The objective of this study was to assess the influence of clay minerals on the morphology of mycelial pellets produced under submerged conditions as a prelude to the development of biomineral sorbents for toxic metals. The macro- and microscopic morphology of fungal pellets of melanin-containing microfungi Cladosporium cladosporioides, C. herbarum and Humicola grisea grown in liquid clay-containing medium was studied using scanning electron microscopy. It was found that the inclusion of clay minerals (bentonite, palygorskite and kaolinite) in the liquid medium influenced size, shape and structure of the mycelial pellets produced. In general, a reduction of pellet size, an increase in the length of surface hyphae of the pellets (except for H. grisea), and a reduction in exopolymer production were observed with increasing clay mineral concentrations up to 5% (w/v). For C. herbarum, an increasing concentration of bentonite changed the normal pelleted growth form to diffuse star-like growth, which was also observed for C. cladosporioides in the presence of kaolinite. The presence of bentonite and kaolinite in the medium for H. grisea and palygorskyte for C. cladosporioides led to the formation of numerous bulbous hyphae in pellets. However, bentonite decreased such bulbous growth in C. herbarum compared to control pellets which grew as bulbous mycelium. The dynamics of C. cladosporioides growth and pellet formation in the presence of bentonite (0·5% (w/v)) was investigated. It was found that the clay particles were involved in the formation of pellet structure at all stages of fungal growth. The porosity of a growing pellet (evaluated as the ratio of surface hyphae length to pellet radius) increased during early growth (17–24 h) and then decreased up to the stationary phase. A general model of the structure of a fungal pellet grown in clay-containing medium is proposed. The pellets consist of three main layers: a central core, with densely packed mycelium aggregated with solid clay minerals or a matrix of clay/polysaccharides; a middle layer with looser mycelium mixed with clay mineral flakes; and an outer, or ‘hairy’ zone, with loose hyphae surrounded by clay mineral flakes. The relevance of these studies to fungal growth and morphology, and to the development of biomineral sorbents for metal removal is discussed.

A set of aspergillus strains from culture collections and wild-type black aspergilli isolated on non-selective media were used to validate the use of media with 20% tannic acid for exclusive and complete selection of the black aspergilli. The 20% tannic acid medium proved useful for both quantitative and qualitative selection of all different black aspergilli, including all recognized species: A. carbonarius, A. japonicus, A. aculeatus, A foetidus, A. heteromorphus, A. niger, A. tubingensis and A. brasiliensis haplotypes. Even higher concentrations of tannic acid can be utilized by the black aspergilli suggesting a very efficient tannic acid-degrading system. Colour mutants show that the characteristic ability to grow on high tannic acid concentrations is not causally linked to the other typical feature of these aspergilli, i.e. the formation of brown-black pigments. Sequence analysis of the A. niger genome using the A. oryzae tannase gene yielded eleven tannase-like genes, far more than in related species. Therefore, a unique ecological niche in the degradation of tannic acid and connected nitrogen release seems to be reserved for these black-spored cosmopolitans.

Methanol extracts from 24 Trichoderma isolates, selected as biocontrol agents and representating different species and genotypes from three of the four taxonomic sections of this genus (T. sect. Trichoderma, T. sect. Pachybasium and T. sect. Longibrachiatum) were screened for antibacterial, anti-yeast and antifungal activities against a panel of seven bacteria, seven yeasts and six filamentous fungi previously used in similar studies. Two different growth media were tested (potato dextrose broth and CYS80), and all isolates included in the antimicrobial tests showed at least one inhibitory activity against one of the target microorganisms in one of the two culture media. No statistically significant differences were detected in the number of active strains between the two culture media, but the highest number of inhibitory strains against bacteria and fungi were found in strains from Trichoderma sect. Pachybasium, whereas strains from T. sect. Longibrachiatum showed the highest anti-yeast values. In all cases, a correlation was found between the strains that were active against yeasts and fungi. However, some degree of variability was detected for strains within the same taxonomic section. In general terms, strains from T. asperellum (mainly in CYS80 medium), and T. longibrachiatum gave the best non-enzymatic antimicrobial profiles.

