We compared the ability of five strains of the ericoid mycorrhizal fungus Hymenoscyphus ericae to utilise glutamine, ammonium or nitrate at high or low carbon (C) availability. The pattern of intraspecific variation in growth was affected by C availability. When C supply was high, growth differences between strains were explained by the total amount of nitrogen (N) taken up, suggesting variation in uptake kinetics. Under C-limiting conditions, strain differences were linked with their nitrogen use efficiency, implying intraspecific differences in N metabolism. The relationship between growth on glutamine and pH shifts in the media indicated that there was intraspecific variation in glutamine transporters. In addition, the correlation between pH changes and the amount of glutamine-N recovered as ammonium in the media indicated that there were intraspecific variations within the enzymatic pathways involved in glutamine metabolism. Our findings, compared with those of a previous study involving the same ericoid strains, draw attention to the temporal variation in nitrogen source utilisation by ericoid mycorrhizal fungi when maintained in axenic culture.

A prerequisite for symbiotic phosphate transfer in an ectomycorrhizal (ECM) association is hypothesized to be conditions in the interface between both symbiotic partners, that either promote the release of inorganic phosphate (P) from the Hartig net into the interfacial apoplast and/or decrease the fungal reabsorption from this location. To get more information about conditions, which might be involved in the regulation of P efflux or P reabsorption, the effect of various external conditions on 33P-orthophosphate (33P) uptake or efflux by axenic cultures of the ECM basidiomycetes Hebeloma crustuliniforme, Amanita muscaria and Laccaria laccata was analysed. In short-time experiments the following external conditions were analysed: an external supply of (1) P in the preculture, (2) cations (0.1–100 mM K, 0.1–50 mM Na, Mg and Ca), and (3) carbohydrates (0.5–50 mM glucose, fructose or sucrose).

The P absorption was generally reduced in cultures previously supplied with an abundant P supply and with increased P concentrations in their tissues. The P uptake was also affected by an external supply of cations, whereas carbohydrates had only a slight effect. Compared to Na, Mg and Ca, the P absorption by H. crustuliniforme and L. laccata was increased by 0.1 mM K in the labelling solution but decreased after a supply of 100 mM K and then did not differ from the other cation treatments. Compared to other cations, an addition of 50 mM Ca led to a decrease of P absorption by A. muscaria, whereas 50 mM Mg increased the P uptake by H. crustuliniforme.

The P efflux from the fungi was affected by both the cation and carbohydrate concentration of the bathing solution. High concentrations of the monovalent cations K and Na (5 mM or 50 mM) in the bathing solution increased the P efflux by H. crustuliniforme (only Na) and L. laccata (K and Na), but had little effects on A. muscaria. By contrast, the same concentrations of the divalent cation Mg reduced the P efflux from all fungal basidiomycetes. The P efflux from hyphae of H. crustuliniforme and A. muscaria was also affected by the carbohydrate concentration in the bathing solution. Compared to the hexoses, glucose and fructose, the P efflux by both basidiomycetes was stimulated even by low concentrations of sucrose (0.5 mM) in the bathing solution. The effect of carbohydrates on P efflux especially from hyphae of H. crustuliniforme was clearly concentration-dependent and the maximal efflux rates were observed after a supply of 2.5 mM sucrose or fructose or 5 mM glucose in the bathing solution. A further rise of the carbohydrate concentration led to a progressive decrease of P efflux from hyphae of H. crustuliniforme. The results are discussed with respect to the transfer processes between both symbiotic partners in a mycorrhizal association and a possible regulation of these exchange processes.

Two dark-spored agaric species from Asia are placed in the genus Anamika (Agaricales or euagarics clade). This result is supported by ITS and nLSU-rDNA sequences with strong measures of branch support, in addition to several morphological and ecological similarities. An inclusive ITS study was performed using a mixed model Bayesian analysis that suggests the derived status of Anamika within Hebeloma, thereby rendering Hebeloma a paraphyletic genus. However, the monophyly of Hebeloma cannot be rejected outright given ITS and nLSU-rDNA data. Thus, we propose two new Asian species in Anamika: A. angustilamellata sp. nov. from dipterocarp and fagaceous forests of southwestern China and northern Thailand; and A. lactariolens comb. nov., a Japanese species originally described in the genus Alnicola. A complete description of A. angustilamellata, including illustrations, is provided.

