[Subcellular localization and resistance to Gibberella fujikuroi of AtELHYPRP2 in transgenic tobacco].

The subcellular localization and the resistance to fungal pathogen Gibberella fujikuroi of the protein encoded by Arabidopsis AtELHYPRP2 (EARLI1-LIKE HYBRID PROLINE-RICH PROTEIN 2, AT4G12500) were investigated using transgenic tobacco plants. The coding sequence of AtELHYPRP2 was amplified from genomic DNA of Col-0 ecotype. After restriction digestion, the PCR fragment was ligated into pCAMBIA1302 to produce a fusion expression vector, pCAMBIA1302-AtELHYPRP2-GFP. Then the recombinant plasmid was introduced into Agrobacterium tumefaciens strain LBA4404 and transgenic tobacco plants were regenerated and selected via leaf disc transformation method. RT-PCR and Western blotting analyses showed that AtELHYPRP2 expressed effectively in transgenic tobacco plants. Observation under laser confocal microscopy revealed that the green fluorescence of AtELHYPRP2-GFP fusion protein could overlap with the red fluorescence came from propidium iodide staining, indicating AtELHYPRP2 is localized to cell surface. Antimicrobial experiments exhibited that the constitutive expression of AtELHYPRP2 could enhance the resistance of tobacco to fungal pathogen G. fujikuroi and the infection sites could accumulate H2O2 obviously. The basal expression levels of PR1 and the systemic expression levels of PR1 and PR5 in transgenic tobacco plants were higher than that of the wild-type plants, suggesting AtELHYPRP2 may play a role in systemic acquired resistance.

Recent advances in genes involved in secondary metabolite synthesis, hyphal development, energy metabolism and pathogenicity in Fusarium graminearum (teleomorph Gibberella zeae).

The ascomycete fungus, Fusarium graminearum (teleomorph Gibberella zeae), is the most common causal agent of Fusarium head blight (FHB), a devastating disease for cereal crops worldwide. F. graminearum produces ascospores (sexual spores) and conidia (asexual spores), which can serve as disease inocula of FHB. Meanwhile, Fusarium-infected grains are often contaminated with mycotoxins such as trichothecenes (TRIs), fumonisins, and zearalenones, among which TRIs are related to the pathogenicity of F. graminearum, and these toxins are hazardous to humans and livestock. In recent years, with the complete genome sequencing of F. graminearum, an increasing number of functional genes involved in the production of secondary metabolites, hyphal differentiation, sexual and asexual reproduction, virulence and pathogenicity have been identified from F. graminearum. In this review, the secondary metabolite synthesis, hyphal development and pathogenicity related genes in F. graminearum were thoroughly summarized, and the genes associated with secondary metabolites, sexual reproduction, energy metabolism, and pathogenicity were highlighted.

Mitochondrial carnitine-dependent acetyl coenzyme A transport is required for normal sexual and asexual development of the ascomycete Gibberella zeae.

Fungi have evolved efficient metabolic mechanisms for the exact temporal (developmental stages) and spatial (organelles) production of acetyl coenzyme A (acetyl-CoA). We previously demonstrated mechanistic roles of several acetyl-CoA synthetic enzymes, namely, ATP citrate lyase and acetyl-CoA synthetases (ACSs), in the plant-pathogenic fungus Gibberella zeae. In this study, we characterized two carnitine acetyltransferases (CATs; CAT1 and CAT2) to obtain a better understanding of the metabolic processes occurring in G. zeae. We found that CAT1 functioned as an alternative source of acetyl-CoA required for lipid accumulation in an ACS1 deletion mutant. Moreover, deletion of CAT1 and/or CAT2 resulted in various defects, including changes to vegetative growth, asexual/sexual development, trichothecene production, and virulence. Although CAT1 is associated primarily with peroxisomal CAT function, mislocalization experiments showed that the role of CAT1 in acetyl-CoA transport between the mitochondria and cytosol is important for sexual and asexual development in G. zeae. Taking these data together, we concluded that G. zeae CATs are responsible for facilitating the exchange of acetyl-CoA across intracellular membranes, particularly between the mitochondria and the cytosol, during various developmental stages.

