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De 88 coletas em 13 áreas de Mata Atlântica em Alagoas, Paraíba, Pernambuco, Rio Grande do Norte e Sergipe entre setembro/2000 e junho/2002, nove famílias, 65 gêneros e 134 espécies de Aphyllophorales foram identificados. Seis espécies são novas para a ciência, 17 novos registros para o Brasil e 43 para o Nordeste. Espécies coletadas em Alagoas, Rio Grande do Norte e Sergipe representam novas ocorrências para esses estados. Polyporaceae e Schizophyllaceae apresentaram, respectivamente, maior e menor número de espécies e gêneros. Ganodermataceae e Hydnaceae não foram encontradas no Rio Grande do Norte e em Sergipe, respectivamente. As demais famílias estiveram presentes em todos os estados pesquisados. Nenhuma espécie ocorreu em todas as áreas de estudo, mas cinco ocorreram em 12 das 13 localidades e 14 estiveram presentes em todos os estados. Pernambuco apresentou maior diversidade específica (88) e maior número de espécies exclusivas (18), enquanto a RPPN Fazenda São Pedro (AL) foi a área com maior diversidade específica (57) e maior número de espécies exclusivas (14). Aphyllophorales foram coletados com maior freqüência em novembro/2001, janeiro e março/2002; mostraram tendência aos basidiomas se encontrarem muito agregados; preferência por ambientes com exposição intermediária à luz e por substratos degradados a muito degradados. As espécies tenderam a ocupar substratos mais degradados em ambiente menos expostos à luz. O número de espécies coletadas e a similaridade entre as áreas foram afetados positivamente pelo grau de conservação. A similaridade também foi afetada positivamente pela extensão das trilhas e negativamente pela distância entre as áreas

[EN] Descriptions 01 eleven species of fungi found recently in Spain are presented here. Among the most interesting are the following ones: Hymenogaster decoros and Urnula crateriurn, which represent the second record in the country; Peziza gerardii, Picoa lefebvrei. Trichophaea woolopeia, Hymenogaster rnuticus and Melanogaster tuberíforrnis, as the third record in Spain. New data on the chorology and ecology 01 the rest of species are added., [IT] Sono descrifte 11 specie di funghi appartenenti agli Ascomiceli o Basidiomiceti trovate recentemente in Spagna. I funghi descritti nel presente lavoro sono stati raccolti nella provincia di Valladolid. Si tratta di specie per la maggior parte rare; infatti per Hymenogaster decorosus e Urnula crateriurn si tratta della seconda segnalazione in territorio spagnolo, per Peziza gerardii, Picoa lefebvrei, Trichophaea woolopeia, Hyrnenogaster rnuticus e Melanogaster tuberíforrnis del terzo ritovamento. Di ogni specie e con maggiore dettaglio nel caso di specie rare, vengono descritte le caratteristiche macro-e microscopiche con immagini delle spore elfettuate al SEM; viene inoltre brevemente descritto l'habitat di ritrovamento.

[EN] Buglossoporus quercinus is mentioned for the first time in Spain. Macro-and microscopic features are described and compared with other species of the genus. Notes on its taxonomy, ecology and chorology are also added., [ES] Se menciona Buglossoporus quercinus por primera vez en España y se incluye una descripción completa del material estudiado, comparando con especies afines. Tambien se añaden datos sobre su taxonomía, ecología y corología.

[EN]Lycoperdon collections recorded from acidic, dry, mossy habitats on rocks or on sand in Fennoscandia and the Northeastern of Spain are here described. These collections are close to L. ericaeum but differs in its protruding ostiole and its subelastic capil1itium., [ES]Se han recolectado basidiomas de Lycoperdon entre musgos en terrenos acidófilos or en zonas arenosas en Fennoscandia y en el noreste de España. Estas recolecciones presentan caracteres similares a los de L. ericaeum, pero difieren en su ostíolo prominente y el capilicio subelàstico.

Rhizopogon abietis A.H. Smith and R. ochracorubens A. H. Smith are recorded for the first time for Finland. Both species should be considered as threatened in Europe. New collections of R. roseolus (Corda) Th. Fr. Have been studied.

This article 'Variability of rice koji for enzyme activities using Basidiomycetes' appeared in the International Rice Research Notes series, created by the International Rice Research Institute (IRRI) to expedite communication among scientists concerned with the development of improved technology for rice and rice-based systems. The series is a mechanism to help scientists keep each other informed of current rice research findings. The concise scientific notes are meant to encourage rice scientists to communicate with one another to obtain details on the research reported.

[EN] The first record of Boletus poikilochromus in Spain ispublished here.A complete description of the studied material, so as comments on its relatioships with close species, taxonomy, ecology and chorology are also added., [FR] Boletus poikilochromus est mentionnée pour la premiére fois en Espagne, avec une description complete du matériel espagnol et commetaires sur les espéces les plus proches. Données sur la taxonomie, ecologie et distribution geograpfique de cette espece sont ajoutées., [ES] Boletus poikiloch romus se menciona por primera vez en España. Se aporta una descripción completa del material español estudiado, con comentarios sobre otras especies próximas y se dan notas sobre su taxonomía, ecología y corología.

[EN] The first record of Boletus poikilochromus in Spain ispublished here.A complete description of the studied material, so as comments on its relatioships with close species, taxonomy, ecology and chorology are also added. [FR] Boletus poikilochromus est mentionnée pour la premiére fois en Espagne, avec une description complete du matériel espagnol et commetaires sur les espéces les plus proches. Données sur la taxonomie, ecologie et distribution geograpfique de cette espece sont ajoutées. [ES] Boletus poikiloch romus se menciona por primera vez en España. Se aporta una descripción completa del material español estudiado, con comentarios sobre otras especies próximas y se dan notas sobre su taxonomía, ecología y corología.

