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Thirty deoxynivalenol-producing F. culmorum strains, isolated from wheat grains, were incubated in vitro and analyzed for trichothecene production. Seventeen strains produced more than 1 ppm of deoxynivalenol and acetyldeoxynivalenol and were considered high-deoxynivalenol-producing strains, whereas 13 F. culmorum strains produced less than 0.07 ppm of trichothecenes and were considered low-deoxynivalenol-producing strains. For all strains, a 550-base portion of the trichodiene synthase gene (tri5) was amplified and sequenced. According to the tri5 data, the F. culmorum strains tested clustered into two groups that correlated with in vitro deoxynivalenol production. For three high-producing and three low-producing F. culmorum strains, the tri5-tri6 intergenic region was then sequenced, which confirmed the two separate clusters within the F. culmorum strains. According to the tri5-tri6 sequence data, specific PCR primers were designed to allow differentiation of high-producing from low-producing F. culmorum strains.

A worldwide collection, of 76 F. oxysporum f.sp. elaeidis isolates (Foe), and of 21 F. oxysporum isolates from the soil of several palm grove was analysed by RFLP. As a probe, we used a random DNA fragment (probe 46) from a genomic library of a Foe isolate. This probe contains two different types of sequence, one being repeated and dispersed in the genome "Palm", the other being a single-copy sequence. All F. oxysporum isolates from the palm-grove soils were non-pathogenic to oil palm. They all had a simple restriction pattern with one band homologous to the single-copy sequence of probe 46. All Foe isolates were pathogenic to oil palm and they all had complex patterns due to hybridization with "Palm". This repetitive sequence reveals that Foe isolates are distinct from the other F. oxysporum palm-grove soils isolates. The sequence can reliably discriminate pathogenic from non-pathogenic oil palm isolates. Based on DNA fingerprint similarities, Foe populations were divided into ten groups consisting of isolates with the same geographic origin. Isolates from Brazil and Ecuador were an exception to that rule as they had the same restriction pattern as a few isolates from the Ivory Coast, suggesting they may originated from Africa.

The Fusarium oxysporum gene nia, encoding nitrate reductase (NR), was isolated from a cosmid library by direct complementation of an F. oxysporum nia- mutant. The gene specifies a protein of 905 amino acids and contains a 57-bp intron. Comparison of the deduced aa sequence with NR of other fungi revealed a high degree of similarity and conservation in the intron position. The cloned nia made it possible to develop the first homologous transformation system for this fungus. Transformation frequencies of up to 600 transformants per microgram of DNA were achieved. Gene replacement, single-copy homologous integrations and integrations at non-homologous sites were observed. Direct comparison between plasmids and cosmids carrying the same gene showed a higher frequency of targeted transformation using cosmid vectors. Gene replacement events were observed in about 50% of the transformants analysed with each type of vector used. This high frequency of substitution offers new applications for the transformation system in F. oxysporum.

We report here the isolation of Foret1, a repeated DNA sequence cloned from the fungal plant pathogen Fusarium oxysporum. This clone exhibits a high degree of sequence similarity with the retroviral pol genes. Sequences homologous to protease, reverse transcriptase, ribonuclease H are found in that order. The overall structure is homologous to the 'gypsy' class of LTR-retrotransposons. Its similarity to elements present in widely different organisms may result from its horizontal transmission in recent evolutionary time.

We report here the discovery of a family of transposable elements, which we refer to as Fot1 elements, in the fungal plant pathogen Fusarium oxysporum. The first element was identified as an insertion in the gene encoding nitrate reductase. It is 1928 bp long, has 44 bp inverted terminal repeats, contains a large open reading frame and is flanked by a 2 bp (TA) target site duplication. This element shares significant structural similarities with a class of transposons that includes Tc1 from Caenorhabditis elegans and therefore represents a new class of transposable elements in fungi.

We have isolated strains of Fusarium oxysporum carrying mutations conferring a phenotype characteristic of a loss of function in the regulatory gene of nitrate assimilation (nirA in Aspergillus nidulans, nit-4 in Neurospora crassa). One of these nir- mutants was successfully transformed with a plasmid containing the nirA gene of A. nidulans. The nitrate reductase of the transformants is still inducible, although the maximum activity is lower than in the wild type. Single and multiple integration events were found, as well as a strict correlation between the presence of the nirA gene and the Nir+ phenotype of the F. oxysporum transformants. We also investigated how the A. nidulans structural gene (niaD) is regulated in F. oxysporum. Enzyme assays and Northern experiments show that the niaD gene is subject to nitrate induction and that it responds to nitrogen metabolite repression in a F. oxysporum genetic background. This indicates that both the mechanisms of specific induction, mediated by a gene product isofunctional to nirA, and nitrogen metabolite repression, presumably mediated by a gene product isofunctional to the homologous gene of A. nidulans, are operative in F. oxysporum.

Fifty-two strains from eight species of Fusarium were analyzed by rapid rRNA sequencing. Two highly variable stretches (138 and 214 nucleotides) of the 5' end of the 28S-like rRNA molecule were sequenced. Such stretches permit evaluation of the divergence between closely related species and even between varieties within a species. The phylogenetic tree computed from the number of nucleotide differences shows seven Fusarium species to be more closely related to one another than the eighth species, F. nivale, is to them. On the basis of these data, we discuss both the phylogenetic value of taxonomical criteria and the impact of our findings on the demarcation of the genus Fusarium. We conclude that this method is suitable for establishing a precise phylogeny between closely related species within a genus.

An heterologous transformation system for the phytopathogenic fungus Fusarium oxysporum has been developed based on the use of the Aspergillus nidulans nitrate reductase gene (niaD). F. oxysporum nia- mutants were easily selected by chlorate resistance. The A. nidulans niaD gene was isolated from a gene library by complementation of an A. nidulans niaD mutant. The cloned gene is capable of transforming F. oxysporum nia- mutants at a frequency of up to ten transformants per microgram of DNA. Southern analysis of the DNA of the F. oxysporum transformants showed that transformation resulted in integration of one or more copies of the vector DNA into the genome.