiting proteins (PGIPs) from the monocot asparagus and leek plants, but were inhibited to variable extents by bean PGIP. PGs from F. verticillioides, F. nygamai and one strain of F. proliferatum were barely inhibited. Residue 97 within PG was demonstrated to contribute to the different levels of inhibition. Together these findings provide new insights into the structural and functional relationships between the PG from the species of the G. fujikuroi complex and the plant PGIP.     

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.     

Fumonisin production and other traits of Fusarium moniliforme strains from maize in northeast Mexico.

Strains of Fusarium moniliforme from maize seed collected in four fields in northeast Mexico were tested for fumonisin production in culture, for sexual compatibility, and for vegetative compatibility by using non-nitrate-utilizing mutants. The test results indicate that a diverse population of fumonisin-producing strains of F. moniliforme (Gibberella fujikuroi) mating population A predominates and that a potential exists for production of fumonisins in Mexican maize.     

Fusarium subglutinans f. sp. Pini represents a distinct mating population in the gibberella fujikuroi species complex

Fusarium strains in the Gibberella fujikuroi species complex cause diseases on a variety of economically important plants. One of these diseases, pitch canker of Pinus spp., is caused by strains identified as Fusarium subglutinans f. sp. pini. Fertile crosses were detected between F. subglutinans f. sp. pini strains from South Africa, California, and Florida. F. subglutinans f. sp. pini strains were not cross-fertile with the standard tester strains of six of the seven other mating populations of G. fujikuroi. Sporadic perithecia with ascospores were obtained in two crosses with the mating population B tester strains. These perithecia were homothallic, and the ascospores derived from these perithecia were vegetatively compatible with the mating population B tester strain parent. We concluded that fertile F. subglutinans f. sp. pini isolates represent a new mating population (mating population H) of G. fujikuroi and that they belong to a unique biological species in a distinct taxon.     

Segregation of secondary metabolite biosynthesis in hybrids of Fusarium fujikuroi and Fusarium proliferatum

Fusarium fujikuroi and Fusarium proliferatum are two phylogenetically closely related species of the Gibberella fujikuroi species complex (GFC). In some cases, strains of these species can cross and produce a few ascospores. In this study, we analyzed 26 single ascospore isolates of an interspecific cross between F. fujikuroi C1995 and F. proliferatum D4854 for their ability to produce four secondary metabolites: gibberellins (GAs), the mycotoxins fusarin C and fumonisin B(1), and a family of red polyketides, the fusarubins. Both parental strains contain the biosynthetic genes for all four metabolites, but differ in their ability to produce these metabolites under certain conditions. F. fujikuroi C1995 produces GAs and fusarins, while F. proliferatum D4854 produces fumonisins and fusarubins. The segregation amongst the progeny of these traits is not the expected 1:1 Mendelian ratio. Only eight, six, three and three progeny, respectively, produce GAs, fusarins, fumonisin B(1) and fusarubins in amounts similar to those synthesized by the producing parental strain. Beside the eight highly GA(3)-producing progeny, some of the progeny produce small amounts of GAs, predominantly GA(1), although these strains contain the GA gene cluster of the non-GA-producing F. proliferatum parental strain. Some progeny had recombinant secondary metabolite profiles under the conditions examined indicating that interspecific crosses can yield secondary metabolite production profiles that are atypical of the parent species.     

Zearalenone Production by Fusarium Species

One-hundred-and-thirteen isolates of Fusarium were tested for their ability to produce zearalenone on autoclaved corn. They belonged to the following species (number of producers per number tested): F. epispheria, (0/1); F. moniliforme, (0/8); Gibberella fujikuroi, (0/3); F. nivale, (0/7); F. oxysporum, (0/15); F. roseum, (31/51); F. solani, (0/9); F. tricinctum (3/19). The isolates of individual species produced the following amounts of zearalenone per gram of corn: 3 isolates of F. roseum (0.6 to 119 mug), 3 of F. roseum "Culmorum" (1 to 210 mug), 3 of F. roseum "Equiseti" (0.6 to 2.0 mug), F. roseum "Gibbosum" (115 to 175 mug), 21 of F. roseum "Graminearum" (0.2 to 230 mug), and 3 of F. tricinctum (0.2 to 6.0 mug). All isolates of F. roseum "Graminearum" which formed the perithecial stage of G. zeae (G. roseum) produced zearalenone. Production occurred by the wild but not the appressed cultural type. Zearalenone production by F. tricinctum was confirmed by a mouse bioassay.     