Functional analyses of the nitrogen regulatory gene areA in Gibberella zeae.

Fusarium head blight caused by Gibberella zeae is a prominent disease of cereal crops that poses serious human health concerns due to the contamination of grains with mycotoxins. In this study, we deleted an orthologue of areA, which is a global nitrogen regulator in filamentous fungi, to characterize its functions in G. zeae. The areA deletion resulted in an inability to use nitrate as a sole nitrogen source, whereas urea utilization was partially available. The virulence of ΔareA strains on wheat heads was markedly reduced compared with the wild-type strain. The areA mutation triggered loss of trichothecene biosynthesis but did not affect zearalenone biosynthesis. The ΔareA strains showed immaturity of asci and did not produce mature ascospores. Chemical complementation by urea restored normal sexual development, whereas the virulence and trichothecene production were not affected by urea addition. GFP-AreA fusion protein was localized to nuclei, and its expression increased in response to nitrogen-limiting conditions. These results suggest that areA-dependent regulation of nitrogen metabolism is required for vegetative growth, sexual development, trichothecene biosynthesis, and virulence in G. zeae.

A putative transcription factor MYT1 is required for female fertility in the ascomycete Gibberella zeae.

Gibberella zeae is an important pathogen of major cereal crops. The fungus produces ascospores that forcibly discharge from mature fruiting bodies, which serve as the primary inocula for disease epidemics. In this study, we characterized an insertional mutant Z39P105 with a defect in sexual development and identified a gene encoding a putative transcription factor designated as MYT1. This gene contains a Myb DNA-binding domain and is conserved in the subphylum Pezizomycotina of Ascomycota. The MYT1 protein fused with green fluorescence protein localized in nuclei, which supports its role as a transcriptional regulator. The MYT1 deletion mutant showed similar phenotypes to the wild-type strain in vegetative growth, conidia production and germination, virulence, and mycotoxin production, but had defect in female fertility. A mutant overexpressing MYT1 showed earlier germination, faster mycelia growth, and reduced mycotoxin production compared to the wild-type strain, suggesting that improper MYT1 expression affects the expression of genes involved in the cell cycle and secondary metabolite production. This study is the first to characterize a transcription factor containing a Myb DNA-binding domain that is specific to sexual development in G. zeae.

Proteomic profiling of two maize inbreds during early gibberella ear rot infection.

Fusarium graminearum is the causal agent of gibberella ear rot in maize ears, resulting in yield losses due to mouldy and mycotoxin-contaminated grain. This study represents a global proteomic approach to document the early infection by F. graminearum of two maize inbreds, B73 and CO441, which differ in disease susceptibility. Mock- and F. graminearum-treated developing kernels were sampled 48 h post-inoculation over three field seasons. Infected B73 kernels consistently contained higher concentrations of the mycotoxin deoxynivalenol than the kernels of the more tolerant inbred CO441. A total of 2067 maize proteins were identified in the iTRAQ analysis of extracted kernel proteins at a 99% confidence level. A subset of 878 proteins was identified in at least two biological replicates and exhibited statistically significantly altered expression between treatments and/or the two inbred lines of which 96 proteins exhibited changes in abundance >1.5-fold in at least one of the treatments. Many proteins associated with the defense response were more abundant after infection, including PR-10 (PR, pathogenesis-related), chitinases, xylanase inhibitors, proteinase inhibitors, and a class III peroxidase. Kernels of the tolerant inbred CO441 contained higher levels of these defense-related proteins than B73 kernels even after mock treatment, suggesting that these proteins may provide a basal defense against Fusarium infection in CO441.

Trichothecene profiling and population genetic analysis of Gibberella zeae from barley in North Dakota and Minnesota.

