To assess the potential for mating in several Fusarium species with no known sexual stage, we developed degenerate and semidegenerate oligonucleotide primers to identify conserved mating type (MAT) sequences in these fungi. The putative ␣ and high-mobility-group (HMG) box sequences from Fusarium avenaceum, F. culmorum, F. poae, and F. semitectum were compared to similar sequences that were described previously for other members of the genus. The DNA sequences of the regions flanking the amplified MAT regions were obtained by inverse PCR. These data were used to develop diagnostic primers suitable for the clear amplification of conserved mating type sequences from any member of the genus Fusarium. By using these diagnostic primers, we identified mating types of 122 strains belonging to 22 species of Fusarium. The ␣ box and the HMG box from the mating type genes are transcribed in F. avenaceum, F. culmorum, F. poae, and F. semitectum. The novelty of the PCR-based mating type identification system that we developed is that this method can be used on a wide range of Fusarium species, which have proven or expected teleomorphs in different ascomycetous genera, including Calonectria, Gibberella, and Nectria.The genus Fusarium contains filamentous ascomycete fungi with a worldwide distribution. Fusarium species can parasitize cultivated plants (1) and/or produce mycotoxins that pose serious hazards to human and animal health (9, 18). Species of Fusarium can grow successfully on a variety of substrates, can tolerate diverse environmental conditions, and have high levels of intraspecific genetic and genotypic diversity (for examples, see references 8, 12, 17, and 26). Neither the origins of this diversity nor the mechanisms that maintain it are well understood.Meiotic recombination can generate and maintain genotypic variation and result in the reassortment of genes that govern traits such as virulence or toxin production (7). The sexual spores (ascospores) produced by some Fusarium species also may function as infectious propagules (11,19). Although several Fusarium species have a known sexual cycle, i.e., they mate in either a homothallic or heterothallic manner followed by subsequent meiosis and the production of ascospores, important pathogenic species, including Fusarium avenaceum, Fusarium cerealis, Fusarium culmorum, Fusarium equiseti, Fusarium poae, and Fusarium sporotrichioides, have no known sexual stage.Assessing the potential for mating by toxigenic strains of Fusarium would increase our understanding of the genetic mechanisms that maintain intraspecific diversity and biological and evolutionary species integrity. The frequency of sexual reproduction is also an important parameter for the design of strategies to control plant pathogens, since these strategies are often different for clonally and sexually reproducing organisms. High levels of race-specific resistance can be developed in plant cultivars against clonally reproducing organisms, whereas horizontal resistance could be more effective against pathogen...

A homologue of the adenylyl cyclase (AC) gene of Neurospora crassa, named Fpacy1 was cloned from the genomic library of Fusarium proliferatum ITEM 2287 by screening the library with a DNA fragment amplified by using PCR primers designed from conserved sequences of the catalytic domain of AC genes from other fungi. The deduced FPACY1 protein had 53-77% identity with the AC proteins of other fungi. DeltaFpacy1 mutants obtained by targeted gene disruption showed retarded vegetative growth, increased conidiation and delayed conidial germination. Colonization capability of the mutants, assessed on maize seedlings and tomato fruits also was adversely affected. In sexual crosses the AC mutants retained full male fertility, but their female fertility decreased significantly. Disruption of Fpacy1 abolished vegetative self-incompatibility, suggesting that the AC gene is involved in multiple developmental processes related to vegetative growth, as well as sexual and parasexual events. The elevated thermo- and H(2)O(2)-tolerance of the DeltaFpacy1 mutants was coupled to an increased sensitivity towards Cd and Cu, indicating that the cAMP signaling pathway may have both negative and positive regulatory roles on the stress response mechanisms of fungal cells. When grown under nitrogen limitation conditions, the DeltaFpacy1 mutants produced an average of approximately 274 microg g(-1) bikaverin, whereas only traces of this metabolite was detected in the wild type. This finding provides further evidence of the role of the cAMP-PKA pathway in regulating bikaverin production.

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