Spore germination in Rhizopogon abietis, R. luteolus, R. roseolus and R. villosulus was induced in the presence of Rhodotorula glutinis and activated charcoal, in agar medium (N6:5). In one R. roseolus sample, 51% of spores germinated within 35 days, allowing observation of the course of spore germination and the different developmental patterns of homokaryotic mycelia. In these plates, spores showed two times of germination. The spores tha germinated early produced an apical germ tube. Later other spores germinated in proximity to young mycelium, by forming a germ vesicle. One of the hyphal growth patterns obtained (interruption-swelling-ramification)is similar to that reported for other fungi. With this technique for inducing spore germination, it is possible to obtain enough monosporic cultures to perform mating tests.

Ligninolytic Basidiomycetes have been reported to produce a wide variety of chloroaromatic compounds as secondary metabolites, which are structurally similar to environmental pollutants. Among these are chlorinated hydroquinone metabolites (CHM), such as 2-chloro-1,4-dimethoxybenzene (2Cl-14DMB), 2,6-dichloro-1,4-dimethoxybenzene (26DCl-14DMB), tetrachloro-1,4-dimethoxybenzene and tetrachloro-4-methoxyphenol, which are synthesized by 11 genera of Basidiomycetes.The biosynthesis of these chlorinated metabolites is not just a biological accident. An important physiological role of chloroaromatics, including CHM, is their antibiotic function protecting fungi from other organisms. However, since many ligninolytic white rot fungi synthesize CHM, these metabolites might also be involved in lignin degradation.The lignin degrading system of white rot fungi is composed of peroxidases, oxidases and secondary metabolites, such as veratryl alcohol (VA) and anisyl alcohol (AA). VA is an important fungal metabolite in the catalytic cycle of lignin peroxidase (LiP), one of the main ligninolytic enzymes of white rot fungi. Three functions of VA have been described: (1) VA is a redox mediator for LiP-catalyzed oxidations via intermediate formation of a cation radical (2) VA is a cofactor necessary to close the catalytic cycle of LiP (3) VA prevents the inactivation of LiP by H 2 O 2 .1,4-dimethoxybenzene (14DMB) can replace VA as a cofactor and/or redox mediator in the catalytic cycle of LiP; however, 14DMB is not a fungal metabolite. Nevertheless, 14DMB is structurally related to CHMs, which are fungal metabolites. This structural relationship between 14DMB and CHM implies that CHM may also replace VA in the catalytic cycle of LiP.The objectives of this PhD study were to determine the extent of biosynthesis of CHM among white rot fungi and to elucidate their possible physiological role in lignin degradation.For this purpose, 92 ligninolytic Basidiomycetes were screened for their ability to produce the CHMs tetrachloro-4-methoxyphenol (drosophilin A, DA) and tetrachloro-1,4-dimethoxybenzene (drosophilin A methyl ether, DAME). Five fungal strains (two strains of Bjerkandera adusta , Agaricus arvensis, Peniophora pseudopini and Phellinus fastuosus ) produced these metabolites in detectable amounts. Furthermore, the CHM production coincided with the expression of ligninolytic activity.P. fastuosus ATCC26.125 was used for further studies to elucidate if the ligninolytic enzymes from this fungus were able to use DA and DAME as substrates. The dominant ligninolytic peroxidase produced by P. fastuosus was a manganese independent peroxidase (MIP), which efficiently oxidizes phenols but not veratryl alcohol nor Mn(II). Purified MIP isozymes were able to oxidize chlorophenolic compounds, including the natural DA and the anthropogenic pentachlorophenol. MIP could not oxidize DAME or other (chlorinated) 1,4-dimethoxybenzenes.The CHMs, 2Cl-14DMB, 26DCl-14DMB, DAME and DA were examined as substrates for LiP, which is an important ligninolytic enzyme from Bjerkandera . Our results indicated that 14DMB, 2Cl-14DMB, and DA were substrates for LiP; however, 26DCl-14DMB and DAME were not. According to these results, we concluded that 26DCl-14DMB and DAME do not have a physiological role in lignin degradation, but primarily function as antibiotic agents.2Cl-14DMB, which is a LiP substrate, is synthesized by several fungi including the well studied fungus Bjerkandera sp. BOS55. We showed that 2Cl-14DMB, like 14DMB, can replace VA as a cofactor and redox mediator in the catalytic cycle of LiP.We demonstrated that LiP oxidized 2Cl-14DMB to 2-chloro-1,4-benzoquinone and two different dimers. Furthermore, EPR results showed the formation of a long-lived 2Cl-14DMB cation radical. The dimer formation and EPR results suggest that the 2Cl-14DMB cation radical is stable enough enabling it to function as a diffussible redox mediator, which can oxidize substrates at a distance of the enzyme. Our results indeed showed that 2Cl-14DMB acts as a redox mediator in the LiP-catalyzed oxidation of the polymeric dye Poly R-478, 4-methoxymandelic acid and oxalate.We examined whether 2Cl-14DMB could also replace VA as a cofactor in the LiP-catalyzed oxidation of AA. 2Cl-14DMB enormously stimulated the oxidation of AA, compared to VA. Although our results supported the theory that 2Cl-14DMB closed the catalytic cycle of LiP, we could not directly explain the high molar ratio of p-anisaldehyde formed to 2Cl-14DMB consumed. This high molar ratio indicated that the 2Cl-14DMB cation radical must be recycled back to 2Cl-14DMB. In support of this observation, AA was found to reduce the 2Cl-14DMB cation radical back to 2Cl-14DMB.Furthermore, the presence of AA to reduce the 2Cl-14DMB cation radical prevents LiP from H 2 O 2 -inactivation. According to our observations, a mechanism for the behavior of 2Cl-14DMB in the catalytic cycle of LiP and its role in AA oxidation was proposed. In the absence of AA or at low 2Cl-14DMB concentrations, a 2Cl-14DMB cation radical - LiP compound II complex is formed which ultimately results in LiP inactivation. Addition of enough reducing substrate, AA or excess 2Cl-14DMB, reduces the 2Cl-14DMB cation radical - LiP II complex releasing LiP compound II and thereby preventing the inactivation of LiP.In conclusion, CHMs were observed to be substrates for ligninolytic enzymes suggesting purposeful roles of these chlorinated metabolites. 2Cl-14DMB was identified as a stable diffussible redox mediator which could have potential application in the improvement of biobleaching and biopulping.