Gibberella zeae, the principal cause of Fusarium head blight (FHB) of barley, contaminates grains with several mycotoxins, which creates a serious problem for the malting barley industry in the United States, China, and Europe. However, limited studies have been conducted on the trichothecene profiles and population genetic structure of G. zeae isolates collected from barley in the United States. Trichothecene biosynthesis gene (TRI)-based polymerase chain reaction (PCR) assays and 10 variable number tandem repeat (VNTR) markers were used to determine the genetic diversity and compare the trichothecene profiles of an older population (n = 115 isolates) of G. zeae collected in 1997 to 2000 with a newer population (n = 147 isolates) collected in 2008. Samples were from across the major barley-growing regions in North Dakota and Minnesota. The results of TRI-based PCR assays were further validated using a subset of 32 and 28 isolates of G. zeae by sequence analysis and gas chromatography, respectively. TRI-based PCR assays revealed that all the G. zeae isolates in both populations had markers for deoxynivalenol (DON), and the frequencies of isolates with a 3-acetyldeoxynivalenol (3-ADON) marker in the newer population were ≈11-fold higher than those among isolates in the older population. G. zeae populations from barley in the Midwest of the United States showed no spatial structure, and all the isolates were solidly in clade 7 of G. zeae, which is quite different from other barley-growing areas of world, where multiple species of G. zeae are commonly found in close proximity and display spatial structure. VNTR analysis showed high gene diversity (H = 0.82 to 0.83) and genotypic diversity but low linkage disequilibrium (LD = 0.02 to 0.07) in both populations. Low genetic differentiation (F(ST) = 0.013) and high gene flow (Nm = 36.84) was observed between the two populations and among subpopulations within the same population (Nm = 12.77 to 29.97), suggesting that temporal and spatial variations had little influence on population differentiation in the Upper Midwest. Similarly, low F(ST) (0.02) was observed between 3-ADON and 15-acetyldeoxynivalenol populations, indicating minor influence of the chemotype of G. zeae isolates on population subdivision, although there was a rapid increase in the frequencies of isolates with the 3-ADON marker in the Upper Midwest between the older collection made in 1997 to 2000 and the newer collection made in 2008. This study provides information to barley-breeding programs for their selection of isolates of G. zeae for evaluating barley genotypes for resistance to FHB and DON accumulation.

Histidine kinase two-component response regulator proteins regulate reproductive development, virulence, and stress responses of the fungal cereal pathogens Cochliobolus heterostrophus and Gibberella zeae.

Histidine kinase (HK) phosphorelay signaling is a major mechanism by which fungi sense their environment. The maize pathogen Cochliobolus heterostrophus has 21 HK genes, 4 candidate response regulator (RR) genes (SSK1, SKN7, RIM15, REC1), and 1 gene (HPT1) encoding a histidine phosphotransfer domain protein. Because most HKs are expected to signal through RRs, these were chosen for deletion. Except for pigment and slight growth alterations for rim15 mutants, no measurable altered phenotypes were detected in rim15 or rec1 mutants. Ssk1p is required for virulence and affects fertility and proper timing of sexual development of heterothallic C. heterostrophus. Pseudothecia from crosses involving ssk1 mutants ooze masses of single ascospores, and tetrads cannot be found. Wild-type pseudothecia do not ooze. Ssk1p represses asexual spore proliferation during the sexual phase, and lack of it dampens asexual spore proliferation during vegetative growth, compared to that of the wild type. ssk1 mutants are heavily pigmented. Mutants lacking Skn7p do not display any of the above phenotypes; however, both ssk1 and skn7 mutants are hypersensitive to oxidative and osmotic stresses and ssk1 skn7 mutants are more exaggerated in their spore-type balance phenotype and more sensitive to stress than single mutants. ssk1 mutant phenotypes largely overlap hog1 mutant phenotypes, and in both types of mutant, the Hog1 target gene, MST1, is not induced. ssk1 and hog1 mutants were examined in the homothallic cereal pathogen Gibberella zeae, and pathogenic and reproductive phases of development regulated by Ssk1 and Hog1 were found to mirror, but also vary from, those of C. heterostrophus.

Variation and transgression of aggressiveness among two Gibberella zeae crosses developed from highly aggressive parental isolates.

