Mycelial interactions were studied between 28 isolates of Amylostereum areolatum and 15 of A. chailletii from wounded spruce in 10 sample plots in Sweden and Lithuania. Based on somatic incompatibility, nine vegetative compatibility groups (VCGs) of A. areolatum were detected. Five of these VCGs were isolated from two or more trees and included 24 (86%) of all isolates. In A. chailletii, 12 VCGs were found and three contained two isolates each. Grouping of isolates into VCGs was more pronounced in A. areolatum. Two of the A. areolatum VCGs had wide geographical distribution. VCG A2 was isolated from 14 different trees in two Swedish and four Lithuanian sample plots. VCG A1 was found in one plot in Sweden and in two in Lithuania. VCGs of A. chailletii had a local distribution and were confined to a single sample plot or to a forest stand. In A. areolatum occurrence of compatible pairings between isolates was uniform in all spatial scales of investigation: within a plot, within each country and among both countries. The proportion of somatically compatible A. chailletii isolates was significantly lower and decreased with geographic distance.

Several varieties have been distinguished within the phytopathogenic pycnidial fungus Phoma exigua on the basis of cultural characteristics. These varieties have been stated to correlate with differences in ability to attack different host plants. Molecular discrimination of the varieties was investigated using amplified fragment length polymorphism (AFLP) and sequence analysis of the ITS region of the rDNA cluster on a set of 43 strains and 7 outgroup strains. The ITS sequences of the 43 different P. exigua strains were highly similar and revealed no subgroups within P. exigua while the AFLP fingerprint patterns of two primer combinations showed clear clustering of most varieties.

Rhodotorula mucilaginosa produces the siderophore rhodotorulic acid (RA) when grown in iron-limited conditions. R. mucilaginosa grew at rates between 0.10 and 0.19 h−1 in iron-restricted conditions, depending on the carbon source, and at 0.23 h−1 in iron-sufficient conditions. In bioreactors inoculated with iron-starved pre-cultures, initial specific growth rates in batch culture were dependent on the iron concentration. The critical dilution rate (Dcrit, at which steady state cultures cannot be sustained) in continuous cultures was also dependent on the iron concentration and was lower than μmax in batch culture. Sucrose was the best carbon source for RA production [287±11 μmol (g biomass)−1] and production could be further increased by supplementing the medium with the precursors acetate [460±13 μmol (g biomass)−1], ornithine [376±6 μmol (g biomass)−1], or both [539±15 μmol (g biomass)−1]. Citric acid was an effective suppresser of RA production. RA was produced in a growth rate dependent manner and was optimally produced at pH 6.5.

During surveys of woodlands in Taiwan a previously undescribed fungus Dryadomyces amasae gen. sp. nov., was found in the sapwood of dead angiosperm timbers in the gallery systems of the scolytine ambrosia beetle Amasa concitatus. The fungus grows predominantly in the immediate vicinity of the feeding beetle larvae and serves as a nutritional ambrosia fungus. The transmission of D. amasae to new breeding substrates is ensured by an oral mycetangium, a paired organ which was found in A. concitatus and A. aff. glaber near the mandibles and contained multiple cells of the fungus. Morphologically D. amasae resembles species of Ambrosiella. However, phylogenetic analysis based on partial nucSSU rDNA placed the fungus with certain species of the genus Ambrosiella within the Ophiostomatales, whereas the type species of Ambrosiella, A. xylebori, was assigned to the Microascales. A. xylebori, as well as A. ferruginea and A. hartigii, demonstrated phialidic conidial development, leading to an emendation of the description of the genus Ambrosiella. In contrast, Dryadomyces exhibited conidial development by apical, sympodial wall formation with prominent denticles bearing conidia. Other, previously described Ambrosiella species exhibited non-phialidic conidiogenesis, but lacked denticles on the conidiophores. Consequently, their classification needs further revision.

Strains of Heterobasidion annosum belonging to the S and P intersterility groups (IGs) were compared in respect of laccase activity on various substrates. Biomass production was significantly lower in P cultures than in S cultures. By contrast, laccase activity, measured in relation to growth rate, was significantly higher for the P strains tested, compared with a similar number of S strains, particularly in substrates, rich in carbohydrates as well as in inorganic and organic nitrogen. Using the Bradford assay method, P cultures were shown to contain significantly higher amounts of protein than S. The pH optimum of laccase for both IGs was 4·5 with guaiacol as substrate and 5·3 with syringaldazine. Isozyme patterns varied greatly, depending on strain, substrate and incubation time. In P cultures 4–5 bands were obtained, whereas S strain laccase was mostly separated into 2–3 isozyme bands. Only one was significantly different in position between the IGs. The results may help to explain why P strains are more aggressive than S strains as pathogens and wood decayers.