Aspergillus fumigatus is saprotrophic with an unusual ability to colonize the respiratory tract. The mechanisms that permit pathogenicity may have evolved to adapt the fungus to life as a saprobe. To define the nature of these adaptations and identify common themes in fungal pathogenesis to vertebrates, insects and plants, we compared A. fumigatus with a plant pathogen (Haematonectria haematococca) and an insect pathogen (Metarhizium anisopliae) in their abilities to degrade and utilize host-derived macromolecules (horse lung polymers, porcine mucin, hyaluronic acid, alfalfa cell walls and cockroach cuticle). Each fungus produced a similar range of proteases on mucin and lung polymers, and high levels of several glycosidic enzymes on mucin and plant cell walls, which contain inductive carbohydrate substrates. Following 18 h of growth by A. fumigatus at pH 4 or pH 8, the degradation of mucin carbohydrates and mucin protein were approximately 40% and 75% respectively, suggesting that the aspartyl proteases (produced at pH 4) and the subtilisin proteases (produced at pH 8) are more important than carbohydrases for degrading mucin. The highly glycosylated mucin residue remaining after 18 h growth resisted further degradation, in part due to bound sialic acid as A. fumigatus secretes a sulphatase but not sialidase. Hyaluronidase activity (an important virulence factor in bacteria) was not produced by A. fumigatus, M. anisopliae or H. haematococca, but each fungus secreted a range of other enzymes (phospholipase A2, phospholipase C, acid phosphatase, alkali phosphatase, phosphodiesterase and esterase) that are common toxic components of bacteria as well as reptilia and invertebrate venoms. Thus thermotolerant opportunists such as A. fumigatus may sustain themselves and cause disease in human hosts using depolymerases that are widely distributed in fungi and that provide them with the versatility to exploit many environments.

PCR was used to amplify ribosomal internal transcribed spacer and 5·8 S DNA from type material of Rhizopogon colossus var. colossus, R. hawkerae, R. parksii, R. reticulatus and R. villosulus plus a number of collections belonging to the same section (Sect. Villosuli). Different restriction enzymes were used to digest these amplified rDNAs to find polymorphisms useful in identification. On the basis of these studies and our previous morphological observations, the conclusion is that the names proposed by Smith & Zeller for collections studied, as well as R. reticulatus, represent only one species, R. villosulus.

Mycelial interactions on Hagem agar between 156 isolates of Stereum sanguinolentum obtained from wounded Picea abies or from slash in 12 sample plots located in Sweden, Finland and Lithuania revealed 97 vegetative compatibility groups (VCGs). Of the 23 VCGs that consisted of two or more (maximum, 23) isolates, 13 came from different sample plots, and sometimes from two or all three countries, whereas 10 were confined to a single sample plot. Some variation was observed in cultural characteristics among isolates belonging to the same VCG. Within a sample plot compatible isolates were randomly distributed indicating that distances up to 180 m in a forest stand have little influence on spread of S. sanguinolentum VCGs. Proportions of compatible pairings decreased among isolates collected within the bigger spatial scale. Occurrence of compatibility and consequent grouping of the isolates into VCGs was more pronounced in Swedish populations then Finnish and Lithuanian populations.

