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PubMed2001

Investigation of the response of wood-rotting fungi to copper stress by size-exclusion chromatography and capillary zone electrophoresis with ICP MS detection.

A method based on the coupling of size-exclusion chromatography (SEC) with inductively coupled plasma mass spectrometry (ICP MS) was developed for screening the changes in the bioligand composition of wood-rotting fungi as a function of their exposure to copper stress. Strains of four different species of wood-rotting fungi: Phanerochaete chrysosporium, Schizophyllum commune, Daedalea quercina and Pleurotus ostreatus were examined. Only one, namely Ph. chrysosporium, showed any significant difference in terms of the fingerprint of Cu-binding ligands between control and exposed cells which suggest trapping of Cu(II) by cell walls as the only resistance mechanisms. In the case of Ph. chrysosporium the bioinduction of a new Cu-binding ligand was demonstrated. The presence of a new compound in the SE chromatographic fraction of interest was confirmed by CZE-ICP MS. Attempts to identify the new compound by electrospray MS/MS failed because of insufficient sensitivity.

Basidiomycota↗
PubMed2005

Fungi evolution revisited: application of the penalized likelihood method to a Bayesian fungal phylogeny provides a new perspective on phylogenetic relationships and divergence dates of Ascomycota groups.

The depiction of evolutionary relationships within phylum Ascomycota is still controversial because of unresolved branching orders in the radiation of major taxa. Here we generated a dataset of 166 small subunit (18S) rDNA sequences, representative of all groups of Fungi and used as input in a Bayesian phylogenetic analysis. This phylogeny suggests that Discomycetes are a basal group of filamentous Ascomycetes and probably maintain ancestor characters since their representatives are intermingled among other filamentous fungi. Also, we show that the evolutionary rate heterogeneity within Ascomycota precludes the assumption of a global molecular clock. Accordingly, we used the penalized likelihood method, and for calibration we included a 400 million-year-old Pyrenomycete fossil considering two distinct scenarios found in the literature, one with an estimated date of 1576 Myr for the plant-animal-fungus split and the other with an estimated date of 965 Myr for the animal-fungus split. Our data show that the current classification of the fossil as a Pyrenomycete is not compatible with the second scenario. Estimates under the first scenario are older than dates proposed in previous studies based on small subunit rDNA sequences but support estimates based on multiprotein analysis, suggesting that the radiation of the major Ascomycota groups occurred into the Proterozoic era.

Ascomycota↗
PubMed2002

Two endophytic fungi in different tissues of scots pine buds (Pinus sylvestris L.).

Two fungal species were isolated with different frequencies from pine tissue cultures originating from buds. One species was detected in 33.1% of the cultures initiated in March, and another was present in 1.7% of cultures initiated in June. Based on analyses of phylogenetic and physiological characteristics these fungi were identified as Hormonema dematioides (isolated in March) and Rhodotorula minuta (isolated in June). Probes targeted towards the 18S rRNA of H. dematioides and R. minuta were made. When in situ hybridizations were performed on pine bud tissue, R. minuta was detected inside the cells of meristematic tissue in 40% of the samples, in contrast to H. dematioides, which was not found in this tissue. Using light microscopy, H. dematioides was found to be localized in the scale tissues of the buds. Fungal endophytes have previously been detected in scale tissues, but not in the meristematic tissues of buds. The habitats of these fungi may reflect their different roles in the plant.

Ascomycota↗
PubMed2006

Genetic diversity of naturally established ectomycorrhizal fungi on Norway spruce seedlings under nursery conditions.

We have assessed ectomycorrhizal fungi colonizing Norway spruce (Picea abies L.) seedlings in nine forest nurseries using restriction fragment length polymorphism (RFLP) and sequencing analyses of the internal transcribed spacers (ITS1-5.8S-ITS2) amplicons. Restriction analysis of the amplified DNA fragments with HinfI, MboI, and TaqI enzymes allowed the definition of 17 RFLP genotypes; five of them could be unambiguously assigned to Thelephora terrestris, Hebeloma longicaudum, H. crustuliniforme, Tricharina ochroleuca, and Cenococcum geophilum species by comparison with the sporocarp RFLP-pattern database. The remaining genotypes have been sequenced and compared with sequences deposited in the GenBank database. The phylogenetic analysis of resulting sequences and their identified matches indicated that isolated genotypes have formed seven clades. The ascomycetes were predominant: we have determined eight species--Wilcoxina mikolae, Phialophora finlandia, Tuber sp., Cenococcum geophilum, Tricharina ochroleuca, Pulvinula constellatio, and two unidentified ascomycetes--whereas the basidiomycetes were less common (four species denoted: Amphinema byssoides, Hebeloma crustuliniforme, H. longicaudum, and Thelephora terrestris). Wilcoxina mikolae and Phialophora finlandia were the most frequent fungi. Analysis of variance revealed that ascomycetes abundance was higher in nurseries that used organic fertilizer.