The genetic relationships of 56 isolates from the taxa traditionally grouped in Fusarium sections Fusarium (syn. Discolor) and Roseum were studied using the Restriction Fragment Length Polymorphism (RFLP) technique, by Southern hybridisation with random genomic and mitochondrial DNA probes originating from Fusarium species. Pairwise distances between taxa were calculated from the 957 RFLP bands scored on autoradiograms, using Dice's coefficient in the RAPDistance computer program. A strong genetic relationship was observed between F. graminearum, F. culmorum and F. crookwellense. However, the morphologically similar taxon, F. pseudograminearum shared only approximately 40% of genomic DNA RFLP bands with these taxa, demonstrating affinity, but less direct genetic similarity. Of all taxa examined F. pseudograminearum and F. graminearum shared the greatest similarity in mitochondrial DNA RFLP patterns. F. avenaceum subsp. avenaceum, subsp. aywerte and subsp. nurragi displayed very little genetic resemblance with each other, or with F. heterosporum and the cereal infecting pathogens. F. torulosum was closest genetically to subsp. avenaceum, with approximately 22% of genomic DNA RFLP bands in common. On the basis of these results F. avenaceum subsp. aywerte and subsp. nurragi are elevated to species rank, as F. aywerte comb.. nov. and F. nurragi comb. nov., and F. avenaceum subsp. avenaceum is returned to its former species status.

The nucleotide sequence of the internal transcribed spacer region of the ribosomal RNA gene (rDNA-ITS) was obtained for strains belonging to 10 special forms (f. spp.) of the phytopathogenic fungus Fusarium solani (teleomorph Haematonectria haematococca). Phylogenetic analysis of nucleotide sequences of the rDNA-ITS region showed that most clusters were composed of strains belonging to the same special form. F. solani f. sp. eumartii and f. sp. phaseoli were excluded from the cluster composed of all other special forms. In order to use the molecular data to identify each special form, several restriction enzymes specific for each special form were selected from the mapping data. The digestion pattern of the rDNA-ITS region with these restriction enzymes was able to distinguish F. solani f. spp. phaseoli, cucurbitae race 1, batatas, piperis, eumartii, and xanthoxyli. Although f. spp. mori, robiniae and pisi were not differentiated by restriction analysis, they were separated in the phylogenetic tree.

In the Pacific Northwest, yellow chanterelles have long been referred to as Cantharellus cibarius, synonymous with the European yellow chanterelle. Broad scale genetic surveys of North American chanterelles with C. cibarius-like morphology have demonstrated that the nrDNA internal transcribed spacer exhibits length variability, suggesting that this common morphology masks a species complex. Recently researchers have used morphological and genetic data to identify the yellow chanterelle most frequently harvested from American Pacific Northwest forests as C. formosus, a species once thought to be rare in the region. We present three genetic data sets and one morphological data set that characterize a previously undescribed, species of yellow chanterelle from the central Cascade Mountains of Oregon. Phylogenetic analyses of the nrDNA large subunit and ITS regions show that C. cascadensis sp. nov., along with two other yellow chanterelle taxa (C. cibarius var. roseocanus and European C. cibarius), are more closely related to white chanterelles (C. subalbidus) than they are to C. formosus. Data from five microsatellite loci provide evidence that C. formosus, C. subalbidus, and C. cascadensis sp. nov. do not interbreed when they co-occur spatially and temporally in Douglas fir-western hemlock forests. This demonstrates that these three sympatric chanterelles are biological species with boundaries congruent with those delineated by nrDNA phylogenetic clades. Morphological data indicate that the colour of the pileus and shape of the stipe can be used to separate fresh collections of the two yellow species now known to co-occur in Douglas fir-western hemlock forests in Oregon.

The European Union (EU) has introduced statutory limits for patulin in fruit products. Species definitions need to be unambiguous for this capability, especially to determine ‘weak spots’ in food, drink and feed production. Fungi were analysed for the isoepoxydon dehydrogenase (IDH) gene of the patulin metabolic pathway to indicate potential patulin production. In several cases inhibition of PCR product formation was indicated. Dilution as a means of overcoming PCR inhibition and to determine optimal concentrations was assessed and the ramifications for nucleic acid analysis in general are highlighted. The inhibitors may be secondary metabolites. However, inhibition was not involved obviously for Penicillium brevicompactum. The gene was detected frequently from Aspergillus section Clavati and Penicillium subgenus Penicillium species. Some strains within certain species were negative for the gene. Detection of the gene product in Byssochlamys is reported.

