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The reproductive genes of fungi, like those of many other organisms, are thought to diversify rapidly. This phenomenon could be associated with the formation of reproductive barriers and speciation. Ascomycetes produce two classes of mating type-specific peptide pheromones. These are required for recognition between the mating types of heterothallic species. Little is known regarding the diversity or the extent of species specificity in pheromone peptides among these fungi. We compared the putative protein-coding DNA sequences of the 2 pheromone classes from 70 species of Ascomycetes. The data set included previously described pheromones and putative pheromones identified from genomic sequences. In addition, pheromone genes from 12 Fusarium species in the Gibberella fujikuroi complex were amplified and sequenced. Pheromones were largely conserved among species in this complex and, therefore, cannot alone account for the reproductive barriers observed between these species. In contrast, pheromone peptides were highly diverse among many other Ascomycetes, with evidence for both positive diversifying selection and relaxed selective constraint. Repeats of the α-factor-like pheromone, which occur in tandem arrays of variable copy number, were found to be conserved through purifying selection and not concerted evolution. This implies that sequence specificity may be important for pheromone reception and that interspecific differences may indeed be associated with functional divergence. Our findings also suggest that frequent duplication and loss causes the tandem repeats to experience "birth-and-death" evolution, which could in fact facilitate interspecific divergence of pheromone peptide sequences.

Mutants of Gibberella fujikuroi with different colony characteristics, morphology and pigmentation were generated by exposure to UV radiation. A mutant, Mor-189, was selected based on its short filament length, relatively high gibberellin A(4) (GA(4)) and gibberellin A(3) (GA(3)) production, as well as its lack of pigmentation. Production of GA(4) by Mor-189 was studied using different inorganic and organic nitrogen sources, carbon sources and by maintaining the pH of the fermentation medium using calcium carbonate. Analysis of GA(4) and GA(3) was done by reversed-phase high-performance liquid chromatography and LC-MS. The mutants of G. fujikuroi produced more GA(4) when the pH of the medium was maintained above 5. During shake flask studies, the mutant Mor-189 produced 210 mg l(-1) GA(4) in media containing wheat gluten as the nitrogen source and glucose as the carbon source. Fed-batch fermentation in a 14 l agitated fermenter was performed to evaluate the applicability of the mutant Mor-189 for the production of GA(4). In 7-day fed-batch fermentation, 600 mg l(-1) GA(4) were obtained in the culture filtrate. The concentration of GA(4) and GA(3) combined was 713 mg l(-1), of which GA(4) accounted for 84% of the total gibberellin. These values are substantially higher than those published previously. The present study indicated that, along with maintenance of pH and controlled glucose feeding, the use of wheat gluten as the sole nitrogen source considerably enhances GA(4) production by the mutant Mor-189.

Carotenoids are a structurally diverse class of terpenoid pigments that are synthesized by many microorganisms and plants. In this study, we identified five putative carotenoid biosynthetic genes from the ascomycete Gibberella zeae (GzCarB, GzCarO, GzCarRA, GzCarT, and GzCarX). HPLC showed that the fungus produces two carotenoids: neurosporaxanthin and torulene. We deleted the five genes individually to determine their functions. GzCarB, GzCarRA, and GzCarT were required for neurosporaxanthin biosynthesis, but the deletion of GzCarX or GzCarO (DeltagzcarX or DeltagzcarO) failed to alter the production of neurosporaxanthin or torulene. DeltagzcarRA and DeltagzcarB did not produce neurosporaxanthin or torulene. DeltagzcarB led to the accumulation of phytoene, which is an intermediate in carotenoid biosynthesis, but DeltagzcarRA did not. DeltagzcarT produced torulene but not neurosporaxanthin. Based on these functional studies and similarities to carotenoid biosynthesis genes in other fungi, we deduced the functions of the three genes and propose the carotenoid biosynthetic pathway of G. zeae.

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.

We constructed a genetic linkage map of Gibberella zeae (Fusarium graminearum) by crossing complementary nitrate-nonutilizing (nit) mutants of G. zeae strains R-5470 (from Japan) and Z-3639 (from Kansas). We selected 99 nitrate-utilizing (recombinant) progeny and analyzed them for amplified fragment length polymorphisms (AFLPs). We used 34 pairs of two-base selective AFLP primers and identified 1048 polymorphic markers that mapped to 468 unique loci on nine linkage groups. The total map length is approximately 1300 cM with an average interval of 2.8 map units between loci. Three of the nine linkage groups contain regions in which there are high levels of segregation distortion. Selection for the nitrate-utilizing recombinant progeny can explain two of the three skewed regions. Two linkage groups have recombination patterns that are consistent with the presence of intercalary inversions. Loci governing trichothecene toxin amount and type (deoxynivalenol or nivalenol) map on linkage groups IV and I, respectively. The locus governing the type of trichothecene produced (nivalenol or deoxynivalenol) cosegregated with the TRI5 gene (which encodes trichodiene synthase) and probably maps in the trichothecene gene cluster. This linkage map will be useful in population genetic studies, in map-based cloning, for QTL (quantitative trait loci) analysis, for ordering genomic libraries, and for genomic comparisons of related species.

