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.

Characteristics of hyphal septation, cellular interaction, sporulation, cultures and/or nucleotide sequences from the 5′ terminal domain of the nuclear large subunit rRNA gene of some species of Exobasidium, Microstroma and Muribasidiospora were examined and compared. Our analyses show that the order Microstromatales comprises Microstroma, Muribasidiospora triumfetticola and the anamorphic species Rhodotorula bacarum, R. phylloplana and Sympodiomycopsis paphiopedili, whereas Muribasidiospora hesperidium and Muribasidiospora indica are members of the Exobasidiales. Because of the high degree of morphological divergence between Muribasidiospora triumfetticola and Microstroma, a new genus, Volvocisporium, and a new family, Volvocisporiaceae, of the Microstromatales are proposed. Apart from the phylogenetic implications, the most interesting observations in the present work are the unusual development of the basidia, especially of the basidiospores, and the nutrient transport system from the host attacking hyphae to the basidia in Muribasidiospora triumfetticola.

The taxonomy of lichenized fungi (lichens) is relatively well-known compared to that of other fungi because lichens are long-lived and possess a thallus that requires exposure to light. Therefore they are easily observed on their natural substratum and sampling can be carried out much more thoroughly than in other fungal groups. Comprehensive catalogues of lichen taxa known worldwide are available and currently about 13500 species are accepted.

An analysis of selected, recently revised groups suggests that there has been a mean increase in species number of about 25% since 1931. One third of the species recognized before 1931 were withdrawn, and almost half of the currently recognized species have been described after 1931. Some 3000 names seem to be ‘orphaned’, not included in currently accepted genera and not appearing in recent species counts. New species are being described at an increasing rate, so that large numbers of ‘missing lichens’ seem to be present. However, there is no clear indication how numerous they are, only an estimation that they amount to some 25% of all species.

A geographical analysis of checklists suggests that most missing species will be found in the tropics and the southern hemisphere. A taxonomic analysis of recently published species suggests that there is no particular group yielding more novelties than others. Our own fieldwork impressions suggest that tropical primary forests are the most productive in revealing undescribed species. These are found in particular on leaves and on bark high up on the tree trunks. In well-studied areas like western Europe the new discoveries tend to be cryptic species or species rarely producing ascocarps.

In conclusion, the ‘missing’ lichens are estimated at about 4000, and are to be found everywhere and in all taxonomic groups, but predominantly in primary tropical forests. This would result in a total of about 18000 lichen species.

The ectomycorrhizal fungus Hebeloma velutipes consists of two biological species (BSP 16 and 17). Within BSP 17 a dikaryon was found with two divergent types of the ribosomal Internal Transcribed Spacer (ITS1 and 2). The two ITS types segregated in monokaryotic progeny of that dikaryon, showing that these different ITS types represent different alleles at homologous rDNA loci in the two nuclei. RFLP analysis of a number of strains of BSP 17 showed that the polymorphism is widespread in Europe. There was no deficiency of the heterokaryotic type, demonstrating that ITS divergence in this species is not correlated with reduced intercompatibility. A strain from North America, not assigned to a biological species, showed the same polymorphism. Cladistic analysis of the two ITS sequences showed that they were not sister groups. One of the ITS types formed a monophyletic group together with the ITS type of BSP 16, the other type formed a clade with the ITS type of H. incarnatulum (BSP 18). BSP 16 and 17 showed partial intercompatibility. However, several lines of evidence suggest that the polymorphism of BSP 17 is not the result of frequent and continuing hybridisation with BSP 16. Instead, we give arguments for the hypothesis that the polymorphism evolved in allopatry and that the two types have come together relatively recently. The results of the polymorphism indicate a potential problem for molecular identification of fungal species based on ITS fingerprinting. The results also show that no generalisations are possible about the relation of speciation (the formation of BSP) and nuclear ITS divergence.