Ligninolytic Basidiomycetes have been reported to produce a wide variety of chloroaromatic compounds as secondary metabolites, which are structurally similar to environmental pollutants. Among these are chlorinated hydroquinone metabolites (CHM), such as 2-chloro-1,4-dimethoxybenzene (2Cl-14DMB), 2,6-dichloro-1,4-dimethoxybenzene (26DCl-14DMB), tetrachloro-1,4-dimethoxybenzene and tetrachloro-4-methoxyphenol, which are synthesized by 11 genera of Basidiomycetes.The biosynthesis of these chlorinated metabolites is not just a biological accident. An important physiological role of chloroaromatics, including CHM, is their antibiotic function protecting fungi from other organisms. However, since many ligninolytic white rot fungi synthesize CHM, these metabolites might also be involved in lignin degradation.The lignin degrading system of white rot fungi is composed of peroxidases, oxidases and secondary metabolites, such as veratryl alcohol (VA) and anisyl alcohol (AA). VA is an important fungal metabolite in the catalytic cycle of lignin peroxidase (LiP), one of the main ligninolytic enzymes of white rot fungi. Three functions of VA have been described: (1) VA is a redox mediator for LiP-catalyzed oxidations via intermediate formation of a cation radical (2) VA is a cofactor necessary to close the catalytic cycle of LiP (3) VA prevents the inactivation of LiP by H 2 O 2 .1,4-dimethoxybenzene (14DMB) can replace VA as a cofactor and/or redox mediator in the catalytic cycle of LiP; however, 14DMB is not a fungal metabolite. Nevertheless, 14DMB is structurally related to CHMs, which are fungal metabolites. This structural relationship between 14DMB and CHM implies that CHM may also replace VA in the catalytic cycle of LiP.The objectives of this PhD study were to determine the extent of biosynthesis of CHM among white rot fungi and to elucidate their possible physiological role in lignin degradation.For this purpose, 92 ligninolytic Basidiomycetes were screened for their ability to produce the CHMs tetrachloro-4-methoxyphenol (drosophilin A, DA) and tetrachloro-1,4-dimethoxybenzene (drosophilin A methyl ether, DAME). Five fungal strains (two strains of Bjerkandera adusta , Agaricus arvensis, Peniophora pseudopini and Phellinus fastuosus ) produced these metabolites in detectable amounts. Furthermore, the CHM production coincided with the expression of ligninolytic activity.P. fastuosus ATCC26.125 was used for further studies to elucidate if the ligninolytic enzymes from this fungus were able to use DA and DAME as substrates. The dominant ligninolytic peroxidase produced by P. fastuosus was a manganese independent peroxidase (MIP), which efficiently oxidizes phenols but not veratryl alcohol nor Mn(II). Purified MIP isozymes were able to oxidize chlorophenolic compounds, including the natural DA and the anthropogenic pentachlorophenol. MIP could not oxidize DAME or other (chlorinated) 1,4-dimethoxybenzenes.The CHMs, 2Cl-14DMB, 26DCl-14DMB, DAME and DA were examined as substrates for LiP, which is an important ligninolytic enzyme from Bjerkandera . Our results indicated that 14DMB, 2Cl-14DMB, and DA were substrates for LiP; however, 26DCl-14DMB and DAME were not. According to these results, we concluded that 26DCl-14DMB and DAME do not have a physiological role in lignin degradation, but primarily function as antibiotic agents.2Cl-14DMB, which is a LiP substrate, is synthesized by several fungi including the well studied fungus Bjerkandera sp. BOS55. We showed that 2Cl-14DMB, like 14DMB, can replace VA as a cofactor and redox mediator in the catalytic cycle of LiP.We demonstrated that LiP oxidized 2Cl-14DMB to 2-chloro-1,4-benzoquinone and two different dimers. Furthermore, EPR results showed the formation of a long-lived 2Cl-14DMB cation radical. The dimer formation and EPR results suggest that the 2Cl-14DMB cation radical is stable enough enabling it to function as a diffussible redox mediator, which can oxidize substrates at a distance of the enzyme. Our results indeed showed that 2Cl-14DMB acts as a redox mediator in the LiP-catalyzed oxidation of the polymeric dye Poly R-478, 4-methoxymandelic acid and oxalate.We examined whether 2Cl-14DMB could also replace VA as a cofactor in the LiP-catalyzed oxidation of AA. 2Cl-14DMB enormously stimulated the oxidation of AA, compared to VA. Although our results supported the theory that 2Cl-14DMB closed the catalytic cycle of LiP, we could not directly explain the high molar ratio of p-anisaldehyde formed to 2Cl-14DMB consumed. This high molar ratio indicated that the 2Cl-14DMB cation radical must be recycled back to 2Cl-14DMB. In support of this observation, AA was found to reduce the 2Cl-14DMB cation radical back to 2Cl-14DMB.Furthermore, the presence of AA to reduce the 2Cl-14DMB cation radical prevents LiP from H 2 O 2 -inactivation. According to our observations, a mechanism for the behavior of 2Cl-14DMB in the catalytic cycle of LiP and its role in AA oxidation was proposed. In the absence of AA or at low 2Cl-14DMB concentrations, a 2Cl-14DMB cation radical - LiP compound II complex is formed which ultimately results in LiP inactivation. Addition of enough reducing substrate, AA or excess 2Cl-14DMB, reduces the 2Cl-14DMB cation radical - LiP II complex releasing LiP compound II and thereby preventing the inactivation of LiP.In conclusion, CHMs were observed to be substrates for ligninolytic enzymes suggesting purposeful roles of these chlorinated metabolites. 2Cl-14DMB was identified as a stable diffussible redox mediator which could have potential application in the improvement of biobleaching and biopulping.