Mycelium grew from a spore-mycelial inoculum of Aspergillus oryzae added to ultra-pure water (upw) containing silicon compounds, but did not grow in upw alone. Growth of other fungi also occurred in upw only when silicon compounds were added. Increased growth of A. oryzae, and other fungi, also followed the addition of silicic acid and other silicon compounds to Czapek Dox. Aspergillus oryzae solubilized silicon compounds in both upw and nutrient-rich media. Although interactions between microorganisms and silicon have been generally neglected, the results show that silicon compounds can increase fungal growth under both oligotrophic and nutrient-rich conditions.

Cladistic analysis of characters derived from nuclear-encoded 18S rDNA sequences was used to infer the phylogenetic placement of ambrosial species of the anamorph genus Raffaelea among perithecial ascomycetes. Of eight species in the genus investigated, seven resolve as a monophyletic lineage that forms a sister group to the sexual genus Ophiostoma. Raffaelea hennebertii, the single species excluded from this lineage, appears to be allied phylogenetically with species of Melanospora. These data are discussed in relation to the monophyly of Raffaelea and its relationship with other sexual and asexual fungal associates of wood-boring Coleoptera. In addition, we report the presence of significant length variation in PCR-amplified 18S rDNA fragments for three of the Raffaelea species studied.

Nine ascomycetes, 1 basidiomycete and 15 mitosporic fungi are reported from submerged wood, collected from a single English river site. Ascotaiwania pallida, Neta angliae and Trichocladium englandense spp. nov. and Frigidispora colnensis gen. & sp. nov. are described and illustrated. Most of the species present on the submerged wood appear to have been identified, for the single date and location sampled, because the asymptote of the accumulative curve was reached between 80–100 substrata. The results of this study are compared with those from collections, using similar collection techniques in tropical regions. Results suggest that temperate streams appear to have a lower fungal diversity than in the tropics. Only Acrogenospora sphaerocephala was common to tropical (Seychelles) and temperate (U.K., River Coln) sites.

Entonaema, Pulveria, Phylacia, Rhopalostroma, Sarcoxylon and Thamnomyces are relatively small and poorly studied genera of Xylariaceae. Their affinities to the mainstream of the family largely remain to be evaluated. Secondary metabolite profiles of type materials, recently collected specimens, and cultures, were generated to address this problem from a chemotaxonomic point of view. Micro-scale extraction and subsequent analytical HPLC with UV/visual (diode array) and ms detection in the positive and negative Electrospray mode were carried out, employing spectral libraries and standardised gradients that had been optimised to detect characteristic pure compounds in species of allied genera. Surprisingly, the characteristic metabolites had frequently remained stable even in specimens collected up to 190 years ago. Hence, this methodology not only proved valuable to establish the conspecificity of type materials with recent records, but also revealed some interesting correlations: (1) Stromatal pigments of Entonaema cinnabarina, E. globosum and E. liquescens are mitorubrins and other characteristic compounds also prevailing in particular species of Hypoxylon; (2) Rhopalostroma, Phylacia, Pulveria and Thamnomyces contain binapththalenes and other compounds typical of Daldinia and Hypoxylon; (3) Sarcoxylon, as well as E. dengii, E. moluccanum and E. pallida, contained none of these pigments, but characteristic yet unknown lipophilic metabolites were detected in their stromatal extracts; (4) Cultures of E. cinnabarina and Rhopalostroma indicum, obtained for the first time, produced essentially the same secondary metabolites that are also typical of Daldinia, but absent in Biscogniauxia, Hypoxylon and other xylariaceous genera. Hence, chemotaxonomic characters reflected the adaptive radiation of this family. Correlations between the evolution of morphological/anatomical characters are discussed.

Thirty 3–4 yr-old twigs were collected at each of three sampling dates between May 1995 and May 1996 from each of ten approximately 120 yr-old European beech trees in the Tuscan-Emilian Apennines to examine the bark for the presence of endophytic fungi. Five trees with low crown transparency ([les ]5%) and five trees with high crown transparency ([ges ]25%) were compared. Almost all of the 900 examined bark samples were colonized by endophytic fungi. More than 30% of the twigs appeared to host three or more fungal species. Forty-four endophyte species were detected. An Aposphaeria and a Cryptosporiopsis species, Botryosphaeria quercuum, Discula umbrinella and Neohendersonia kickxii occurred most frequently in all of the three samplings and seemed to play a dominant role as endophytes in beech bark. Significant differences in endophyte assemblages between trees with low and trees with high crown transparency could be detected only with respect to the Aposphaeria species. The colonization of tissues by this fungus, possibly a weak pathogen, was predominant on trees with high crown transparency.