Auxarthron conjugatum (Onygenaceae) and Myxotrichum deflexum (Myxotrichaceae) are distantly related cleistothecial (gymnothecial) ascomycetes that form ascomata with strikingly similar peridia in which rigid, branched and anastomosed, thick-walled hyphae create a cage- or mesh-like enclosure (reticuloperidium). We tested the hypothesis that the reticuloperidium plays a role in dispersal mediated by arthropods by enclosing ascomata of these fungi together with flies from the family Sarcophagidae. Gymnothecia of both fungi were picked up easily when the stiff hairs of the flies impaled the ascomata by passing through the interhyphal spaces of the reticuloperidium. Ascospore release from the gymnothecia then occurred during grooming activities during which the limbs of the flies caught the ascoma appendages causing the peridium to be torn apart. This adaptation to arthropod morphology and behaviour is interpreted as the driving force behind the evolution of reticuloperidia in unrelated groups of cleistothecial ascomycetes.

Immunogold labelling and electron microscopy were used to investigate β-1,3-glucanase secretion by Claviceps purpurea during ergot disease of rye in situ. Molecular cytology allowed us to explore the hypothesis that this enzyme might degrade host phloem callose to maintain flow of assimilates for fungal nutrition and therefore β-1,3-glucanase could play a role in pathogenicity. An extracellular endo-β-1,3-glucanase was purified from axenic culture and an antibody was raised. Enzyme activity staining and immunoblotting showed that the antibody was monospecific for β-1,3-glucanase present in fungal protein populations. Mycelia printings of plate-grown cultures displayed spot-like and streaky immuno-signals suggesting a secretion of β-1,3-glucanase at hyphal tips and young hyphae. The enzyme was immuno gold localized predominantly in cell walls of mycelia from axenic culture. In Western blots of honeydew fractions, one β-1,3-glucanase was immunoreactive. In inoculated plants, immunogold labelling was found in all infection stages and limited to the host-pathogen interface. Gold labelling was detected over fungal protoplasts in vacuoles and in tubular-vesicular complexes and multivesicular bodies, which fused with the fungal plasma membrane, indicating that they are part of the secretion pathway. The labelling of the fungal secretory organelles and lack of labelling in any host area apart from the interface verified the fungal origin of β-1,3-glucanase immuno-detected in infected ovaries. Antigenic sites were located in external, subcuticular, penetrating and intercellular hyphae, indicating that the enzyme was secreted throughout the colonization process in planta. Intense labelling regularly associated with fungal cell walls extended into adjacent host walls, indicating a migration of the fungal β-1,3-glucanase into the host apoplast. The gold labelling over host periplasmic spaces showed that the enzyme reached the deposition sites of callose, pointing to an enzymatic suppression of putative plant defense reactions. The host phloem was colonized inter- and intracellularly. Hyphae penetrated into the pectic middle lamella of sieve plates and intense immuno-labelling for β-1,3-glucanase in this area supports a phloem unblocking hypothesis.

Proliferation of intraradical hyphae of the arbuscular mycorrhizal fungus Glomus fistulosum was observed and measured in surface disinfected mycorrhizal maize root segments in an aseptic cultivation system. The maximum growth of hyphae proliferating from the root segments was observed at pH 6·3, and low concentrations of buffering substances stabilized the pH of the incubation medium (0·9 mm MES, 1 mm BIS-TRIS). The proliferation of intraradical hyphae was not influenced but the germination of chlamydospores of the mycorrhizal fungus was inhibited by an artificial inorganic soil solution at lowest dilutions. The percentage of root segments bearing proliferating hyphae and the growth of hyphae decreased with increasing concentration of phosphate in the incubation medium. All reduced sulphur-containing compounds tested (sulphite, dithionite and metabisulphite) suppressed the proliferation of hyphae completely.

As part of an investigation into the factors influencing the colonization of the rhizosphere and the root tissues of host plant, by pathogenic and non-pathogenic strains of Fusarium oxysporum, the effect of a range of carbon (C) sources on the growth habits of five strains of F. oxysporum were compared. The strains used were two F. oxysporum f. sp. lycopersici (Fol strains), two F. oxysporum f. sp. radicis-lycopersici (Forl strains) pathogenic on tomato and strain Fo47, a non-pathogenic strain of F. oxysporum, currently used as a biocontrol agent to reduce severity of fusarium diseases on several crops. Radial extension rates on solid media were measured using soluble saccharides (glucose, xylose and D galacturonic acid), polysaccharides (carboxymethyl cellulose, xylan and pectin) and soluble and insoluble extracts of tomato roots as C sources. Growth parameters were estimated by fitting a logistic equation to biomass data recorded from liquid culture using the same C sources.