Gibberella zeae (anamorph: Fusarium graminearum) is the most common cause of Fusarium head blight (FHB) of wheat (Triticum aestivum) worldwide. Aggressiveness is the most important fungal trait affecting disease severity and stability of host resistance. Objectives were to analyze in two field experiments (i) segregation for aggressiveness among 120 progenies from each of two crosses of highly aggressive parents and (ii) stability of FHB resistance of seven moderately to highly resistant winter wheat cultivars against isolates varying for aggressiveness. Aggressiveness was measured as FHB severity per plot, Fusarium exoantigen absorbance, and deoxynivalenol content. In the first experiment, mean FHB ratings were 20 to 49% across environments and progeny. Significant genotypic variation was detected in both crosses (P < 0.01). Isolate-environment interaction explained approximately half of the total variance. Two transgressive segregants were found in cross B across environments. Traits were significantly (P < 0.05) intercorrelated. In the second experiment, despite significant (P < 0.05) genotypic variance for cultivar and isolate, no significant (P > 0.05) interaction was observed for any trait. In conclusion, progeny of highly aggressive parents might exhibit increased aggressiveness due to recombination and may, therefore, adapt nonspecifically to increased quantitative host resistance.

Fusarium spp. associated with rice Bakanae: ecology, genetic diversity, pathogenicity and toxigenicity.

African and Asian populations of Fusarium spp. (Gibberella fujikuroi species complex) associated with Bakanae of rice (Oryzae sativa L.) were isolated from seeds and characterized with respect to ecology, phylogenetics, pathogenicity and mycotoxin production. Independent of the origin, Fusarium spp. were detected in the different rice seed samples with infection rate ranges that varied from 0.25% to 9%. Four Fusaria (F. andiyazi, F. fujikuroi, F. proliferatum and F. verticillioides) were found associated with Bakanae of rice. While three of the Fusaria were found in both African and Asian seed samples, F. fujikuroi was only detected in seed samples from Asia. Phylogenetic studies showed a broad genetic variation among the strains that were distributed into four different genetic clades. Pathogenicity tests showed that all strains reduced seed germination and possessed varying ability to cause symptoms of Bakanae on rice, some species (i.e. F. fujikuroi) being more pathogenic than others. The ability to produce fumonisins (FB(1) and FB(2)) and gibberellin A3 in vitro also differed according to the Fusarium species. While fumonisins were produced by most of the strains of F. verticillioides and F. proliferatum, gibberellin A3 was only produced by F. fujikuroi. Neither fumonisin nor gibberellin was synthesized by most of the strains of F. andiyazi. These findings provide new information on the variation within the G. fujikuroi species complex associated with rice seed and Bakanae disease.

Roles of the glyoxylate and methylcitrate cycles in sexual development and virulence in the cereal pathogen Gibberella zeae.

The glyoxylate and methylcitrate cycles are involved in the metabolism of two- or three-carbon compounds in fungi. To elucidate the role(s) of these pathways in Gibberella zeae, which causes head blight in cereal crops, we focused on the functions of G. zeae orthologs (GzICL1 and GzMCL1) of the genes that encode isocitrate lyase (ICL) and methylisocitrate lyase (MCL), respectively, key enzymes in each cycle. The deletion of GzICL1 (DeltaGzICL1) caused defects in growth on acetate and in perithecium (sexual fruiting body) formation but not in virulence on barley and wheat, indicating that GzICL1 acts as the ICL of the glyoxylate cycle and is essential for self-fertility in G. zeae. In contrast, the DeltaGzMCL1 strains failed to grow on propionate but exhibited no major changes in other traits, suggesting that GzMCL1 is required for the methylcitrate cycle in G. zeae. Interestingly, double deletion of both GzICL1 and GzMCL1 caused significantly reduced virulence on host plants, indicating that both GzICL1 and GzMCL1 have redundant functions for plant infection in G. zeae. Thus, both GzICL1 and GzMCL1 may play important roles in determining major mycological and pathological traits of G. zeae by participating in different metabolic pathways for the use of fatty acids.

Gibberella zeae chitin synthase genes, GzCHS5 and GzCHS7, are required for hyphal growth, perithecia formation, and pathogenicity.