The sexual system of Heterobasidion insulare was elucidated and 40 specimens originating from the mainland of China, Taiwan, and Japan were studied with the aid of mating tests. Most of the material was also investigated with DNA fingerprinting using random amplified microsatellite (RAMS) and M13 minisatellite primers. Denaturing gradient gel electrophoresis (DGGE) and sequencing were applied for detecting variation in the internally transcribed spacer (ITS) region of the ribosomal DNA cluster. H. insulare proved to be a species complex with bipolar mating system. Clamp connections occurred in both heterokaryotic and homokaryotic mycelia. Three intersterility groups were found: the ‘T group’ included specimens from Taiwan and Guizhou Province (southern China), the ‘N group’ specimens from northern China and Japan, and the ‘Y group’ specimens from Yunnan (south-west China). The T group seems to be totally intersterile with the two other groups, and was grouped separately in neighbour joining analysis based on DNA fingerprints. As concluded from mating experiments, groups N and Y are partially intersterile, but they are able to produce hybrid heterokaryons, no group specific DNA markers were found, and they were grouped together in neighbour joining analysis. The identity of H. insulare sensu stricto was studied from the type collections. It seems that none of the above-presented taxa can be referred to it, and we propose the term H. insulare sensu lato to be used in future studies.

Three hypotheses of the phylogeny of the lichenized Parmeliaceae are constructed, using sequences of the nuclear ribosomal gene cluster (SSU, ITS, 5·8S rDNA) from Bryoria capillaris, Cetraria islandica, Evernia prunastri, Hypogymnia physodes, Parmelia saxatilis, Platismatia glauca, Pleurosticta acetabulum, Usnea florida, Vulpicida juniperina, V. pinastri and Xanthoparmelia conspersa. The first analysis, based on SSU sequences, shows the representatives of the Parmeliaceae to form a monophyletic group within the order Lecanorales. Usnea florida and Bryoria capillaris, sometimes classified in the families Usneaceae and Alectoriaceae, respectively, are also members of the Parmeliacceae. Cetraria and Vulpicida form a well-supported monophyletic group together with Usnea, but there is little support for any other groupings within Parmeliaceae. The second analysis is based on ITS an 5·8S sequence data of the representatives of the Parmeliaceae included in the first analysis, and supports the close relation of Cetraria and Vulpicida. Other groupings are not supported, and the topology also varies greatly with minor alignment changes. This is attributed to the included taxa being too distantly related to each other, causing alignment difficulties of the variable ITS region when a relatively small number taxa are included. It is predicted that a larger sample of phylogenetically intermediate taxa will overcome this problem. When combining the two datasets, the results are similar to the ITS results. The present study is one of the first phylogenetic studies utilizing the information in more than one gene alone in lichenized ascomycetes.

Visual markers detectable by histochemical staining have been developed for analysing the time course and tissue specificity of maize infections by Fusarium moniliforme. Three F. moniliforme strains, RRC 374, MRC 826 and RRC PAT, were transformed with a plasmid, pHPG, containing the gusA reporter gene which codes for β-glucuronidase (GUS) and the hph gene for hygromycin resistance as the selectable marker. Introduction of plasmid DNA into germinating conidia yielded 1·2×10−7 transformants per conidium; expression of both gusA and hph was however, transient. Stable transformants were obtained using protoplasts as the recipient, but transformation frequency was reduced. Southern blot and PCR analyses confirmed incorporation of pHPG into the genome of all three F. moniliforme strains with gusA properly inserted in MRC 826 and RRC PAT, but apparently disrupted in RRC 374. The growth pattern for transformed F. moniliforme isolates and the parental wild types followed a sigmoid curve on minimal and enriched media. Hygromycin totally inhibited growth for wild type isolates, but not of transformants. Transformed isolates maintained the ability to infect the maize plant. Thus, this study is the first report of F. moniliforme transformed with a visibly detectable reporter gene to use for analysing this endophyte-host interaction of world-wide importance to animal and human health.