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.     

Gibberella konza (Fusarium konzum) sp. nov. from prairie grasses, a new species in the Gibberella fujikuroi species complex

The Gibberella fujikuroi species complex (Fusarium section Liseola and allied taxa) is composed of an increasingly large number of morphological, biological and phylogenetic species. Most of the known species in this group have been isolated from agricultural ecosystems or have been described from a small number of isolates. We sampled Fusarium communities from native prairie grasses in Kansas and recovered a large number of isolates that superficially resemble F. anthophilum. We used a combination of morphological, biological and molecular characters to describe a new species, Gibberella konza (Gibberella fujikuroi mating population I [MP-I]), from native prairie grasses in Kansas. Although female fertility for field isolates of this species appears to be low, G. konza is heterothallic, and we developed reliably female fertile mating population tester strains for this species. The F. konzum anamorph is differentiated from F. anthophilum and from other Fusarium species in section Liseola by mating compatibility, morphology, AFLP fingerprint profile and differences in β-tubulin DNA sequence.     

Gibberellin biosynthesis and gibberellin oxidase activities in Fusarium sacchari,Fusarium konzum and Fusarium subglutinans strains

Several isolates of three Fusarium species associated with the Gibberella fujikuroi species complex were characterized for their ability to synthesize gibberellins (GAs): Fusarium sacchari (mating population B), Fusarium konzum (mating population I) and Fusarium subglutinans (mating population E). Of these, F. sacchari is phylogenetically related to Fusarium fujikuroi and is grouped in the Asian clade of the complex, while F. konzum and F. subglutinans are only distantly related to Fusarium fujikuroi and belong to the American clade. Variability was found between the different F. sacchari strains tested. Five isolates (B-12756; B-1732, B-7610, B-1721 and B-1797) were active in GA biosynthesis and accumulated GA₃ in the culture fluid (2.76–28.4 μg/mL), while two others (B-3828 and B-1725) were inactive. GA₃ levels in strain B-12756 increased by 2.9 times upon complementation with ggs2 and cps-ks genes from F. fujikuroi. Of six F. konzum isolates tested, three (I-10653; I-11616; I-11893) synthesized GAs, mainly GA₁, at a low level (less than 0.1 μg/mL). Non-producing F. konzum strains contained no GA oxidase activities as found for the two F. subglutinans strains tested. These results indicate that the ability to produce GAs is present in other species of the G. fujikuroi complex beside F. fujikuroi, but might differ significantly in different isolates of the same species.     

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.     

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.     

Dimorphic fungus characteristic of fumonisin-producing strains of Fusarium moniliforme from Zhejiang

Fusarium moniliforme and its fumonisins have been shown to be carcinogenic in lab animals and have been linked to high incidences of human esophageal cancer. In this study we report the dimorphic fungus characteristic of fumonisin-producing strains of F. moniliforme from foodstuffs in Zhejiang, China. All of the twenty strains of F. moniliforme shown produce fumonisin B₁ 475.9–6322.2 μg/g in corn medium. These strains of F. moniliforme form yeast-like colonies in Sabouraud's agar plates contained 9% NaCl at 37 °C incubator and shows mostly budding reproduction. In blood agar plates these strains of F. moniliforme appear grass-green haemolytic reactions. This is the first report that yeast-like growth, dimorphic pathogenic fungus feature is found in F. moniliforme. These results suggest that it is also important to program epidemiological surveys of F. moniliforme as a primary pathogenic fungus, while proceeding to produce mycotoxins of F. moniliforme in food hygiene. This revised version was published online in August 2006 with corrections to the Cover Date.     