International audience; For about 50 years, scientists have studied the biological activities of Macromycetes on different pathologies. In this article different fungal activities that reduce the effects of risk factors for cardiovascular diseases are reviewed. Among the 17 species tested on animals, 16 demonstrated at least one of the following activities: ability to reduce hypercholesterolemia or to cure dyslipoproteinemia, possibility to decrease arterial hypertension or hyperglycemia, and ability to cure disturbances in platelet aggregation.

The aim of this thesis is studying the genetics of Phaffia and to develop a genetic transformation system for this yeast. The genetic properties of Phaffia were studied on the gene and genome level.As a first step the molecular structure of the Phaffia actin gene was analyzed. Actin genes are highly conserved throughout nature, and as such they have been used for the classification of significantly diverging eukaryotic groups, like (in-)vertebrates, plants and fungi.We anticipated that the analysis of the primary structure of Phaffia actin gene and comparison with the actin genes from fungi, including 2 ascomycetous filamentous fungi, 2 basidiomycetous yeasts and 5 ascomycetous yeasts would provide further phylogenetic information on this yeast.It was found that the Phaffia actin gene encoded a protein consisting of 375 amino acids. In addition 4 (non-coding) intervening sequences were present. Comparison of both the coding DNA sequence and its predicted protein product with their fungal counterparts, revealed that least homology was found with the ascomycetous yeasts, like Saccharomyces cerevisiae and Kluyveromyces lactis . It was also shown, that based on these comparisons Phaffia is closer related to the filamentous ascomycetous fungi Thermomyces lanuginosus and Aspergillus nidulans , whereas most homology was found with the basidiomycetous yeast Filobasidiella neoformans (perfect stage of Cryptococcus neoformans ).In addition to the phylogenetic analysis of the actin exons, the architecture of the introns (splice site consensus sequences, size, position in the gene) was compared. it was shown that the Phaffia introns most resembled that of Filobasidiella neoformans where as least resemblance occurred with the ascomycetous yeasts. This result was in agreement with the actin exon homology studies. Furthermore, the presence of multiple introns in the Phaffia actin gene resembled the situation in the actin genes from F. neoformans and the filamentous fungi, whereas the ascomycetous yeasts only carry one intron in their actin genes.Similar results were obtained by (phylo-)genetic analysis of the five introns containing Phaffia glyceraldehyde-3-phosphate dehydrogenase gene.The genomic organization of the multiple rDNA genes in Phaffia was elucidated. It was found that Phaffia carries the rDNA genes in three clusters, of 12, 14 and 35 copies, on three different chromosomes. In the ascomycetous yeasts and fungi the rDNA is mainly present on one chromosome.The significant differences on the gene and genome level with the ascomycetous yeasts affected the strategy for the development of a transformation system for Phaffia . Whereas several marker gene sequences or sequences for plasmid replication and maintenance can be readily interchanged between most ascomycetous species as a result of high homologies, it was shown that this was not the case for Phaffia . Therefore an almost entirely homologous transformation sytem was developed using plasmids carrying the dominant G418 resistance gene (Km R), driven by either the Phaffia actin or the gpd promoter and a Phaffia ribosomal DNA (rDNA) fragment for homologous integration.It was found that the rDNA clusters could serve as a target for high copy number integration. This integrative transformation system was used to determine the ploidy of Phaffia , strain CBS 6938, by monitoiring chromosomal shifts as a result of multiple integrations. It was found that this strain was haploid.Plasmids carrying the gpd promoter driven Km Rgene transformed Phaffia with significant higher efficiencies than constructs with the actin promoter. Furthermore, the plas-mid copy number and transformation efficiencies of the first were found to be influenced by the presence of the gpd terminator downstream the Km Rgene.It was shown that plasmid amplification occurred independent from selection pressure to an extend that appeared to be negatively related to the effectiveness of expression of the Km Rgene. This observation indicated that the rising metabolic burden, as a consequence of amplification, imposes limits to the number of plasmid copies.The effectiveness, stability, and plasmid amplifying properties of the Phaffia transformation system offer possibilities for the use of recombinant DNA technology in developing industrially attractive Phaffia strains.

Fifty-five collections misidentified and stored in various institutional herbaria, under severa1 Rhizopogon species narnes, are revised. Among these, twelve belong to Ascomycotina and 9 were identified to species level; the other collections, immature fruitbodies wcre impossible to identify with the data available. Forty-two collections are Basidiomycotina and only an immature Scleroderma remain without identification to the species level.