Analysis of Variance↗
PubMed2000

Spatiotemporal Patterns of Laccase Activity in Interacting Mycelia of Wood-Decaying Basidiomycete Fungi.

A BSTRACTInterspecific fungal interactions are important ecological processes, whereas their physiological mechanisms are little understood. The aim of this work was to study how activity of fungal extracellular laccase was changed across mycelia during interactions between white- and brown-rot basidiomycetes from different wood decay stages. Qualitative assay of eight species interacting with each other in all combinations showed four spatial patterns of laccase activity: (I) laccase activity present both in contact zone and mycelium, (II) laccase activity only in contact zone, (III) laccase activity in mycelium but not in contact zone, (IV) no laccase activity. Presence of laccase activity only in the contact zone was more frequent than expected from random samples associated with mycelia that replaced other ones. On the other hand, the presence of laccase activity in the mycelium but not in the contact zone was only attributed to fungal species that were replaced by their antagonists. After one month, laccase activity was distributed over mycelia more homogeneously than after 6 days of interactions. In interacting mycelia, laccase activity was higher than in control and increasing with time. Saprotrophic fungi from late successional stages of wood decay generally had higher laccase activity than early succession saprotrophic and pathogenic fungi. The qualitative assays were confirmed by quantitative assay of total laccase activity. Significance of the results in antagonistic fungal interactions as well as in the processes of hyphal tip growth and mycelium senescence is discussed.

Journal Article↗
PubMed2004

Degradation of acrylic copolymers by white-rot fungi.

Various water-soluble homopolymers and copolymers of acrylamide (AAm) and acrylic acid (AA) which contained phenolic sites, such as guaiacol, lignin sulfonate (LS) and 3,4-dihydroxybenzoic acid (3,4-DHBA), were tested with regard to their degradability by white-rot fungi. Compared with Phanerochaete chrysosporium, Pleurotus ostreatus caused a significantly higher decrease in the average molecular weight ( Mw) of most of the copolymers and the homopolymer under the applied culture conditions. However, the Mw of poly(guaiacol/AAm) increased significantly during incubation with Pl ostreatus. P. chrysosporium was able to reduce only the Mw of the poly(LS/AA) to a significant degree and not that of the other polymers. The mineralization rate of AAm and AA copolymers and terpolymers of AAm, AA and phenolics (LS, 3,4-DHBA, guiacol), which were tested with P. ostreatus and Trametes versicolor, turned out to be low (0.8-3.2%). While the rates of mineralization were similar among all polymers, the decrease in radioactivity from the culture media was higher with the terpolymers bearing phenolic sites. UV spectra of the culture media revealed that the phenolic sites in the terpolymers were significantly degraded by both fungi. Obviously, the degradation of phenolics within the polymer chain caused a higher decrease in Mw but did not significantly increase the mineralization rate.

Acrylamides↗
PubMed2004

The ability of white-rot fungi to degrade the endocrine-disrupting compound nonylphenol.

Phanerochaete chrysosporium, Pleurotus ostreatus, Trametes versicolor and Bjerkandera sp. BOL13 were tested for their ability to degrade the endocrine-disrupting compound nonylphenol at an initial concentration of 100 mg l-1. The highest removals were achieved with T. versicolor and Bjerkandera sp. BOL13, which were able to degrade 97 mg l-1 and 99 mg l-1 of nonylphenol in 25 days of incubation, respectively. Nonylphenol removal was associated with the production of laccase by T. versicolor, but the levels of laccase, manganese peroxidase and lignin peroxidase produced by Bjerkandera sp. BOL13 were very low. At 14 degrees C, T. versicolor and Bjerkandera sp. BOL13 sustained the removal of 88 mg l-1 and 79 mg l-1 of nonylphenol, respectively. No pollutant removal was recorded at 4 degrees C, although both fungi could grow at this temperature in the absence of nonylphenol. A microtoxicity assay showed that the fungi produced compounds that were toxic to Vibrio fischerii; and thus a reduction in toxicity could not be correlated with nonylphenol metabolism. T. versicolor and Bjerkandera sp. BOL13 were capable of colonizing soil artificially contaminated with 430 mg kg-1 of nonylphenol. Only 1.3+/-0.1% of nonylphenol remained in the soil after 5 weeks of incubation.