PCR-based identification of MAT-1 and MAT-2 in the Gibberella fujikuroi species complex

All sexually fertile strains in the Gibberella fujikuroi species complex are heterothallic, with individual mating types conferred by the broadly conserved ascomycete idiomorphs MAT-1 and MAT-2. We sequenced both alleles from all eight mating populations, developed a multiplex PCR technique to distinguish these idiomorphs, and tested it with representative strains from all eight biological species and 22 additional species or phylogenetic lineages from this species complex. In most cases, either an approximately 800-bp fragment from MAT-2 or an approximately 200-bp fragment from MAT-1 is amplified. The amplified fragments cosegregate with mating type, as defined by sexual cross-fertility, in a cross of Fusarium moniliforme (Fusarium verticillioides). Neither of the primer pairs amplify fragments from Fusarium species such as Fusarium graminearum, Fusarium pseudograminearum, and Fusarium culmorum, which have, or are expected to have, Gibberella sexual stages but are thought to be relatively distant from the species in the G. fujikuroi species complex. Our results suggest that MAT allele sequences are useful indicators of phylogenetic relatedness in these and other Fusarium species.     

Production of gibberellic acids by an orchid-associated Fusarium proliferatum strain

The rice pathogen Fusarium fujikuroi is well known for its ability to produce the plant hormones gibberellins (GAs). However, the majority of closely related Fusarium species is unable to produce GAs although the GA gene cluster is present in their genomes. In this study, we analyzed five orchid-associated Fusarium isolates for their capacity to produce GAs. Four of them did not produce any GAs and were shown not to contain any GA biosynthetic genes. However, the fifth isolate, which has been identified as F. proliferatum based on five molecular markers, produced significant amounts of GAs in contrast to previously characterized F. proliferatum strains. We focused on the molecular characterization of two GA-specific genes, ggs2 and cps/ks, both inactive in F. proliferatum strain D-02945. Complementation of a F. fujikuroi Deltaggs2 mutant with the ET1 ggs2 gene fully restored GA biosynthesis, confirming that the orchid-associated isolate contains an active gene copy. A possible correlation between GA production and their role in plant-fungal interactions is discussed.     

Fusarium temperatum sp. nov. from maize, an emergent species closely related to Fusarium subglutinans

A large number of Fusarium isolates closely related to F. subglutinans were collected from maize in Belgium. We used a robust polyphasic approach to describe a new biological species, Fusarium temperatum, within the Gibberella fujikuroi species complex. F. temperatum can be distinguished from F. subglutinans and from other Fusarium species within the Gibberella fujikuroi species complex with AFLP fingerprint profile, differences in the translation elongation factor 1-α and β-tubulin DNA sequence and interspecies mating compatibility analyses. Intraspecies mating compatibility suggests that sexual reproduction might be common for field isolates of F. temperatum, and reliable female fertile mating population tester strains were proposed for this heterothallic species.     

Genetic variation in Fusarium section Liseola from no-till maize in Argentina

Strains of Fusarium species belonging to section Liseola cause stalk and ear rot of maize and produce important mycotoxins, such as fumonisins. We isolated two species, Fusarium verticillioides (Gibberella fujikuroi mating population A) and Fusarium proliferatum (G. fujikuroi mating population D) from maize cultivated under no-till conditions at five locations in the Córdoba province of Argentina. We determined the effective population number for mating population A (N(e)) and found that the N(e) for mating type was 89% of the count (total population) and that the N(e) for male or hermaphrodite status was 36%. Thus, the number of strains that can function as the female parent limits N(e), and sexual reproduction needs to occur only once every 54 to 220 asexual generations to maintain this level of sexual fertility. Our results indicate that the fungal populations isolated from no-till maize are similar to those recovered from maize managed with conventional tillage. We placed 36 strains from mating population A into 28 vegetative compatibility groups (VCGs). Of the 13 strains belonging to five multimember VCGs, only 2 isolates belonging to one VCG were clones based on amplified fragment length polymorphism (AFLP) fingerprints. Members of the other four multimember VCGs had an average similarity index of 0.89, and members of one VCG were no more closely related to other members of the same VCG than they were to other members of the population as a whole. This finding suggests that the common assumption that strains in the same VCG are either clonal or very closely related needs to be examined in more detail. The variability observed with AFLPs and VCGs suggests that sexual reproduction may occur more frequently than estimated by N(e).