The aim of this thesis is studying the genetics of Phaffia and to develop a genetic transformation system for this yeast. The genetic properties of Phaffia were studied on the gene and genome level.As a first step the molecular structure of the Phaffia actin gene was analyzed. Actin genes are highly conserved throughout nature, and as such they have been used for the classification of significantly diverging eukaryotic groups, like (in-)vertebrates, plants and fungi.We anticipated that the analysis of the primary structure of Phaffia actin gene and comparison with the actin genes from fungi, including 2 ascomycetous filamentous fungi, 2 basidiomycetous yeasts and 5 ascomycetous yeasts would provide further phylogenetic information on this yeast.It was found that the Phaffia actin gene encoded a protein consisting of 375 amino acids. In addition 4 (non-coding) intervening sequences were present. Comparison of both the coding DNA sequence and its predicted protein product with their fungal counterparts, revealed that least homology was found with the ascomycetous yeasts, like Saccharomyces cerevisiae and Kluyveromyces lactis . It was also shown, that based on these comparisons Phaffia is closer related to the filamentous ascomycetous fungi Thermomyces lanuginosus and Aspergillus nidulans , whereas most homology was found with the basidiomycetous yeast Filobasidiella neoformans (perfect stage of Cryptococcus neoformans ).In addition to the phylogenetic analysis of the actin exons, the architecture of the introns (splice site consensus sequences, size, position in the gene) was compared. it was shown that the Phaffia introns most resembled that of Filobasidiella neoformans where as least resemblance occurred with the ascomycetous yeasts. This result was in agreement with the actin exon homology studies. Furthermore, the presence of multiple introns in the Phaffia actin gene resembled the situation in the actin genes from F. neoformans and the filamentous fungi, whereas the ascomycetous yeasts only carry one intron in their actin genes.Similar results were obtained by (phylo-)genetic analysis of the five introns containing Phaffia glyceraldehyde-3-phosphate dehydrogenase gene.The genomic organization of the multiple rDNA genes in Phaffia was elucidated. It was found that Phaffia carries the rDNA genes in three clusters, of 12, 14 and 35 copies, on three different chromosomes. In the ascomycetous yeasts and fungi the rDNA is mainly present on one chromosome.The significant differences on the gene and genome level with the ascomycetous yeasts affected the strategy for the development of a transformation system for Phaffia . Whereas several marker gene sequences or sequences for plasmid replication and maintenance can be readily interchanged between most ascomycetous species as a result of high homologies, it was shown that this was not the case for Phaffia . Therefore an almost entirely homologous transformation sytem was developed using plasmids carrying the dominant G418 resistance gene (Km R), driven by either the Phaffia actin or the gpd promoter and a Phaffia ribosomal DNA (rDNA) fragment for homologous integration.It was found that the rDNA clusters could serve as a target for high copy number integration. This integrative transformation system was used to determine the ploidy of Phaffia , strain CBS 6938, by monitoiring chromosomal shifts as a result of multiple integrations. It was found that this strain was haploid.Plasmids carrying the gpd promoter driven Km Rgene transformed Phaffia with significant higher efficiencies than constructs with the actin promoter. Furthermore, the plas-mid copy number and transformation efficiencies of the first were found to be influenced by the presence of the gpd terminator downstream the Km Rgene.It was shown that plasmid amplification occurred independent from selection pressure to an extend that appeared to be negatively related to the effectiveness of expression of the Km Rgene. This observation indicated that the rising metabolic burden, as a consequence of amplification, imposes limits to the number of plasmid copies.The effectiveness, stability, and plasmid amplifying properties of the Phaffia transformation system offer possibilities for the use of recombinant DNA technology in developing industrially attractive Phaffia strains.

[EN]The possibility of characterizing Rhizopogon by thin-layer chromatography was investigated. Fifty-eight fruitbodies were sampled. The most detailed chromatograms were obtained from ethanol extracts and plates running with n-butanol-water-acetic acid (4:1:1), examined with ultra-violet light and marking out fluorescent spots. This preliminary study showed 17 chromatogram patterns, ten of which were clearly different and characteristic of only one current species (R. evadens, R. occidentalis, R. pachydermus, R. pannous, R. rocabrunae, R. roseolus, R. subsalmonius, R. verii, R. villosulus and R. vinicolor). The chromatogram patterns may be a great aid in differentiating Rhizopogon species., [ES] Se investigó la posibilidad de caracterizar las especies de Rhizopogon mediante cromatografia en capa fina. Se utilizaron 58 muestras de herbario. Los cromatogramas que mostraron mejor resolución fueron los obtenidos a partir de extractes en etanol utilizando n-butanol-agua- àcido acético (4:1:1) como eluente, examinando las placas con luz ultravioleta. Este estudio preliminar mostró 17 modelos cromatogràficos, de los cuales 10 fueron claramente diferentes y característicos para una única especie (R. evadens, R. occidentalis, R. pa chydermus, R. pannosus, R. rocabrunae, R. roseolus , R. subsalmonius, R. verii, R. villosulus y R. vinicolor) . Los modelos cromatogràficos pueden ser pues, de gran ayuda para diferenciar especies de Rhizopogon.

This is the second contribution to the study of lignicolous marine fungi from the coast of Mar del Plata city, Argentina. Twelve species were collected from driftwood, intertidal wood and drift culms found in rocks and sand beaches. Ten species of Ascomycotina are described: Buergenerula spartinae Kohlm. & R. V. Gessner, Chaetosphaeria chaetosa Kohlm., Corollospora maritima Werderm., Halosarpheia trullifera (Kohlm.) E. B. G. Jones, S. T. Moss & Cuomo, H. viscosa (I. Schmidt) Shearer & J. L. Crane ex Kohlm. & Volkm-Kohlm., Halosphaeriopsis mediosetigera (Cribb & J. W. Cribb) T. W. Johnson, Wettsteinina marina (H. Ellis & Everh.) Shoemaker & C. E. Babc., Leptosphaeria oraemaris Linder, Passeriniella obiones (P. Crouan & H. Crouan) K. D. Hyde & Mouzouras, Remispora maritima Linder; one species of Deuteromycotina: Phoma glomerata Wollenw. & Hochapfel and one species of Basidiomycotina: Digitatispora marina Doguet. Seven are new records for Argentina and four of them also for South America.

[EN]A rare and interesting hypogeous fungi, Gautieria trabutii (Chat.) Pat. (Gasteromycetes), recorded for the first time in the Iberian Peninsula, is described and discussed., [ES]Se describe y comenta un raro e interesante hongo hipogeo, Gautieria trabutii (Chat.) Pat. (Gasteromycetes), recolectado por primera vez en la Península Ibérica.