Gibberella zeae causes Fusarium head blight of cereal crops, and sexual spores of the fungus play an important role as primary inocula. We isolated a restriction enzyme-mediated integration (REMI) transformant, ZH431, of G. zeae with defects in perithecia formation and virulence. Integration of the REMI vector resulted in disruption of GzCHS7 gene, which encodes a putative class VII chitin synthase with high similarity to Fusarium oxysporum ChsVb. A second chitin synthase, GzCHS5, is adjacently located in a head-to-head configuration with GzCHS7, and its deduced protein sequence showed similarity with a class V chitin synthase in F. oxysporum. Neither DeltaGzChs5 nor DeltaGzChs7 mutants produced perithecia or caused disease on barley heads. Microscopic observation revealed that both mutants formed balloon-shaped hyphae and intrahyphal hyphae and that cell wall rigidity of the mutants was weaker than that of the wild-type strain. Transcription profiles of GzCHS5 and GzCHS7 were not altered in DeltaGzChs7 and DeltaGzChs5, respectively, suggesting that transcription regulations of the genes are independent of each other. Our results demonstrate that GzCHS5 and GzCHS7 are indispensable for perithecia formation and pathogenicity as well as normal septa formation and hyphal growth in G. zeae.

Fusarium head blight severity and deoxynivalenol concentration in wheat in response to Gibberella zeae inoculum concentration.

The relationship between inoculum dose and resulting disease levels and deoxynivalenol (DON) accumulation in the Fusarium head blight (FHB) of wheat pathosystem was examined under controlled conditions. Greenhouse-grown spring wheat plants were inoculated at flowering with suspensions that varied in Gibberella zeae macroconidia concentration. The spikes were bagged for 72 h to promote infection and plants were then kept under ambient greenhouse conditions and disease allowed to develop. Spikes were rated at 15 days after inoculation for disease incidence and severity, removed from the greenhouse, and dried. DON concentration was determined in grain-only and whole-spike samples for each inoculation treatment. Regression analysis was used to evaluate the mathematical relationship between inoculum dose and the (i) disease metrics or (ii) DON concentration. Both disease incidence and severity were found to increase sharply in relation to inoculum concentration until an asymptote was reached. In both instances, a negative exponential function was found to best explain this relationship. By contrast, DON concentration in both grain-only and whole-spike tissues increased with additional inoculum. These relationships were best explained with linear functions for both sample types, although DON accumulation increased at a greater rate in whole-spike tissue. The functions were evaluated further using data collected from unrelated field studies and, although not particularly consistent, provided reasonably accurate predictions in growing seasons when the environment was only moderately favorable for FHB.

GzSNF1 is required for normal sexual and asexual development in the ascomycete Gibberella zeae.

The sucrose nonfermenting 1 (SNF1) protein kinase of yeast plays a central role in the transcription of glucose-repressible genes in response to glucose starvation. In this study, we deleted an ortholog of SNF1 from Gibberella zeae to characterize its functions by using a gene replacement strategy. The mycelial growth of deletion mutants (DeltaGzSNF1) was reduced by 21 to 74% on diverse carbon sources. The virulence of DeltaGzSNF1 mutants on barley decreased, and the expression of genes encoding cell-wall-degrading enzymes was reduced. The most distinct phenotypic changes were in sexual and asexual development. DeltaGzSNF1 mutants produced 30% fewer perithecia, which matured more slowly, and asci that contained one to eight abnormally shaped ascospores. Mutants in which only the GzSNF1 catalytic domain was deleted had the same phenotype changes as the DeltaGzSNF1 strains, but the phenotype was less extreme in the mutants with the regulatory domain deleted. In outcrosses between the DeltaGzSNF1 mutants, each perithecium contained approximately 70% of the abnormal ascospores, and approximately 50% of the asci showed unexpected segregation patterns in a single locus tested. The asexual spores of the DeltaGzSNF1 mutants were shorter and had fewer septa than those of the wild-type strain. The germination and nucleation of both ascospores and conidia were delayed in DeltaGzSNF1 mutants in comparison with those of the wild-type strain. GzSNF1 expression and localization depended on the developmental stage of the fungus. These results suggest that GzSNF1 is critical for normal sexual and asexual development in addition to virulence and the utilization of alternative carbon sources.

A putative pheromone signaling pathway is dispensable for self-fertility in the homothallic ascomycete Gibberella zeae.

