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Research has been conducted on deoxynivalenol-producing Fusarium culmorum stains. The genetic relationship between the strains have been investigated via the use of analysis of gene and intergenetic region, and the results are reported.

The nuclear ribosomal DNA of the entomopathogenic fungus Beauveria brongniartii is polymorphic in terms of both restriction site and length. Insertions of 350-450 bp long, identified as group-I introns, were detected in the 28s rDNA. A panel of 47 strains of B. brongniartii,two B. bassiana and one Metarhizium anisopliae of various geographical and biological origins were found to contain 14 variant forms of intron differing in size and restriction pattern, at four different positions. Twelve types of ribosomal large subunit were defined on the basis of variant distribution and compared with strain clustering based on internal transcribed spacers analysis. There was a correlation between the characteristic introns and isolates collected from the sugar cane pest Hoplochelus marginalis. Primers for polymerase chain reaction amplification were chosen from these variants, and used to develop a specific method for detecting strains pathogenic towards Hoplochelus. [ABSTRACT FROM AUTHOR]

While the extent and impact of horizontal transfers in prokaryotes are widely acknowledged, their importance to the eukaryotic kingdom is unclear and thought by many to be anecdotal. Here we report multiple recent transfers of a huge genomic island between Penicillium spp. found in the food environment. Sequencing of the two leading filamentous fungi used in cheese making, P. roqueforti and P. camemberti, and comparison with the penicillin producer P. rubens reveals a 575 kb long genomic island in P. roqueforti--called Wallaby--present as identical fragments at non-homologous loci in P. camemberti and P. rubens. Wallaby is detected in Penicillium collections exclusively in strains from food environments. Wallaby encompasses about 250 predicted genes, some of which are probably involved in competition with microorganisms. The occurrence of multiple recent eukaryotic transfers in the food environment provides strong evidence for the importance of this understudied and probably underestimated phenomenon in eukaryotes.

Molecular microbial ecology has revealed remarkable biodiversity - prokaryotic and eukaryotic - in numerous soil environments. However, no culture-independent surveys of the termitosphere exists, although termites dominate tropical rainforests. Here, we focused on soil feeders, building nests with their soil-born faeces, enriched with clay-organic complexes, thus contributing to the improvement of soil fertility. In order to assess the fungal community composition of these termitaries compared with soils not foraged by termites, samples of the two types were collected in the Lopé rainforest, Gabon, and processed for generation of fungal internal transcribed spacer (ITS) clone libraries. Although primers were universal, most of the recovered sequences represented Ascomycete that were previously uncharacterized and the proportions of which reached 72.5% in soils and 80% in termitaries. Their affiliation with identified fungi was analysed in performing a phylogenetic tree based on 5.8S rDNA. Furthermore, the ascomycete communities of soil-feeding termitaries and soils shared only 6.3% of sequences. This discrepancy of composition between soil and nest may result from the building behaviour of termites, as the organic matter in the nest is chemically modified, and some vacant ecological microniches are available for more specialized fungi.

Calcineurin phosphatase and cyclophilin A are cellular components involved in fungal morphogenesis and virulence. Their roles were investigated in the phytopathogenic fungus Botrytis cinerea using gene inactivation, drug inhibition and cDNA macroarrays approaches. First, the BCP1 gene coding for cyclophilin A was identified and inactivated by homologous recombination. The bcp1Delta null mutant obtained was still able to develop infection structures but was altered in symptom development on bean and tomato leaves. Opposite to this, calcineurin inhibition using cyclosporin A (CsA) modified hyphal morphology and prevented infection structure formation. CsA drug pattern signature on macroarrays allowed the identification of 18 calcineurin-dependent (CND) genes among 2839 B. cinerea genes. Among the co-regulated CND genes, three were shown to be organized as a physical cluster that could be involved in secondary metabolism. The signature of BCP1 inactivation on macroarrays allowed the identification of only three BCP1 cyclophilin-dependent (CPD) genes that were different from CND genes. Finally, no CsA drug pattern signature was observed in the bcp1Delta null mutant which provided a molecular target validation of the drug.

A purified glycoprotein from Botrytis cinerea (strain T4), identified as endopolygalacturonase 1 (T4BcPG1) by mass spectrometry analysis, has been shown to activate defense reactions in grapevine (Vitis vinifera cv. Gamay). These reactions include calcium influx, production of active oxygen species, activation of two mitogen-activated protein kinases, defense gene transcript accumulation, and phytoalexin production. Most of these defense reactions were also activated in grapevine in response to purified oligogalacturonides (OGA) with a degree of polymerization of 9 to 20. In vivo, these active OGA might be a part of the released products resulting from endopolygalacturonase activity on plant cell walls. Nevertheless, the intensity and kinetics of events triggered by OGA were very different when compared with T4BcPG1 effects. Moreover, chemical treatments of T4BcPG1 and desensitization assays have allowed us to discriminate enzymatic and elicitor activities, indicating that elicitor activity was not due to released oligogalacturonides. Thus, BcPG1 should be considered as both an avirulence and a virulence factor. The role of the secreted BcPG1 in the pathogenicity of Botrytis cinerea is discussed.