The major structural elements of wood and other vascular tissues are cellulose, hemicellulose and generally 20-30% lignin. Lignin gives the plant strength, it serves as a barrier against microbial attack and it acts as a water impermeable seal across cell walls of the xylem tissue. However, the presence of lignin has practical drawbacks for some of the applications of lignocellulosic materials. First, lignin has to be removed for the production of high quality pulps. Second, lignin reduces the digestibility of lignocellulosic materials. High quality pulps can be produced with chemical methods, however the abundant use of chemicals and energy, and the formation of an enormous waste stream has led scientists to investigate the possibilities of biodelignification. White-rot fungi give the most rapid and extensive degradation and have become subject of intensive research. Results obtained with the model organism Phanerochaete chrysosporium and other strains have revealed that lignin biodegradation is an extracellular, oxidative and non-specific process. This unique biodegradative potential has been considered for broader applications such as waste water treatment and the degradation of xenobiotic compounds. The research described in this thesis concentrates on the function of aryl alcohols in fungal physiology.Aryl alcohols In the physiology of white-rot fungi. White-rot fungi have a versatile machinery of enzymes, including peroxidases and oxidases, which work in harmony with secondary aryl alcohol metabolites to degrade the recalcitrant, aromatic biopolymer lignin. In chapter 2 literature concerning the important physiological roles of aryl (veratryl, anisyl and chlorinated anisyl) alcohols in the ligninolytic enzyme system has been reviewed. Their functions include stabilization of lignin peroxidase, charge-transfer reactions and as substrate for oxidases generating extracellular H 2 O 2 .The experimental research described in this thesis was initiated to evaluate the possibilities of white-rot fungi in the biopulping of hemp stem wood. Sixty-seven basidiomycetes were isolated and screened for high peroxidative activity (chapter 3). Several of the new isolates were promising manganese peroxidase-producing white-rot fungi. Enzyme assays indicated that for the production of H 2 O 2 either extracellular glyoxal or aryl alcohol oxidase were present. In contrast, lignin peroxidase was only detected in P. chrysosporium , despite attempts to induce this enzyme in other strains with oxygen and oxygen/veratryl alcohol additions. A highly significant correlation was found between two ligninolytic indicators: ethene formation fromα-keto-γ- methylthiolbutyric acid and the decolorization of a polymeric dye, Poly R-478. Three of the new isolates had significantly higher Poly R decolorizing activities compared to P. chrysosporium .One of the best Poly R decolorizing strains, Bjerkandera sp. BOS55 was selected for further characterization. A novel enzyme activity (manganese independent peroxidase) was detected in the extracellular fluid of Bjerkandera sp. BOS55 (chapter 4). The purified enzyme could oxidize several compounds like phenol red, 2,6-dimethoxyphenol (DMP), Poly R-478, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and guaiacol with H 2 O 2 as an electron acceptor. In contrast, veratryl alcohol was not a substrate. This enzyme also had the capacity to oxidize DMP in the absence of H 2 O 2 . Bjerkandera sp. BOS55 also produced de novo several aromatic metabolites. Besides veratryl alcohol and veratraldehyde, compounds which are known to be involved in the ligninolytic system of several other white-rot fungi (chapter 2), other metabolites were formed. These included anisaldehyde, 3-chloro-anisaldehyde, 3,5-dichloro-anisaldehyde and small amounts of the corresponding anisyl, 3-chloro-anisyl and 3,5-dichloro-anisyl alcohol (chapters 5 and 6). This was the first report of de novo biosynthesis of simple chlorinated aromatic compounds by a white-rot fungus. These unexpected findings led us investigate the physiological role(s) of the denovo biosynthesized chlorinated anisyl alcohols (chapter 6). All metabolites were produced simultaneously with the extracellular ligninolytic enzymes. The monoand dichlorinated anisyl alcohols appeared to be excellent substrates for the extracellular aryl alcohol oxidases. The formed aldehydes were readily recycled via reduction by washed fungal mycelium, thus creating an extracellular H 2 O 2 production system regulated by intracellular enzymes. Lignin peroxidase does not oxidize the chlorinated anisyl alcohols both in the absence and in the presence of veratryl alcohol. It was therefore concluded that the chlorinated anisyl alcohols are well protected against the fungus's own aggressive ligninolytic enzymes. The relative amounts of veratryl alcohol and the chlorinated anisyl alcohols differ significantly depending on the growth conditions, indicating that the production of veratryl alcohol and the (chlorinated) anisyl metabolites are independently regulated.It was concluded that the chlorinated anisyl metabolites, biosynthesized by the white-rot fungus Bjerkandera sp. BOS55, are purposeful for ecologically significant processes such as lignin degradation. These results made us speculate if a significant biogenesis of chlorinated aromatics by fungi occurs in natural environments (chapter 7). Many common wood- and forest litter-degrading fungi were indeed detected that produced chlorinated anisyl metabolites (CAM). These compounds, which are structurally related to xenobiotic chloroaromatics, were present in the environment and occur at high concentrations of approximately 75 mg CAM kg -1wood or litter. The ubiquity among common fungi to produce large amounts of chlorinated aromatic compounds in the environment leads to the conclusion that these kind of compounds can no longer be considered to originate from anthropogenic sources only.Degradation of aryl alcohols by fungi. In chapter 2 the anabolic and catabolic routes of aryl alcohols by white-rot fungi has been reviewed. These fungi can not use veratryl alcohol as sole source of carbon and energy. However, several bacteria, yeasts and fungi were selectively isolated from paper mill waste water that grew on veratryl alcohol (chapter 8). Penicilliumsimplicissimum was selected for the characterization of the veratryl alcohol degradation route. P. simplicissimum oxidized veratryl alcohol via a NAD(P) +-dependent veratryl alcohol dehydrogenase to veratraldehyde which was further oxidized to veratric acid in a NAD(P) +-dependent reaction. Veratric acid-grown cells contained NAD(P)H-dependent O -demethylase activity for veratrate, vanillate and isovanillate. Ring-cleavage of protocatechuate was by a protocatechuate 3,4-dioxygenase. An interesting aspect of P. simplicissimum is the production of vanillyl alcohol oxidase with covalently bound FAD (chapter 9). The intracellular enzyme was purified 32-fold. SDS-PAGE of the purified enzyme revealed a single fluorescent band of 65 Kda. Gel filtration and sedimentation-velocity experiments indicated that the purified enzyme exists in solution as an octamer, containing 1 molecule flavin/subunit. The covalently bound prosthetic group of the enzyme was identified as 8α-(N 3-histidyl)FAD from pH dependent fluorescence quenching (p Ka = 4.85) and no decrease in fluorescence upon reduction with sodium borohydride. The enzyme showed a narrow and rather peculiar substrate specificity. In addition to vanillyl alcohol and 4-hydroxybenzyl alcohol, eugenol and chavicol are substrates for the enzyme (chapter 10). The formed products, coniferyl and coumaryl alcohol are the natural precursors of lignin in plants. This reaction has a potential application to produce coniferyl alcohol and subsequent synthetic lignin (DHP) from the inexpensive precursor eugenol.