Aliivibrio fischeri↗
PubMed2005

Biofiltration of waste gases with the fungi Exophiala oligosperma and Paecilomyces variotii.

Two biofilters fed toluene-polluted air were inoculated with new fungal isolates of either Exophiala oligosperma or Paecilomyces variotii, while a third bioreactor was inoculated with a defined consortium composed of both fungi and a co-culture of a Pseudomonas strain and a Bacillus strain. Elimination capacities of 77 g m(-3) h(-1) and 55 g m(-3) h(-1) were reached in the fungal biofilters (with removal efficiencies exceeding 99%) in the case of, respectively, E. oligosperma and Paecilomyces variotii when feeding air with a relative humidity (RH) of 85%. The inoculated fungal strains remained the single dominant populations throughout the experiment. Conversely, in the biofilter inoculated with the bacterial-fungal consortium, the bacteria were gradually overgrown by the fungi, reaching a maximum elimination capacity around 77 g m(-3) h(-1). Determination of carbon dioxide concentrations both in batch assays and in biofiltration studies suggested the near complete mineralization of toluene. The non-linear toluene removal along the height of the biofilters resulted in local elimination capacities of up to 170 g m(-3) h(-1) and 94 g m(-3) h(-1) in the reactors inoculated, respectively, with E. oligosperma and P. variotii. Further studies with the most efficient strain, E. oligosperma, showed that the performance was highly dependent on the RH of the air and the pH of the nutrient solution. At a constant 85% RH, the maximum elimination capacity either dropped to 48.7 g m(-3) h(-1) or increased to 95.6 g m(-3) h(-1), respectively, when modifying the pH of the nutrient solution from 5.9 to either 4.5 or 7.5. The optimal conditions were 100% RH and pH 7.5, which allowed a maximum elimination capacity of 164.4 g m(-3) h(-1) under steady-state conditions, with near-complete toluene degradation.

Air Pollution↗
PubMed2004

Gibberellin biosynthesis in fungi: genes, enzymes, evolution, and impact on biotechnology.

Gibberellins (GAs) constitute a large family of tetracyclic diterpenoid carboxylic acids, some members of which function as growth hormones in higher plants. As well as being phytohormones, GAs are also present in some fungi and bacteria. In recent years, GA biosynthetic genes from Fusarium fujikuroi and Arabidopsis thaliana have been cloned and well characterised. Although higher plants and the fungus both produce structurally identical GAs, there are important differences indicating that GA biosynthetic pathways have evolved independently in higher plants and fungi. The fact that horizontal gene transfer of GA genes from the plant to the fungus can be excluded, and that GA genes are obviously missing in closely related Fusarium species, raises the question of the origin of fungal GA biosynthetic genes. Besides characterisation of F. fujikuroi GA pathway genes, much progress has been made in the molecular analysis of regulatory mechanisms, especially the nitrogen metabolite repression controlling fungal GA biosynthesis. Basic research in this field has been shown to have an impact on biotechnology. Cloning of genes, construction of knock-out mutants, gene amplification, and regulation studies at the molecular level are powerful tools for improvement of production strains. Besides increased yields of the final product, GA3, it is now possible to produce intermediates of the GA biosynthetic pathway, such as ent-kaurene, ent-kaurenoic acid, and GA14, in high amounts using different knock-out mutants. This review concentrates mainly on the fungal biosynthetic pathway, the genes and enzymes involved, the regulation network, the biotechnological relevance of recent studies, and on evolutionary aspects of GA biosynthetic genes.

Biotechnology↗
PubMed2001

Screening of micro-organisms for decolorization of melanins produced by bluestain fungi.

A total of 17 fungi and four bacteria were screened for their ability to decolorize melanin, using isolated extracellular melanin of the bluestain fungus Aureobasidium pullulans as substrate. On agar media, decolorization was observed by four fungal strains: Bjerkandera adusta VTT-D-99746, Galactomyces geotrichum VTT-D-84228, Trametes hirsuta VTT-D-95443 and Trametes versicolor VTT-D-99747. The four fungi were more efficient on nitrogen-limited medium than on complete medium. The melanin-decolorizing activity of G. geotrichum appeared to be located on the mycelium and could be liberated into the medium enzymatically.

Ascomycota↗
PubMed1999

The NADP-dependent glutamate dehydrogenase gene from Penicillium chrysogenum and the construction of expression vectors for filamentous fungi.