The strains were characterized by means of the pattern of radial extension rates on the various C sources, and the two Forl strains were discriminated further from the other strains. The growth parameters were unique features of each F. oxysporum strains, whatever the C source. The pathogenic strains did not exhibit particular abilities in degrading the cell wall components. It was concluded that growth habits related to carbohydrate utilization are unique to each strain of F. oxysporum and that these traits are not related to pathogenicity.

A transformation system is described for the edible mushrooms Pleurotus ostreatus and Volvariella volvacea. The system developed is based on a positive selection strategy using the trp3iar gene from Coprinus cinereus, which confers resistance to the antimetabolite 5-fluoroindole. Transformation frequencies were low in both species. Southern blot analysis confirmed the integration of transforming DNA into the genome of transformants and indicated the presence of tandemly duplicated copies of the plasmid in some of these transformants. The system has potential for introducing beneficial traits such as enhanced substrate bioconversion and faster sporophore development.

The phylogenetic relationships of several smut fungi parasitic on dicotyledons were analysed. Parsimony analysis was performed based on the sequences of the ITS regions of the rDNA genes. Three genera were considered: Microbotryum, Sphacelotheca and Ustilago. The cladogram showed a dichotomy: the species of Microbotryum and Ustilago parasitic on dicotyledons (dicot Ustilago) were found to be divided into two independent taxa among the Microbotryaceae. A divergent mechanism of evolution of the species of each of these two clades with their respective hosts could be involved in this dichotomy. According to our results, Microbotryum appears monophyletic and restricted to the anthericolous smuts on Caryophyllaceae. However, no morphological characters have yet been found to support this distinction, and we refute the denomination Bauhinus as defined by Moore to describe the group of ‘dicot Ustilago’ as it leads to controversial determinations. Sphacelotheca belongs to Microbotryales, but could not be synonymised with Microbotryum as S. polygoni-persicariae is in an independent clade. Lastly, U. duriaeana is independent of the ‘dicot Ustilago’ clade; the position of this species among Microbotryales is still uncertain.

The succession of microfungal communities on Myrtus communis leaf litter was monitored from Jan. 1993 to Nov. 1994 using the litter bag method. Three main groups of fungal colonizers were identified, whose presence is correlated with the successive decomposition stages of the substrate and with seasonal variations.

Cladistic analysis was performed on partial sequences from the nuclear-encoded small subunit ribosomal DNA. Four Halosarpheia spp., Lignincola laevis and Nais inornata were examined to determine the placement of these genera within the unitunicate ascomycetes. Two equally most parsimonious cladograms resulted from the analysis of the data. In a strict consensus of the two cladograms the taxa sampled did not form a monophyletic group, and Halosarpheia was shown to be polyphyletic. The marine Halosphaeriales formed a subclade with taxa from the Microascales. Data indicate that L. laevis and N. inornata may be congeneric, but other species of both genera require further study.

Six species of Mycosphaerella and eight anamorphs with unknown Mycosphaerella states occurring on myrtaceous hosts other than Eucalyptus are treated in the present study. Eight additional species that have recently been described elsewhere are discussed, while a further six species remain unconfirmed, and two are excluded from Mycosphaerella. New species are described in Mycosphaerella, Mycovellosiella, Passalora, Pseudocercospora and Stenella, and new combinations proposed in Eruptio and Microthyrium. Keys are provided to distinguish the accepted species occurring on Myrtaceae other than Eucalyptus, while their morphological similarities with taxa known from this host are discussed.