The Botrytis cinerea homolog (Bc-hch) of Nc-het-c and Pa-hch (vegetative incompatibility loci of Neurospora crassa and Podospora anserina respectively) was cloned and sequenced. The gene structure of Bc-hch is very close to those of Nc-het-c and Pa-hch. A PCR-RFLP approach on a 1171 bp fragment was used to screen polymorphism at this locus among 117 natural isolates of B. cinerea. Restriction patterns by the restriction enzyme HhaI fell into two allelic types. Moreover, haplotypes at the Bc-hch strictly corresponded to the resistance phenotypes to fenhexamid, a novel Botryticide. The use of Bc-hch as a population marker thus reveals a new structuring of B. cinerea natural populations into two groups (I and II). This result was confirmed by genic differentiation tests performed with five other markers on a sample of 132 B. cinerea isolates from the French region of Champagne.

Telomeric DNA was isolated from the phytopathogenic fungus Botrytis cinerea by PCR using only the oligonucleotide primer (CCCTAA)4. As with other filamentous fungi, B. cinerea has a short TTAGGG telomeric repeat. Telomere-linked restriction fragment length polymorphism (RFLP) was found in strains of B. cinerea isolated from different host plants collected from different regions at different periods. Almost every strain had a specific RFLP pattern, including those collected from the same plant one month apart. Thus, this marker appears to be an excellent tool to show the great polymorphism of B. cinerea strains by fingerprinting. The Southern blots of some strains of B. cinerea showed one band which was much more intense than the others, suggesting that the majority of telomere-associated sequences have the same sequence.

The nitrate reductase (niaD) gene was isolated from the phytopathogenic ascomycete Botrytis cinerea using a probe obtained by a polymerase chain reaction (PCR) with degenerate oligonucleotides corresponding to domains conserved among three fungal nitrate reductases. The B. cinerea niaD gene encodes a predicted protein of 907 amino acids and contains no intron. Nitrate reductase-deficient mutants of B. cinerea have been isolated. One of them was transformed with the niaD genes of Fusarium oxysporum f.sp. melonis and B. cinerea. The transformation was always ectopic when the donor DNA originated from F. oxysporum, but there was 80% gene replacement when the donor DNA originated from B. cinerea.

The nuclear ribosomal DNA of the entomopathogenic fungus Beauveria brongniartii is polymorphic in terms of both restriction site and length. Insertions of 350-450 bp long, identified as group-I introns, were detected in the 28s rDNA. A panel of 47 strains of B. brongniartii, two B. bassiana and one Metarhizium anisopliae of various geographical and biological origins were found to contain 14 variant forms of intron differing in size and restriction pattern, at four different positions. Twelve types of ribosomal large subunit were defined on the basis of variant distribution and compared with strain clustering based on internal transcribed spacers analysis. There was a correlation between the characteristic introns and isolates collected from the sugar cane pest Hoplochelus marginalis. Primers for polymerase chain reaction amplification were chosen from these variants, and used to develop a specific method for detecting strains pathogenic towards Hoplochelus.

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.

Recombinant clones from a cDNA library of an Aphanocladium album chitinase-overproducing mutant strain were isolated by screening with antiserum against a 39 kDa chitinase purified from this hyperparasitic fungus. Analysis of the isolated positive clones indicated that most of them carried the same cDNA. A cDNA from this group was used as a hybridization probe to isolate an 8 kb DNA fragment from a genomic library of the wild-type strain. The chitinase 1 gene was mapped to this fragment by two independent approaches. Its partial DNA sequence was in perfect agreement with an amino-terminal peptide sequence obtained by sequencing 23 amino acids of the 39 kDa chitinase. Its transfer in Fusarium oxysporum resulted in a transformant producing both a protein of about 39 kDa that cross-reacted with the chitinase antiserum and a chitinase activity that was inhibited by the same antiserum. Northern blot analysis indicates that the cloned chitinase gene was subject to catabolite repression and appeared inducible by chitin.

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.

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.

Whereas each of the two complementary mating types, O and E, of Paramecium tetraulrelia normally shows cytoplasmic inheritance, an abnormal heredity of mating type was observed in the progeny of crosses between two stocks of different geographical origin of Paramecium tetraurelia (stock 51 and stock 32). The modified pattern of mating-type inheritance was shown to result from the interaction of the two wild-type alleles at the locus mtD (mtD51 and mtD32), leading to a new differentiated state O*, different from the normal O and E states observed in both stock 51 and stock 32 cells. The genetic analysis of O* clones showed that the O* phenotype involves both a new heritable cytoplasmic state and possibly a nuclear change which can be transmitted through conjugation and segregates in a Mendelian fashion. All the data can be interpreted if the assumption is made that mating-type determination is achieved only by the commitment or noncommitment to the expression of mating-type E , and that this commitment may simply reflect the activation or nonactivation of the locus mtD, under the influence of one or two "cytoplasmic factors" including the product of the gene mtD itself.