The major structural elements of wood and other vascular tissues are cellulose, hemicellulose and generally 20-30% lignin. Lignin gives the plant strength, it serves as a barrier against microbial attack and it acts as a water impermeable seal across cell walls of the xylem tissue. However, the presence of lignin has practical drawbacks for some of the applications of lignocellulosic materials. First, lignin has to be removed for the production of high quality pulps. Second, lignin reduces the digestibility of lignocellulosic materials. High quality pulps can be produced with chemical methods, however the abundant use of chemicals and energy, and the formation of an enormous waste stream has led scientists to investigate the possibilities of biodelignification. White-rot fungi give the most rapid and extensive degradation and have become subject of intensive research. Results obtained with the model organism Phanerochaete chrysosporium and other strains have revealed that lignin biodegradation is an extracellular, oxidative and non-specific process. This unique biodegradative potential has been considered for broader applications such as waste water treatment and the degradation of xenobiotic compounds. The research described in this thesis concentrates on the function of aryl alcohols in fungal physiology.Aryl alcohols In the physiology of white-rot fungi. White-rot fungi have a versatile machinery of enzymes, including peroxidases and oxidases, which work in harmony with secondary aryl alcohol metabolites to degrade the recalcitrant, aromatic biopolymer lignin. In chapter 2 literature concerning the important physiological roles of aryl (veratryl, anisyl and chlorinated anisyl) alcohols in the ligninolytic enzyme system has been reviewed. Their functions include stabilization of lignin peroxidase, charge-transfer reactions and as substrate for oxidases generating extracellular H 2 O 2 .The experimental research described in this thesis was initiated to evaluate the possibilities of white-rot fungi in the biopulping of hemp stem wood. Sixty-seven basidiomycetes were isolated and screened for high peroxidative activity (chapter 3). Several of the new isolates were promising manganese peroxidase-producing white-rot fungi. Enzyme assays indicated that for the production of H 2 O 2 either extracellular glyoxal or aryl alcohol oxidase were present. In contrast, lignin peroxidase was only detected in P. chrysosporium , despite attempts to induce this enzyme in other strains with oxygen and oxygen/veratryl alcohol additions. A highly significant correlation was found between two ligninolytic indicators: ethene formation fromα-keto-γ- methylthiolbutyric acid and the decolorization of a polymeric dye, Poly R-478. Three of the new isolates had significantly higher Poly R decolorizing activities compared to P. chrysosporium .One of the best Poly R decolorizing strains, Bjerkandera sp. BOS55 was selected for further characterization. A novel enzyme activity (manganese independent peroxidase) was detected in the extracellular fluid of Bjerkandera sp. BOS55 (chapter 4). The purified enzyme could oxidize several compounds like phenol red, 2,6-dimethoxyphenol (DMP), Poly R-478, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and guaiacol with H 2 O 2 as an electron acceptor. In contrast, veratryl alcohol was not a substrate. This enzyme also had the capacity to oxidize DMP in the absence of H 2 O 2 . Bjerkandera sp. BOS55 also produced de novo several aromatic metabolites. Besides veratryl alcohol and veratraldehyde, compounds which are known to be involved in the ligninolytic system of several other white-rot fungi (chapter 2), other metabolites were formed. These included anisaldehyde, 3-chloro-anisaldehyde, 3,5-dichloro-anisaldehyde and small amounts of the corresponding anisyl, 3-chloro-anisyl and 3,5-dichloro-anisyl alcohol (chapters 5 and 6). This was the first report of de novo biosynthesis of simple chlorinated aromatic compounds by a white-rot fungus. These unexpected findings led us investigate the physiological role(s) of the denovo biosynthesized chlorinated anisyl alcohols (chapter 6). All metabolites were produced simultaneously with the extracellular ligninolytic enzymes. The monoand dichlorinated anisyl alcohols appeared to be excellent substrates for the extracellular aryl alcohol oxidases. The formed aldehydes were readily recycled via reduction by washed fungal mycelium, thus creating an extracellular H 2 O 2 production system regulated by intracellular enzymes. Lignin peroxidase does not oxidize the chlorinated anisyl alcohols both in the absence and in the presence of veratryl alcohol. It was therefore concluded that the chlorinated anisyl alcohols are well protected against the fungus's own aggressive ligninolytic enzymes. The relative amounts of veratryl alcohol and the chlorinated anisyl alcohols differ significantly depending on the growth conditions, indicating that the production of veratryl alcohol and the (chlorinated) anisyl metabolites are independently regulated.It was concluded that the chlorinated anisyl metabolites, biosynthesized by the white-rot fungus Bjerkandera sp. BOS55, are purposeful for ecologically significant processes such as lignin degradation. These results made us speculate if a significant biogenesis of chlorinated aromatics by fungi occurs in natural environments (chapter 7). Many common wood- and forest litter-degrading fungi were indeed detected that produced chlorinated anisyl metabolites (CAM). These compounds, which are structurally related to xenobiotic chloroaromatics, were present in the environment and occur at high concentrations of approximately 75 mg CAM kg -1wood or litter. The ubiquity among common fungi to produce large amounts of chlorinated aromatic compounds in the environment leads to the conclusion that these kind of compounds can no longer be considered to originate from anthropogenic sources only.Degradation of aryl alcohols by fungi. In chapter 2 the anabolic and catabolic routes of aryl alcohols by white-rot fungi has been reviewed. These fungi can not use veratryl alcohol as sole source of carbon and energy. However, several bacteria, yeasts and fungi were selectively isolated from paper mill waste water that grew on veratryl alcohol (chapter 8). Penicilliumsimplicissimum was selected for the characterization of the veratryl alcohol degradation route. P. simplicissimum oxidized veratryl alcohol via a NAD(P) +-dependent veratryl alcohol dehydrogenase to veratraldehyde which was further oxidized to veratric acid in a NAD(P) +-dependent reaction. Veratric acid-grown cells contained NAD(P)H-dependent O -demethylase activity for veratrate, vanillate and isovanillate. Ring-cleavage of protocatechuate was by a protocatechuate 3,4-dioxygenase. An interesting aspect of P. simplicissimum is the production of vanillyl alcohol oxidase with covalently bound FAD (chapter 9). The intracellular enzyme was purified 32-fold. SDS-PAGE of the purified enzyme revealed a single fluorescent band of 65 Kda. Gel filtration and sedimentation-velocity experiments indicated that the purified enzyme exists in solution as an octamer, containing 1 molecule flavin/subunit. The covalently bound prosthetic group of the enzyme was identified as 8α-(N 3-histidyl)FAD from pH dependent fluorescence quenching (p Ka = 4.85) and no decrease in fluorescence upon reduction with sodium borohydride. The enzyme showed a narrow and rather peculiar substrate specificity. In addition to vanillyl alcohol and 4-hydroxybenzyl alcohol, eugenol and chavicol are substrates for the enzyme (chapter 10). The formed products, coniferyl and coumaryl alcohol are the natural precursors of lignin in plants. This reaction has a potential application to produce coniferyl alcohol and subsequent synthetic lignin (DHP) from the inexpensive precursor eugenol.