The gdhA gene encoding the NADP-dependent glutamate dehydrogenase activity from Penicillium chrysogenum has been isolated and characterized for its use in gene expression. The nucleotide sequence of a 2816-bp genomic fragment was determined, showing an open reading frame of 1600 bp interrupted by two introns, of 160 bp and 57 bp respectively, with fungal consensus splice-site junctions. The predicted amino acid sequence revealed a high degree of identity to glutamate dehydrogenase enzymes, especially to those from the fungi Aspergillus nidulans (82%) and Neurospora crassa (78%). The gdhA gene was found to be present in a single copy in the genome of several P. chrysogenum strains with different penicillin productivity. The use of the gdhA promoter for homologous and heterologous gene expression in fungi and Escherichia coli was analyzed. Heterologous gene expression was ascertained by the construction of gene fusions with the lacZ gene from E. coli and the bleomycin-resistance determinant (bleR) from Streptoalloteichus hindustanus. Homologous gene expression was shown through the use of the penicillin-biosynthetic genes pchC and penDE from P. chrysogenum and the cephalosporin biosynthetic genes cefEF and cefG from Acremonium chrysogenum.

Amino Acid Sequence↗
PubMed2001

Feasibility of bioremediation by white-rot fungi.

The ligninolytic enzymes of white-rot fungi have a broad substrate specificity and have been implicated in the transformation and mineralization of organopollutants with structural similarities to lignin. This review presents evidence for the involvement of these enzymes in white-rot fungal degradation of munitions waste, pesticides, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, bleach plant effluent, synthetic dyes, synthetic polymers, and wood preservatives. Factors relating to the feasibility of using white-rot fungi in bioremediation treatments for organopollutants are discussed.

Ascomycota↗
PubMed2001

Gibberellin Biosynthesis in Plants and Fungi: A Case of Convergent Evolution?

As well as being phytohormones, gibberellins (GAs) are present in some fungi and bacteria. Indeed, GAs were first discovered in the fungus Gibberella fujikuroi, from which gibberellic acid (GA3) and other GAs are produced commercially. Although higher plants and the fungus produce structurally identical GAs, there are important differences in the pathways and enzymes involved. This has become particularly apparent with the identification of almost all of the genes for GA-biosynthesis in Arabidopsis thaliana and G. fujikuroi, following the sequencing of the Arabidopsis genome and the detection of a GA-biosynthesis gene cluster in the fungus. For example, 3b-hydroxylation occurs early in the pathway in G. fujikuroi and is catalyzed by a cytochrome P450 monooxygenase, whereas it is usually the final step in plants and is catalyzed by 2-oxoglutarate-dependent dioxygenases. Similarly, 20-oxidation is catalyzed by dioxygenases in plants and a cytochrome P450 in the fungus. Even where cytochrome P450s have equivalent functions in plants and Gibberella, they are unrelated in terms of amino acid sequence. These profound differences indicate that higher plants and fungi have evolved their complex biosynthetic pathways to GAs independently and not by horizontal gene transfer.

Journal Article↗
PubMed2006

Pathocycles: Ustilago maydis as a model to study the relationships between cell cycle and virulence in pathogenic fungi.

Activation of virulence in pathogenic fungi often involves differentiation processes that need the reset of the cell cycle and induction of a new morphogenetic program. Therefore, the fungal capability to modify its cell cycle constitutes an important determinant in carrying out a successful infection. The dimorphic fungus Ustilago maydis is the causative agent of corn smut disease and has lately become a highly attractive model in addressing fundamental questions about development in pathogenic fungi. The different morphological and genetic changes of U. maydis cells during the pathogenic process advocate an accurate control of the cell cycle in these transitions. This is why this model pathogen deserves attention as a powerful tool in analyzing the relationships between cell cycle, morphogenesis, and pathogenicity. The aim of this review is to summarize recent advances in the unveiling of cell cycle regulation in U. maydis. We also discuss the connection between cell cycle and virulence and how cell cycle control is an important downstream target in the fungus-plant interaction.

Cell Cycle↗
PubMed2001

FEM1, a Fusarium oxysporum glycoprotein that is covalently linked to the cell wall matrix and is conserved in filamentous fungi.