The creA genes of two plant pathogenic fungi, the gibberellin-producing rice pathogen Gibberella fujikuroi and the gray mold Botrytis cinerea, were isolated and characterized. The deduced amino acid sequences of both glucose repressors are 64% identical to each other and 59% (G. fujikuroi) and 61% (B. cinerea) identical to the CreA protein of Aspergillus nidulans. The zinc finger regions of the Gibberella and Botrytis CreA proteins shared 98% identity with the corresponding zinc finger region of the A. nidulans protein, and studies by complementation of a creA null mutant of A. nidulans showed that the proteins are functional homologues of A. nidulans CreA. Northern blot analysis revealed that creA transcript levels are independent of the carbon source in both fungi.

The idealized individual in many fungal species is a haploid self-sterile hermaphrodite that may be propagated by asexually produced spores or that may reproduce sexually. In field populations, polymorphism occurs for female-sterile/hermaphrodite status, and female-sterile mutants, which function only as males during sexual reproduction, may comprise > 50% of the population. The effective population number may be based on the number of strains of different mating type or the relative frequency of hermaphrodites. The female-sterile mutants are at a selective disadvantage every time sexual reproduction occurs, and must have an advantage during vegetative propagation to persist at a significant frequency. When a high frequency of female-sterile strains is observed in field populations, it indicates that vegetative propagation is a significant component of the fungus' natural history. Depending on the mutation rate to female sterility and the selective advantage of the female-sterile strains during vegetative propagation, the ratio of sexual:asexual generations can range from 1:15 to 1:2300 for species in the Gibberella fujikuroi complex. The relative rarity of sexual reproduction may permit female-sterile strains to accumulate to a level such that local populations could completely lose sexuality and appear as asexual (imperfect) species.

The gibberellin (GA) biosynthetic pathway includes four apparent cytochrome P450-mediated steps that convert kaurene to 7 alpha-hydroxykaurenoic acid. One of these reactions, the hydroxylation of kaurenoic acid to 7 alpha-hydroxykaurenoic acid, is mediated by kaurenoic acid hydroxylase. This reaction can be catalyzed in vitro by microsomal preparations from the fungus Gibberella fujikuroi (Saw.) Wr. and monitored by HPLC. Cultures grown in the presence of 84 microM AMO-1618 (an inhibitor of kaurene synthesis) had reduced levels of GA3 in fungal filtrates and decreased cell-free kaurenoic acid hydroxylase activity. However, the level of hydroxylase activity from AMO-1618-treated cultures could be induced several-fold by growing cultures in the presence of 350 microM kaurene. Since transcripts related to GA biosynthesis might be decreased in AMO-1618-treated cultures, a subtractive hybridization procedure was used to enrich cDNA fragments corresponding to messages that are more abundant in untreated than treated cultures. A fungal cDNA library was screened with the subtraction products and a clone was isolated that corresponds to two down-regulated transcripts in AMO-1618-treated cultures. This cDNA does not encode a cytochrome P450 but may be associated with GA biosynthesis.

We constructed a recombination-based map of the fungal plant pathogen Gibberella fujikuroi mating population A (asexual stage Fusarium moniliforme). The map is based on the segregation of 142 restriction fragment length polymorphism (RFLP) markers, two auxotrophic genes (arg1, nic1), mating type (matA+/matA-), female sterility (ste1), spore-killer (Sk), and a gene governing the production of the mycotoxin fumonisin B1 (fum1) among 121 random ascospore progeny from a single cross. We identified 12 linkage groups corresponding to the 12 chromosome-sized DNAs previously observed in contour-clamped homogeneous electric field (CHEF) gels. Linkage groups and chromosomes were correlated via Southern blots between appropriate RFLP markers and the CHEF gels. Eleven of the 12 chromosomes are meiotically stable, but the 12th (and smallest) is subject to deletions in 3% (4/121) of the progeny. Positive chiasma interference occurred on five of the 12 chromosomes, and nine of the 12 chromosomes averaged more than one crossover per chromosome. The average kb/cM ratio in this cross is approximately 32.