Mycology is changing as an era of extensive genome sequencing comes of age and provides vital information that enables questions to be addressed about fungi in all the major taxonomic groups. As technology transfer facilitates what was once only possible for a very small number of model species, it becomes possible to explore the biology and biodiversity of fungi as a whole. The availability of genome sequence information and reverse genetic technologies allows hypotheses that emerge from biological observations to be tested. Genomic and post-genomic technologies will underline the importance of fungi as excellent models for the study of fundamental biological phenomena. Two enduring areas of research in my own laboratory are described that are now being extended using post-genomic approaches. These projects relate to how fungal hyphae extend and guide their tips and secondly how plant pathogenic oomycete zoospores are guided on their journey to the plant surface.

Sequences of nuclear ITS (nrDNA) regions were obtained from 22 species of Tyromyces s.l., a group of important wood-rotting, poroid holobasidiomycetes, belonging to Coriolaceae. Different reference collections of a species generally produced identical molecular sequences. Parsimony analysis of these sequences resulted in trees compatible with the currently accepted delimitation of genera, but the alignment of sequences has proven to be difficult owing to regions of uncertainty. Analyses based on inclusion and exclusion of ambiguous regions in the alignment demonstrate that closely related taxes are, however, always clustered together, especially at the generic level. It is considered that ITS sequences are reliable in distinguishing genera and species of Tyromyces s.l., but not able to provide sufficient resolution for relationships between some formerly recognized genera. The taxonomic implications of ITS variation are also discussed. Species of Oligoporus and Postia clustered in one clade, indicating a close relationship as shown from morphological research. Species of Skeletocutis are characterized by encrusted hyphal ends in the hymenophore, but the type species of Tyromyces, T. chioneus, is embedded within the Skeletocutis clade, indicating a strong link between these two genera. Species of Antrodia and of Spongipellis included in this study were not well supported as monophyletic groups, although they are clustered together within the trees.

Coarse woody debris is important for mycodiversity in forest ecosystems, but its availability in managed stands is reduced. Leaving dead wood during felling is suggested as an option to sustain and restore the diversity. However, little is known what fungi would colonise freshly cut wood left on managed sites, and how the colonisation process is influenced by ecological factors. During summer and autumn, 120 freshly cut Picea abies stem sections over 8 cm in diameter were placed upright in mapped locations over two discrete plots separated by 100 m in a north-temperate forest. After seven weeks the sections were collected, and isolation and identification of fungi was done from their upper surfaces. In all 943 fungal strains were isolated, representing 97 species. Species richness in the summer survey was 42.5% higher than during the autumn survey. Low species similarity characterized the different seasons (Sorensen indices: SS=0.36 and SN=0.34) and for 21 species (22%) observation frequency was significantly affected by season. As a result, community structures in summer and autumn differed notably (z-test; P<0.001). Species richness between the two plots differed by less than 10%, but there were 64 species (66%) found only in one of them, thus qualitative similarity was low (SS=0.49). Quantitative similarity was higher (SN=0.63), indicating that the dominant species colonised wood to a similar extent in both areas. Fungal community structure differed significantly among the two plots (z-test; P<0.001). Our data showed that freshly cut CWD contributed to mycodiversity in managed north-temperate forest, providing habitats for numerous individuals from over 100 species. The fungal community within a single stand differed markedly both across small distances and over the seasons. In order to sustain and enhance mycodiversity in managed stands, coarse wood should always be left during harvesting. This study also demonstrates the importance of molecular identification and ITS sequence databases for exploring fungal diversity in natural communities.

The entomopathogenic fungus Pandora neoaphidis is an important natural enemy of aphids. ISSR, ERIC (Enterobacterial Repetitive Intergenic Consensus) and RAPD PCR-based DNA fingerprint analyses were undertaken to study intra-specific variation amongst 30 isolates of P. neoaphidis worldwide, together with six closely related species of Entomophthorales. All methods yielded scorable binary characters, and distance matrices were constructed from both individual and combined data sets. Neighbour-joining was used to construct consensus phylogenetic trees which showed that although P. neoaphidis isolates were highly polymorphic they separated into a monophyletic group compared with the other Entomophthorales tested. Three distinct subclades were found, with UK isolates occupying two of these. No specific correlation with aphid host species was established for any of the isolates apart from those in one cluster which contained isolates obtained from nettle aphid, Microlophium carnosum. ERIC, ISSR and RAPD analysis allowed the rapid genetic characterisation and differentiation of isolates with the generation of potential isolate- and cluster specific- diagnostic DNA markers.

