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.     

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.     

Cryptic speciation in Fusarium subglutinans

Fusarium isolates that form part of the Gibberella fujikuroi species complex have been classified using either a morphological, biological, or phylogenetic species concept. Problems with the taxonomy of Fusarium species in this complex are mostly experienced when the morphological and biological species concepts are applied. The most consistent identifications are obtained with the phylogenetic species concept. Results from recent studies have presented an example of discordance between the biological and phylogenetic species concepts, where a group of F. subglutinans sensu stricto isolates, i.e., isolates belonging to mating population E of the G. fujikuroi complex, could be sub-divided into more than one phylogenetic lineage. The aim of this study was to determine whether this sub-division represented species divergence or intraspecific diversity in F. subglutinans. For this purpose, we included 29 F. subglutinans isolates belonging to the E-mating population that were collected from either maize or teosinte, from a wide geographic range. DNA sequence data for six nuclear regions in each of these isolates were obtained and used in phylogenetic concordance analyses. These analyses revealed the presence of two major groups representing cryptic species in F. subglutinans. These cryptic species were further sub-divided into a number of smaller groups that appear to be reproductively isolated in nature. This suggests not only that the existing F. subglutinans populations are in the process of divergence, but also that each of the resulting lineages are undergoing separation into distinct taxa. These divergences did not appear to be linked to geographic origin, host, or phenotypic characters such as morphology.     

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.     

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.     

Complete genetic linkage maps from an interspecific cross between Fusarium circinatum and Fusarium subglutinans

The Gibberella fujikuroi complex includes many plant pathogens of agricultural crops and trees, all of which have anamorphs assigned to the genus Fusarium. In this study, an interspecific hybrid cross between Gibberella circinata and Gibberella subglutinans was used to compile a genetic linkage map. A framework map was constructed using a total of 578 AFLP markers together with the mating type (MAT-1 and MAT-2) genes and the histone (H3) gene. Twelve major linkage groups were identified (n=12). Fifty percent of the markers showed significant deviation from the expected 1:1 transmission ratio in a haploid F(1) cross (P <0.05). The transmission of the markers on the linkage map was biased towards alleles of the G. subglutinans parent, with an estimated 60% of the genome of F(1) individuals contributed by this parent. This map will serve as a powerful tool to study the genetic architecture of interspecific differentiation and pathogenicity in the two parental genomes.     

Fusarium species from nepalese rice and production of mycotoxins and gibberellic acid by selected species

Infection of cereal grains with Fusarium species can cause contamination with mycotoxins that affect human and animal health. To determine the potential for mycotoxin contamination, we isolated Fusarium species from samples of rice seeds that were collected in 1997 on farms in the foothills of the Nepal Himalaya. The predominant Fusarium species in surface-disinfested seeds with husks were species of the Gibberella fujikuroi complex, including G. fujikuroi mating population A (anamorph, Fusarium verticillioides), G. fujikuroi mating population C (anamorph, Fusarium fujikuroi), and G. fujikuroi mating population D (anamorph, Fusarium proliferatum). The widespread occurrence of mating population D suggests that its role in the complex symptoms of bakanae disease of rice may be significant. Other common species were Gibberella zeae (anamorph, Fusarium graminearum) and Fusarium semitectum, with Fusarium acuminatum, Fusarium anguioides, Fusarium avenaceum, Fusarium chlamydosporum, Fusarium equiseti, and Fusarium oxysporum occasionally present. Strains of mating population C produced beauvericin, moniliformin, and gibberellic acid, but little or no fumonisin, whereas strains of mating population D produced beauvericin, fumonisin, and, usually, moniliformin, but no gibberellic acid. Some strains of G. zeae produced the 8-ketotrichothecene nivalenol, whereas others produced deoxynivalenol. Despite the occurrence of fumonisin-producing strains of mating population D, and of 8-ketotrichothecene-producing strains of G. zeae, Nepalese rice showed no detectable contamination with these mycotoxins. Effective traditional practices for grain drying and storage may prevent contamination of Nepalese rice with Fusarium mycotoxins.     

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.     

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.     

Restoration of gibberellin production in Fusarium proliferatum by functional complementation of enzymatic blocks

Nine biological species, or mating populations (MPs), denoted by letters A to I, and at least 29 anamorphic Fusarium species have been identified within the Gibberella fujikuroi species complex. Members of this species complex are the only species of the genus Fusarium that contain the gibberellin (GA) biosynthetic gene cluster or at least parts of it. However, the ability of fusaria to produce GAs is so far restricted to Fusarium fujikuroi, although at least six other MPs contain all the genes of the GA biosynthetic gene cluster. Members of Fusarium proliferatum, the closest related species, have lost the ability to produce GAs as a result of the accumulation of several mutations in the coding and 5' noncoding regions of genes P450-4 and P450-1, both encoding cytochrome P450 monooxygenases, resulting in metabolic blocks at the early stages of GA biosynthesis. In this study, we have determined additional enzymatic blocks at the first specific steps in the GA biosynthesis pathway of F. proliferatum: the synthesis of geranylgeranyl diphosphate and the synthesis of ent-kaurene. Complementation of these enzymatic blocks by transferring the corresponding genes from GA-producing F. fujikuroi to F. proliferatum resulted in the restoration of GA production. We discuss the reasons for Fusarium species outside the G. fujikuroi species complex having no GA biosynthetic genes, whereas species distantly related to Fusarium, e.g., Sphaceloma spp. and Phaeosphaeria spp., produce GAs.     

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.     

Gibberella musae (Fusarium musae) sp. nov., a recently discovered species from banana is sister to F. verticillioides

Several strains of Fusarium isolated from banana were identified previously as F. verticillioides (Sacc.) Nirenberg but described as unable to produce fumonisin. Here we report biochemical and morphological evidence, as well as multilocus phylogenetic analyses based on elongation factor (EF-1α), calmodulin, β-tubulin, and the second largest subunit of RNA polymerase II (RPB2) sequences, indicating that these isolates represent a unique lineage in the Gibberella fujikuroi species complex related to but distinct from F. verticillioides. Together with previous results of molecular studies, as well as with results of metabolite analyses, crossing experiments, pathogenicity tests and morphological characterization, these new data indicate that these strains isolated from banana represent a new species, Gibberella musae Van Hove et al. sp. nov. (anamorph: Fusarium musae Van Hove et al. sp. nov.), which is described herein.     

Fusarium commune is a new species identified by morphological and molecular phylogenetic data

Fusarium commune sp. nov. was isolated from soil and Pisum sativum in Denmark and several widespread locations within the northern hemisphere from diverse substrates including white pine, Douglas fir, carnation, corn, carrot, barley and soil. Fusarium commune is characterized by and distinguished from its putative sister taxon, the F. oxysporum complex, in having long, slender monophialides and polyphialides when cultured in the dark. Based on the combined DNA sequence data from translation elongation factor 1α (EF-1α) and the mitochondrial small subunit ribosomal DNA (mtSSU rDNA), the 15 isolates of F. commune analyzed formed a strongly supported clade closely related to but independent of the F. oxysporum and Gibberella fujikuroi species complexes.     

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.     

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.     

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).     

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.