The development of a PCR assay for the detection of the poplar pathogenic fungi Septoria musiva (teleomorph Mycosphaerella populorum), S. populicola (M. populicola) and S. populi (M. populi) is described. Three pairs of species-specific PCR primers were designed using interspecific polymorphisms in the internal transcribed spacer (ITS) of nuclear ribosomal RNA gene (rDNA) repeats. The specificity of the three primer pairs was successfully tested on a collection of 40 S. musiva, 39 S. populicola and six S. populi isolates. Using stringent PCR conditions, no cross-reaction was observed with any of the isolates tested. The specificity of the PCR assay was further confirmed with DNA extracted from 12 additional Septoria species and 17 other fungal species obtained from stems or leaves of poplars. Specific amplification of the fragments for S. musiva and S. populicola was sensitive relatively to the technique used, detecting as low as 1 pg template DNA, and 10 pg of DNA of the target species in a background of 1 ng of DNA of the other species. Moreover, using DNA purified directly from disrupted conidia, it was possible to detect with a probability of 90%, using one unique PCR assay, the DNA equivalent of 166 conidia per μl of S. musiva and 156 conidia per μl of S. populicola. The procedures developed in this work can thus be applied for rapid and accurate detection and identification of Septoria species from poplars.

For assessments of intraspecific mating using flow cytometry and fluorescence microscopy, two compatible basidiospore-derived isolates were selected from each of four parental basidiomata of North American Biological Species (NABS) X. The nuclear status in NABS X varied with basidiospore-derived isolates. Nuclei within basidiospore-derived isolates existed as haploids, diploids (doubled haploids), or a mixture of haploids and diploids (doubled haploids). Depending on the nuclear status of the basidiospore-derived lines of NABS X, intraspecifically mated cultures can exist as diploids or tetraploids, and possibly triploids or aneuploids under in vitro conditions. Based on previous in vitro mating studies, seven basidiospore isolates were specifically selected to assess rare, interspecific mating among Armillaria cepistipes, A. sinapina, NABS X, and NABS XI. Cultures from basidiospore-derived isolates were paired to produce four interspecifically paired cultures, and matings were assessed using flow cytometry and restriction fragment length polymorphism (RFLP) analyses. Based on flow cytometric analysis, the A. cepistipes isolate exhibited compatibility with a NABS X isolate, and the A. sinapina isolate exhibited compatibility with a NABS X isolate, and the A. sinapina isolates were individually compatible with isolates of NABS X and NABS XI. Mean fluorescence intensities of A. cepistipes × NABS X, A. sinapina × NABS X, and A. sinapina × NABS XI mated cultures revealed a triploid or tetraploid nuclear status compared to the haploid or diploid (doubled haploid) nuclear status of initial basidiospore-derived isolates. Polymerase chain reaction (PCR) and RFLP of the intergenic spacer (IGS) region generated banding patterns for basidiospore-derived isolates and mated cultures. Four species-specific RFLP banding patterns were observed in basidiospore-derived isolates of A. cepistipes, A. sinapina, NABS X, and NABS XI. PCR-RFLP analysis showed combined banding patterns from mated cultures. Flow cytometry and PCR-RFLP analysis are effective tools to assess matings of Armillaria species.

A study of saprobic fungi occurring on the fynbos of the Western Cape Province of South Africa yielded four unknown Anthostomella species. A. proteae from Protea nitida, A. cynaroides from P. cynaroides, A. leucospermi from Leucospermum oleifolium, and A. brabeji from Brabejum stellatifolium are described as new. New records for South Africa include A. conorum from Leucadendron sp., Protea magnifica and P. neriifolia, and A. clypeata from Ischyrolepis subverticellata, Cannomois Virgata, Restio egregius, and R. cfr confusus. A dichotomous key to the Anthostomella species in South Africa is also provided.

Several hyaline-spored coelomycetes placed in Ascochyta, Phoma and Phyllosticta have been recorded from spots on leaves and pods of soybean. Comparison of herbarium material, pathogenicity tests and in vitro studies have demonstrated that Phoma sojicola comb. nov. (syn. Ascochyta sojicola) was predominantly involved as a pathogen of soybean. The diagnostic characters and history of this fungus and other hyaline-spored coelomycetes occurring on soybean are described.