Green moulds caused by Trichoderma harzianum biotypes Th2 and Th4 are responsible for important economic losses on mushroom farms producing Agaricus bisporus, whereas other biotypes present in farms have only slight effects on the cultivated mushroom. Adaptation of Th2 and non-Th2 strains of Trichoderma to grow in compost prepared for mushroom cultivation was compared through extracellular enzyme activities and competition with bacteria isolated from compost. No specific differences between Th2 and non-Th2 strains were observed for the activities of the 17 secreted enzymes tested. Confrontations of 27 bacterial isolates with 4 Trichoderma strains revealed that the aggressive Th2 strains were affected by a lower number of bacterial isolates than the non-Th2 strains. Capacities of T. harzianum, biotype Th2, to colonise mushroom compost are not due to a specific ability to produce enzymes necessary for compost degradation but to their tolerance to the inhibitory effect of bacteria present in compost.

Pleurotus pulmonarius is a species of the oyster mushroom which has become the second most popularly cultivated mushroom in the world. In this study, we show that renewed fruiting from excised stipes can be used as a simple and rapid in vitro bioassay system to detect fruiting modulators. We used this and conventional cultivation techniques to examine the effects of cadmium, a potential contaminant from industrial sources, calcium, which is an ingredient in mushroom compost, and manganese, which has been claimed to improve the yield of P. ostreatus. All the three metallic salts did not affect sporulation. Calcium chloride addition shortened the time taken for the mushroom to fully cover the cultivation compost and improved yield. Insoluble calcium salts at higher concentrations had a similar though less pronounced effect. The calcium and total amino acid contents of fruit bodies also increased. Compost supplementation with calcium is desirable for cultivation of the oyster mushroom but not indispensable since the straw-based cultivation substrate is itself able to provide the required minerals. By contrast, manganese chloride retarded mycelial growth and decreased yield but increased the total amino acid content in the stipe whilst manganese sulphate did not enhance accumulation of manganese into fruit bodies. Excess manganese induced browning of vegetative tissues. Cadmium ions did not kill the oyster mushroom at 4·5 mM but reduced yield by 50%. At this concentration cadmium decreased the total amino acid content and affected the amino acid profile but did not affect the form and shape of the fruit bodies. Pleurotus pulmonarius concentrated cadmium to such an extent that consumption of as little as 20 g (D.W.) of the most contaminated samples would exceed the weekly limit tolerated by humans and thus pose a health hazard. Monitoring the heavy metal contents of mushrooms marketed for food is, therefore, advised as the source of the substrate for cultivation is usually not known.

Diplodia pinea (syn. Sphaeropsis sapinea) is a well-known latent pathogen of Pinus spp. with a worldwide distribution. As such, this fungus is native where pines are endemic in the northern hemisphere and it has been introduced into all countries of the Southern Hemisphere where pines are exotic. The newly described D. scrobiculata (formerly known as the B morphotype of D. pinea) is thought to have a much more limited distribution. D. scrobiculata was first reported as an endophyte and weak pathogen of P. banksiana, where it was found to coexist with D. pinea. Diplodia scrobiculata is now known to have a much broader distribution in Northern America and Europe. In this study, seven Simple Sequence Repeat (SSR) markers were used to evaluate genetic diversity and gene flow between populations of D. scrobiculata. Results indicate a strong geographic isolation between populations of D. scrobiculata from different regions in North America, with unique alleles fixed in the different populations. The data fits the isolation by distance model indicating limited dispersal. Geographic isolation in combination with isolation by distance suggests prolonged reproductive isolation. Intensive collections of endophytes from native P. radiata in California have yielded only D. scrobiculata and not the significantly more pathogenic D. pinea. SSR analysis of three populations of D. scrobiculata from native P. radiata identified many shared alleles among the populations and moderate to high gene flow between them. The three Californian populations are distant and distinct from populations of D. scrobiculata from elsewhere. Under stress conditions, P. radiata is known to be very susceptible to D. pinea in plantations in the Southern Hemisphere. Native P. radiata is currently experiencing severe stress due to pitch canker caused by Fusarium circinatum. Such stress would provide ideal conditions for an associated outbreak of D. pinea. Thus, it is critical to prevent the movement of D. pinea into the last remaining native stands of P. radiata.