Gibberella zeae, a homothallic ascomycetous fungus, does not seek a partner for mating. Here, we focused on the role(s) of putative pheromone and receptor genes during sexual development in G. zeae. Orthologs of two pheromone precursor genes (GzPPG1 and GzPPG2), and their cognate receptor genes (GzPRE2 and GzPRE1) were transcribed during sexual development. The expression of these genes was controlled by the mating-type (MAT) locus and a MAP kinase gene, but not in a MAT-specific manner. Targeted gene deletion and subsequent outcrosses generated G. zeae strains lacking these putative pheromone/receptor genes in various combinations (from single to quadruple deletions). All G. zeae deletion strains were similar to the self-fertile progenitor in both male- and female fertility and other traits. Sometimes, the deletions including DeltaGzPPG1;DeltaGzPRE2 caused increased numbers of immature perithecia. Taken together, it is clear that these putative pheromones/receptors play a non-essential role in the sexual development of G. zeae.

Functional analyses of heterotrimeric G protein G alpha and G beta subunits in Gibberella zeae.

The homothallic ascomycete fungus Gibberella zeae (anamorph: Fusarium graminearum) is a major toxigenic plant pathogen that causes head blight disease on small-grain cereals. The fungus produces the mycotoxins deoxynivalenol (DON) and zearalenone (ZEA) in infected hosts, posing a threat to human and animal health. Despite its agricultural and toxicological importance, the molecular mechanisms underlying its growth, development and virulence remain largely unknown. To better understand such mechanisms, we studied the heterotrimeric G proteins of G. zeae, which are known to control crucial signalling pathways that regulate various cellular and developmental responses in fungi. Three putative Galpha subunits, GzGPA1, GzGPA2 and GzGPA3, and one Gbeta subunit, GzGPB1, were identified in the F. graminearum genome. Deletion of GzGPA1, a homologue of the Aspergillus nidulans Galpha gene fadA, resulted in female sterility and enhanced DON and ZEA production, suggesting that GzGPA1 is required for normal sexual reproduction and repression of toxin biosynthesis. The production of DON and ZEA was also enhanced in the GzGPB1 mutant, suggesting that both Galpha GzGPA1 and Gbeta GzGPB1 negatively control mycotoxin production. Deletion of GzGPA2, which encodes a Galpha protein similar to A. nidulans GanB, caused reduced pathogenicity and increased chitin accumulation in the cell wall, implying that GzGPA2 has multiple functions. Our study shows that G. zeae heterotrimeric G protein subunits can regulate vegetative growth, sexual development, toxin production and pathogenicity.

A guanylyl cyclase-like gene is associated with Gibberella ear rot resistance in maize (Zea mays L.).

Gibberella ear rot, caused by the fungal pathogen Fusarium graminearum Schwabe, is a serious disease of maize (Zea mays L.) grown in northern climates. The infected maize grain contains toxins that are very harmful to livestock and humans. A maize gene that encodes a putative 267-amino acid guanylyl cyclase-like protein (ZmGC1) was characterized and shown to be associated with resistance to this disease. The putative ZmGC1 amino acid sequence is 53% identical and 65% similar to AtGC1, an Arabidopsis guanylyl cyclase. The Zmgc1 coding sequence is nearly identical in a Gibberella ear rot-resistant line (CO387) and a susceptible line (CG62) but several nucleotide sequence differences were observed in the UTRs and introns of the two alleles. Using a 463 bp probe derived from the CG62 allele of Zmgc1 and a recombinant inbred (RI) mapping population developed from a CG62 x CO387 cross, six Zmgc1 restriction fragment length polymorphism (RFLP) fragments (ER1_1, ER1_2, ER1_3, ER1_4, ER1_5, and ER5_1) were mapped on maize chromosomes 2, 3, 7, and 8. Markers ER1_1 and ER5_1 on chromosomes 7 and 8, respectively, were significantly associated with Gibberella ear rot resistance, each in three different environments. The amount of Zmgc1 transcript in ear tissues increased more quickly and to a greater extent in the resistant genotype compared to the susceptible genotype after inoculation with F. graminearum. Zmgc1 is the first guanylyl cyclase gene characterized in maize and the first gene found to be associated with Gibberella ear rot resistance in this plant.