The shrub Prunus serotina , introduced from North America, became a forest pest in the Netherlands. Biological control was considered using the fungus Chondrostereum purpureum , commonly present as a saprophyte and parasite in wood. C. purpureum can cause silverleaf disease in cultivated fruit trees. As biological control of P.serotina was shown to be effective, a risk analysis was performed using a theoretical framework provided by epidemiology and air pollution theory, simulation models, and empirical parametrization of these models. The risk to native non-target trees within the forest due to biological control was negligible, mainly because of their low susceptibility. The risk to fruit trees at 500 m distance from the forest due to biological control was high and at 5000 m distance low. For some areas, the risk to non-target trees, due to biological control, was calculated to be of the same order of magnitude as or less than the risk due to natural infection. A map of the Netherlands showed that only 4 per cent of the hour-squares contained both forests eligible for treatment and orchards possibly endangered by biological control in nearby forests.

The shrub Prunus serotina , introduced from North America, became a forest pest in the Netherlands. Biological control was considered using the fungus Chondrostereum purpureum , commonly present as a saprophyte and parasite in wood. C. purpureum can cause silverleaf disease in cultivated fruit trees. As biological control of P.serotina was shown to be effective, a risk analysis was performed using a theoretical framework provided by epidemiology and air pollution theory, simulation models, and empirical parametrization of these models. The risk to native non-target trees within the forest due to biological control was negligible, mainly because of their low susceptibility. The risk to fruit trees at 500 m distance from the forest due to biological control was high and at 5000 m distance low. For some areas, the risk to non-target trees, due to biological control, was calculated to be of the same order of magnitude as or less than the risk due to natural infection. A map of the Netherlands showed that only 4 per cent of the hour-squares contained both forests eligible for treatment and orchards possibly endangered by biological control in nearby forests.

Des souches d’Ustilago nuda, agent du charbon nu de l’orge, résistantes à la carboxine ont été détectées en France et dans d’autres pays européens. Une résistance croisée positive s’observe entre ce fongicide, le fenfuram, la pyracarbolide et divers autres analogues structuraux ; le mépronil se singularise car il est plus toxique pour les isolats résistants à la carboxine que pour les isolats sensibles. Toutes les souches sont affectées de la même manière par l’antimycine A, l’azide de sodium, le 2,4- dinitrophénol, le fentin-acétate, la 8-hydroxyquinoléine et le thénoyltrifluoroacétone qui, comme la carboxine, agissent au niveau des mitochondries., Strains of Ustitago nuda resistant to carboxin have been detected in France and several other European countries. Positive-cross resistance was found between this fungicide, fenfuram, pyracarbolid and some other related compounds, whereas a situation of negative-cross resistance was observed between carboxin and mepronil. Other inhibitors of mitochondria such as antimycin A, thenoyltrifluoroacetone and sodium azide... had similar toxicity towards all the strains of U. nuda.

Evaluatie van de nevenwerking van een, als biologisch onkruidbestrijdingsmiddel te gebruiken, pathogene inheemse schimmel

The first part of the preliminary checklist of Agaricales from Croatia is given. It comprises the families Pleurotaceae and Tricholomataceae. 252 species (three of them in a broad sense) belonging to 48 genera are presented. Three genera and 35 species are reported for the first time from Croatia. For each species, the published and unpublished sources of data are given, as well as the collections in which the material is deposited.