Analysis of Amplified Fragment Length Polymorphisms (AFLP) in a sample population of Epichloë and Neotyphodium was conducted to evaluate the usefulness of this technique in estimating phylogenetic relationships among these fungi. A total of 963 unique restriction fragments were identified with 13 selective primer combinations. Neighbour-joining and parsimony analysis produced phylogenetic trees with high bootstrap support. All Epichloë festucae isolates were monophyletic, and clades of E. festucae were identified that correspond to the host species from which they were isolated. These groups may indicate the importance of coevolution in the development of the fine fescue-E. festucae symbiosis. The grouping of E. festucae isolates from hard fescue and Chewings fescue into one clade conflicts with the current taxonomy of these host grasses. Sequencing of ribosomal DNA spacer regions ITS1 and ITS2 was conducted for comparisons to the phylogenetic relationships estimated by AFLP. Using the maximum likelihood test, a significant difference was detected between AFLP and rDNA trees due to discrepancies in the association of anamorphs (Neotyphodium) and teleomorphs (Epichloë). This discrepancy may be due to some mechanism of genetic recombination, such as hybridization or parasexual recombination, that occurs in Neotyphodium populations. It is concluded that AFLP markers are useful for producing meaningful phylogenies among a diverse group of fungi and for identifying groups of closely related individuals that are genetically and biologically similar. Further investigation is needed to determine how AFLP markers change over short periods of time in populations and over evolutionary time.

This work tested the hypothesis that the abundance of fungal species on coniferous needles is correlated to climatic factors at a regional scale. The proportions of Pinus sylvestris needles colonised by Lophodermium pinastri, Cyclaneusma minus and Verticicladium trifidum were examined along two altitudinal transects in 2 successive years. L. pinastri and C. minus colonisation increased with altitude, whereas that of V. trifidum decreased. The data are discussed in relation to the importance of climatic controls on species distributions.

Molecular, morphological, and cytological evidence show that the arbuscular mycorrhizal fungus Glomus scintillans is more closely related to the genera Gigaspora and Scutellospora (Gigasporaceae) than it is to Glomus s. str., and consequently it contributes to the non-monophyly of the genus Glomus. We transfer the species to Gerdemannia gen. nov., with consequent autonymy of an appropriate family, Gerdemanniaceae fam. nov. Small subunit (SSU) rDNA analysis places Gerdemannia in a clade sharing common ancestry with the Gigasporaceae, and therefore within the Diversisporales (Glomeromycota). Glomus dominikii is considered to be conspecific with G. scintillans, and these species are synonymised. Glomus chimonobambusae is also transferred to the new genus as Gerdemannia chimonobambusae comb. nov.

The ability of two non-aflatoxin producing strains of Aspergillus flavus to interfere with aflatoxin production by a toxigenic A. flavus strain was examined using a replacement series with suspended disc culture method. Individual glass fiber discs, affixed to a pin suspended from the caps of scintillation vials, were inoculated with medium containing A. flavus conidial mixtures in different proportions (aflatoxin producer[ratio ]non-producer=100[ratio ]0, 80[ratio ]20, 60[ratio ]40, 20[ratio ]80 and 0[ratio ]100 by vol) at a constant total density (1×105 spores ml−1). Reductions in the total conidial density of these strains when grown alone, had little effect on fungal growth (mycelium dry weight) or aflatoxin production. Significant (P<0.0001) reductions in aflatoxin B1 were recorded when non-toxigenic strains represented any proportion of the inoculum mixture. Aflatoxin yield values were less than (P<0.0001) expected from the input ratios for toxigenic vs. non-toxigenic conidial inoculum within the replacement series. Aflatoxin yields were also reduced (P<0.001), with a corresponding increase in fungal growth (P<0.001), when conidia from aflatoxin producing strains were mixed in equal proportions. This suggests that the substantial inhibition of aflatoxin yield for inoculum mixtures results from the failure of spore germlings to establish a cooperative mycelial network.

Close relationship between the Sphaerophoraceae and Austropeltum and Neophyllis, is established by phylogenetic analysis of nuclear SSU rDNA sequences, including newly produced sequences of Austropeltum glareosum, Neophyllis melacarpa and Sphaerophorus globosus. Anatomical, chemical, and ontogenetical characters, and support for this relationship, are discussed, as is the suitability of the nuclear SSU rRNA gene for phylogenetic analyses at this taxonomic level. The analysis gives no support for regarding Austropeltum (presently classified in the Stereocaulaceae) and Neophyllis (presently in the Cladoniaceae) to be closely related to either Stereocaulon ramulosum and Pilophorus acicularis (Stereocaulaceae), or Cladonia bellidiflora (Cladoniaceae), and it is pointed out that the delimitation of all three families needs further investigation. The homology of podetial and pseudopodetial structures is discussed.