Further data on the production of beauvericin, enniatins and fusaproliferin and toxicity to Artemia salina by Fusarium species of Gibberella fujikuroi species complex.

The knowledge of toxigenic profiles of fungal plant pathogens is of extreme importance for evaluating the potential toxicity of infected plant products. Ninety-six fungal isolates belonging to 28 species in the Gibberella fujikuroi complex were studied for the production of beauvericin, enniatins and fusaproliferin in rice cultures. Toxin production ranged from 5 to 3000 microg/g for beauvericin, 2 to 131 microg/g for enniatins, and 4 to 440 microg/g for fusaproliferin. Beauvericin was the most common metabolite produced by 16 species followed by fusaproliferin with 11 species and enniatins with 4 species. The production of beauvericin by F. bulbicola, F. denticulatum, F. lactis, F. phyllophilum, F. pseudocircinatum, and F. succisae and fusaproliferin by F. antophilum, F. begoniae, F. bulbicola, F. circinatum, F. concentricum, F. succisae, and F. udum is reported here for the first time. Brine shrimp larvae were most sensitive to culture extracts of F. acutatum (up to 94+/-3%), F. concentricum (up to 99+/-1%), F. denticuatum (up to 100%) and F. sacchari (up to 100%). Toxicity towards brine shrimp was significantly correlated with the beauvericin content of the fungal extracts with few exceptions. These data indicate that beauvericin and fusaproliferin are common metabolites of species of the G. fujikuroi complex and pose a risk for a possible toxin accumulation in their respective host plant products. However, data from the brine shrimp bioassay showed that further toxic metabolites within this complex need to be characterized.

Evidence for recombination and segregation of virulence to pine in a hybrid cross between Gibberella circinata and G. subglutinans.

Two species associated with the Gibberella fujikuroi species complex, G. circinata (the cause of pitch canker in pines) and G. subglutinans (avirulent on pine), were found to have limited interfertility in hybrid crosses. MAT idiomorphs, polymorphisms in the histone H3 gene, vegetative compatibility, and virulence phenotypes were used to verify recombination. The MAT idiomorphs appeared to be assorting independently, but the histone H3 haplotype was disproportionately represented by that of the G. subglutinans parent. Ninety-eight percent (45/46) of the progeny tested were vegetatively incompatible with both parents. All F(1) progeny were avirulent to pine, but a wide range of virulence was restored through a backcross to the virulent parent (G. circinata). Attempts at hybrid crosses using other isolate combinations were rarely successful (1/26). This limited interfertility supports retention of G. circinata and G. subglutinans as separate species, but offers opportunities to characterize the inheritance of virulence to pine.

A novel F-box protein involved in sexual development and pathogenesis in Gibberella zeae.

Gibberella zeae is an ascomyceteous fungus that causes serious diseases in cereal crops. Severe epidemics require strains that are virulent and that can reproduce sexually. We characterized an insertional mutant (designated ZH436) with a pleiotropic defect in both traits, and identified a novel F-box protein gene encoding FBP1 (F-box protein 1) that is similar to fungal F-box proteins including Saccharomyces cerevisiae Grr1, a well-characterized component of the Skp1-Cullin-F-box protein (SCF(Grr1)) E3 ligase complex required for protein degradation. FBP1 also can bind both S. cerevisiae Skp1 protein, the other component of the SCF(Grr1) complex, and its G. zeae sequence homologue SKP1. Two putative protein interacting domains in FBP1 are essential for in vivo function. FBP1 and ScGRR1 are not so interchangeable between S. cerevisiae and G. zeae, but FBP1 can partially complement several defects of a yeast grr1 deletion mutant. Functional analyses confirmed that FBP1 is required for several phenotypes including both sexual development and virulence in G. zeae; the phenotype of DeltaFBP1 strains is different from those of null mutants for F-box proteins in other filamentous fungi as well as from S. cerevisiae grr1Delta strains. Thus, FBP1 is a versatile F-box protein that presumably participates in the formation of the SCF(FBP1) complex that probably controls the ubiquitin-mediated degradation of proteins involved in sexual reproduction and virulence important for disease development by G. zeae.