A molecular and morphological investigation of 35 collections of Daldinia from 11 plant species revealed five taxonomic entities of Daldinia in northern Europe. These include Daldinia concentrica, D. cf. fissa, D. grandis, D. loculata and D. cf. petriniae. D. cf. fissa, D. grandis and D. loculata were found exclusively on burnt wood, but they do not form a monophyletic unit in a phylogenetic tree derived from analysis of the ITS sequence of the ribosomal DNA.

A PCR assay and a hybridisation assay were developed for identification of Fusarium oxysporum. Based on sequence data from the 28S rDNA, two primers (PFO2 and PFO3) and one 20-mer probe (HFO1) were designed. These oligonucleotides were tested with 16 strains of F. oxysporum and 80 strains of 23 other Fusarium species or of eight species from other fungal genera. A PCR procedure for specific amplification of DNA from the F. oxysporum strains using primers PFO2 and PFO3 was set up. Under the conditions defined, no PCR product was obtained with these primers from the 80 other strains. The oligonucleotide probe HFO1 was labelled using a non-radioactive method and evaluated for specificity in dot blot hybridisation experiments with rDNA amplified by PCR. Under optimised conditions, the probe hybridised exclusively with DNA from F. oxysporum strains. Both PCR and hybridisation procedures were validated with 53 additional isolates of F. oxysporum representative of the populations present in four different soils. These specific assays appear to be reliable tools for the identification of F. oxysporum and may have various applications in ecological studies.

A molecular phylogeny of the Lecanora rupicola group is presented, based on ITS sequence analyses. The study includes saxicolous and corticolous members of the Lecanora rupicola group as well as other Lecanora species with pruinose apothecia. A phylogenetic hypothesis for species in Lecanora s. lat. and various other genera in Lecanoraceae, based on an alignment-free distance estimation technique, shows that the Lecanora rupicola group forms a monophyletic clade within Lecanoraceae. Affinities to the core group of Lecanora are not well supported, likewise the monophyly of Lecanora s. str. with other species groups in Lecanora, such as the lobate taxa (and Rhizoplaca) is not supported. A more detailed analysis involving Lecanora species with pruinose apothecial discs was carried out with model-based Bayesian Markov chain Monte Carlo (B/MCMC) tree sampling. The results suggest the monophyly of the Lecanora species that are characterized by the presence of chromones. Corticolous as well as saxicolous species are included. Lepraria flavescens is closely related to the Lecanora swartzii subgroup, and the new name Lecanora rouxii nom. nov. is introduced for that species. Other Lecanora species with pruinose discs are not closely related to the Lecanora rupicola group.

The internal transcribed spacers, ITS1 and ITS2, and 5.8S region of ribosomal DNA (rDNA) from 20 species of Ascosphaera were amplified by PCR, and their sequences determined and compared. No variation was detected in the sequences from 23 widely distributed isolates of A. apis, in sequences from 11 widely distributed A. atra isolates, in four A. aggregata isolates, or in sequences from two isolates each of A. acerosa, A. duoformis, A. flava, A. larvis, A. pollenicola and A. proliperda. However, the ribosomal sequences from each of these nine species, and from another 11 species of which only a single isolate was examined, differed from one another by 0·18–30·9%. Thus these sequences provide a rapid method for identifying species, and searches using them showed that the sequence of A. apis rDNA recorded in the international databases is, in fact, that of A. atra.

The rDNA sequences also provided data for assessing the relationships of these fungi. Of the rDNA sequences in current international databases, that of Eremascus albus was very close to, but distinct from, those of the Ascosphaera species. Comparisons of the Ascosphaera sequences showed that most formed consistent clusters irrespective of the method of comparison used (distance matrix and parsimony), or whether the ITS1 or ITS2 portions were used; A. acerosa with A. asterophora, A. atra with A. duoformis, A. colubrina closely with A. flava, A. larvis, A. major, A. variegata and more distantly with A. apis and A. celerrima, and, also, A. aggregata with A. subcuticulata, A. proliperda and more distantly with A. solina. The apparent relationships of these clusters were inconsistent, depended on the alignment of several regions of ‘indels’, and could not be resolved. The A. fusiformis, A. naganensis and A. osmophila sequences showed inconsistent relationships with the others, especially that of A. osmophila, which had an A. solina-like ITS2 region, but an atypical ITS1 sequence with a large unique repetitive insertion. The clusters based on gene sequence comparisons clearly correlated with groupings based on ascospore morphology and other characters.