As part of an investigation of the cell wall structure of plant pathogenic, filamentous fungi, we set out to characterize covalently bound cell wall glycoproteins (CWPs) of the tomato pathogen Fusarium oxysporum. N-terminal sequencing of an abundant 60-kDa CWP led to the cloning of the corresponding gene, which we have designated FEM1 (Fusarium extracellular matrix protein). The gene contains an ORF encoding a primary translation product of 212 amino acids, including an N-terminal 17-amino acid secretion signal sequence. Furthermore, FEM1p contains two potential N-glycosylation sites, and is rich in serine and threonine residues (29%) that could serve as O-glycosyl addition sites. At its C-terminus the protein contains a 22-amino acid sequence with the characteristics of a glycosyl-phosphatidylinositol (GPI) anchor addition signal. A mutant FEM1 protein lacking this GPI anchor addition signal is not retained in the fungal cell wall but released into the culture medium, indicating that in the wild-type protein this sequence functions to anchor the protein to the extracellular matrix. Southern analysis shows that FEM1 is present as a single-copy gene in all formae speciales of F. oxysporum tested and in F. solani. Database searches show that FEM1p homologous sequences are present in other filamentous fungi as well.

Amino Acid Sequence↗
PubMed2004

Thuja plicata exclusion in ectomycorrhiza-dominated forests: testing the role of inoculum potential of arbuscular mycorrhizal fungi.

The ability of trees dependent on arbuscular mycorrhizal (AM) fungi to establish in ectomycorrhizal forests is unknown. On northern Vancouver Island, Canada, there are sharp boundaries between mixed red cedar (Thuja plicata)-hemlock (Tsuga heterophylla) (CH) stands, and stands of hemlock and amabilis fir (Abies amabilis) (HA). We tested differences in AM colonization of red cedar between ectomycorrhiza-dominated (HA) stands and stands containing red cedar (CH), across a range of light levels. We used a soil bioassay approach to determine whether there was sufficient AM fungal inoculum in the HA tree stands to colonize red cedar seedlings. Seeds of hemlock and red cedar were sown in forest floor samples collected from the two types of forests, and shade treatments ranging from < 1 to 53% of full sunlight were imposed. After 6 months, seedling survival and root and shoot biomass were quantified, and red cedar seedlings were sampled for AM fungal colonization. Hemlock survival and growth did not differ between soil types, suggesting there was no substrate-associated limitation to its establishment in either forest type. Red cedar colonization by AM fungi was significantly correlated with light levels in CH soils but arbuscular mycorrhizas were absent in roots of red cedar seedlings grown in HA soil. Red cedar survival and relative growth rate were significantly greater in the CH than in HA soil; higher growth was due primarily to greater shoot growth in CH soils at high light levels. The low soil inoculum potential for red cedar in ectomycorrhiza-dominated stands may account for the virtual exclusion of red cedar seedlings from these forests.

Abies↗
PubMed2002

Interactions between indigenous arbuscular mycorrhizal fungi and Aphanomyces euteiches in field-grown pea.

This is the first reported study of the interactions between indigenous arbuscular mycorrhizal fungi (AMF) and Aphanomyces euteiches in pea under field conditions. A. euteiches was applied to the soil by adding oospores produced in vitro. Attempts were made to create a non-mycorrhizal control by incorporating carbendazim (Derosal Fl) in the topsoil before sowing. However, all carbendazim-treated plants showed approximately 20% root colonisation with AMF. Pea plants not treated with carbendazim showed a wide variation in AMF colonisation of 35-70% at the full flowering stage. In these control plots, root length infected with oospores of A. euteiches and colonisation by AMF were negatively correlated. Application of carbendazim increased the percent root length infected with oospores by 50-70%, depending on inoculum density of A. euteiches. Despite the lower levels of AMF colonisation in these treated plots, a negative correlation with oospore-containing root length was still observed. No correlation was found between AMF colonisation and disease severity, disease incidence or pathogen enzymatic activity (glucose-6-phosphate dehydrogenase). Thus, AMF do not seem to influence the vegetative stage of pathogen development during which cortical root rotting takes place, but rather the reproductive stage when oospores are produced. The results of this study underline the importance of field experiments for validating the significance of mycorrhizal fungi for plant health.

Antifungal Agents↗
PubMed2002

Laccases and other polyphenol oxidases in ecto- and ericoid mycorrhizal fungi.

Polyphenol oxidases are known to be produced by a range of ectomycorrhizal (ECM) and ericoid mycorrhizal fungi. These enzymes include laccase (EC 1.10.3.2), catechol oxidase (EC 1.10.3.1) and tyrosinase (EC 1.14.18.1), between which there exists considerable overlap in substrate affinities. In this review we consider the nature and function of these enzymes, along with the difficulties associated with assigning precise enzymatic descriptions. The evidence for production of laccase and other polyphenol oxidases by ECM and ericoid mycorrhizal fungi is critically assessed and their potential significance to the mycorrhizal symbioses discussed.

Ascomycota↗