Fermentation processes for the biochemical reagents cerulenin and helvolic acid employ ‘Cephalosporium caerulens,’ an invalidly published designation that has been used for more than 40 years. However, its identity has never been critically examined because strains were unavailable from major culture collections. An authentic strain of ‘C. caerulens’, derived from the original strain KF-140, was recently found and compared to Sarocladium oryzae, another Acremonium-like fungus which also produces cerulenin and helvolic acid. Morphological comparisons, rDNA sequence data, and chromatography of secondary metabolites established that ‘C. caerulens’ and S. oryzae are conspecific. Sequence data from ribosomal DNA genes indicated S. oryzae belongs to the Hypocreales and is allied with members of the Ceratostomataceae, Scopinella species, Emericellopsis species and certain Acremonium-like anamorphs of uncertain familial relationships. At least two of the isolates of S. oryzae produced titres of cerulenin and helvolic acid similar to those of KF-140. This finding demonstrates that manufacture of cerulenin need not be limited to the original strain.

Fungi of the genus Termitomyces live in an obligate symbiosis with termites of the subfamily Macrotermitinae. Many species of Termitomyces frequently form fruit bodies, which develop from the fungus comb within the nest. In this study, we determined the mating system of a species of Termitomyces associated with the South African termite Macrotermes natalensis. Termite nests were excavated and a Termitomyces sp. was isolated into pure culture from the asexual fruit bodies (nodules) growing in the fungus gardens. For one strain, single basidiospore cultures were obtained from basidiomes growing from the fungus comb after incubation without termites. Using nuclear staining, we show that both comb cultures and single spore cultures have multinucleate cells and that the majority of spores has a single nucleus. However, DNA sequencing of the ITS region in the nuclear RNA gene revealed that the comb mycelium had two different ITS types that segregated in the single spore cultures, which consequently had only a single ITS type. These results unambiguously prove that the strain of Termitomyces studied here has a heterothallic mating system, with the fungus garden of the termite mound being in the heterokaryotic phase. This is the first time the mating system of a Termitomyces species has been studied.

The molecular phylogeny and biogeography of two widely distributed Amanita species, A. muscaria and A. pantherina, were studied based on specimens from diverse localities. Analyses of both a partial sequence of the ITS region of nuclear DNA and a partial sequence of the β-tubulin gene were able to resolve specimens of each species. Analyses revealed a greater divergence of the β-tubulin region than the ITS region. Based on molecular phylogeny of the combination of the ITS and β-tubulin regions, A. muscaria could be separated into at least three groups (Eurasian, Eurasian subalpine, and North American), and A. pantherina could be separated into at least two groups (North American and Eurasian). We hypothesize that the speciation of A. muscaria occurred in Eurasia with subsequent migration to North America via land bridges. However, it is impossible to determine whether A. pantherina moved from Eurasia to North America or vice versa. For both A. muscaria and A. pantherina, the intracontinental relationships of both Eurasia and North America were closer than the relationships between eastern Asia and eastern North America.

The nucleotide sequences of the nuclear ribosomal DNA, including ITS1, ITS2 and the 5·8S rDNA, were determined for 54 specimens representing 13 Leveillula species on 50 different host plant species. The maximum nucleotide sequence diversity among taxa in ITS1-5·8S-ITS2 was 7·1%. Phylogenetic analyses revealed that Leveillula species formed six clades and three basal taxa. The taxonomic positions of several species that are well characterized by morphology of conidia, especially primary conidia, are supported by the present molecular analyses. This result shows that the morphology of primary conidia mostly provides a good criterion to identify Leveillula species. Twenty-six collections of Leveillula taurica recovered from 14 different plant families formed a distinct clade with L. chrozophorae, L. duriaei and L. elaeagni, and showed high homology in the ITS regions. Eight isolates of L. taurica recovered from the Asteraceae, Balsaminaceae, Fabaceae and Campanulaceae showed high sequence diversity, in contrast with other L. taurica specimens, and clustered separately or with other taxa. The result of Kishino-Hasegawa and Templeton tests demonstrated that the possibility of monophyly of L. taurica s. lat. could be significantly rejected, indicating that L. taurica s. lat. is a species complex composed of several biological species.