In Finland and Norway, a uninucleate Rhizoctonia sp. is causing a root dieback disease on nursery-grown Norway spruce and Scots pine seedlings. This Rhizoctonia can be fruited under laboratory conditions and the basidial characters fit well in the species concept of Ceratobasidium bicorne, a species originally described as a moss parasite under forest conditions. Further comparison using traditional methods (cultural morphology, nuclear condition, anastomosis) has not been possible as the forest population of C. bicorne has apparently never been cultured. In the present study, we isolated DNA from a herbarium sample of C. bicorne grown on the moss Polytrichastrum formosum. Sequence analysis of the PCR-amplified rDNA region containing the internal transcribed spacer (ITS) regions and the 5.8S rDNA gene was used to examine the conspecificity of the herbarium sample and the uninucleate Rhizoctonia sp. The nucleotide sequence of the 5.8S rDNA gene was identical between the herbarium sample and five sequenced uninucleate Rhizoctonia strains. Within the uninucleate Rhizoctonia sp., the sequence identity ranged from 96.1 to 100% in ITS1 and from 99.6 to 100% in ITS2. The sequence from the herbarium sample fits well within these limits, strongly suggesting that the uninucleate Rhizoctonia sp. and C. bicorne are conspecific. Interestingly, two of the uninucleate Rhizoctonia strains produced two ITS alleles: the genetic implications are also discussed.

Species belonging to Hypoxylon and related genera are bark pathogens and wood decay agents of angiosperms, sometimes able to live asymptomatically in host tissues. In the present work the ITS regions of the ribosomal DNA from 21 isolates belonging to five species were sequenced and then compared with each other. A phylogenetic parsimony analysis among isolates was carried out. ITS2 appears to be the most informative among the sequenced portions of rDNA. Results are strictly consistent with the latest taxonomic arrangements obtained with classical morphological approaches. The ITS sequence analysis is an efficient and accurate method for discriminating among taxa of Hypoxylon and related genera.

Collybia, as understood by Antonin & Noordeloos, comprises four species: C. racemosa, C. tuberosa, C. cirrhata and C. cookei. Collybia tuberosa, C. cirrhata and C. cookei are morphologically similar and are primarily distinguished from each other by the presence or absence and the colour of sclerotia. All four share a common and unique habitat. Phylogenetic reconstructions using DNA sequences of the ribosomal ITS1–5.8S–ITS2 support four distinct clades, each corresponding to a morphological species, with the C. tuberosa, C. cirrhata and C. cookei clades forming a larger group. Analyses of ribosomal Large Subunit DNA sequences confirmed that Collybia tuberosa, C. cirrhata and C. cookei formed a monophyletic group. In both analyses, the C. racemosa sequence was highly divergent from those of the other three species of the complex and we propose a separate genus name, Dendrocollybia, for this species. Simple diagnostic RFLP patterns were identified for the four species and were used to validate morphological designations and distributions.

The distinctiveness of the European Galerina marginata and the American G. autumnalis is tested with analyses of DNA sequences from the internal transcribed spacer (ITS-2) of the nuclear ribosomal repeat and RFLP analysis of the entire ITS region. European and North American material from six taxa described in Galerina subgenus Naucoriopsis are included along with out-group material of six taxa representing two other subgenera of Galerina. The results do not indicate any genetic difference between species by Smith & Singer (1964) referred to the two ‘stirpes’ Marginata and Autumnalis of Naucoriopsis, i.e. G. autumnalis, G. marginata, G. oregonensis, G. pseudomycenopsis, G. unicolor, and G. venenata and all except G. pseudomycenopsis are considered later synonyms of G. marginata. Galerina badipes (syn. G. cedretorum var. bispora of ‘stirps’ Cedretorum is well supported as a distinct species. Subgenus Naucoriopsis appears as a distinct infrageneric unit well separated from the infrageneric units represented by the out-group species.

Inter simple or short sequence repeat (ISSR) fingerprinting was used to differentiate Botryosphaeria species and related anamorphic fungi. Fifty-one isolates representing 10 different Botryosphaeria species and related taxa, including two RAPD marker groups and one special form of Sphaeropsis sapinea were investigated using five ISSR primers. A total of 171 fingerprint fragments were generated from these isolates, and sizes of fragments were mostly between 200 bp and 1650 bp. Both cluster and principal coordinate analyses were used to examine separately the fingerprint data from the two previously proposed sections Hyala and Brunnea of Botryosphaeria. The results of each analysis indicated that B. dothidea and B. ribis are distinct; B. ribis and B. parva are closely related, but separable; B. mamane is distinct from other Botryosphaeria species with similar morphology or ITS sequences; the A and B RAPD marker groups of S. sapinea and S. sapinea f. sp. cupressi are probably three distinct species; B. obtusa and S. sapinea B group are closely related but distinct; and the name B. stevensii may have been applied to more than one species. This study has shown that ISSR fingerprinting can be a powerful tool for differentiating closely related filamentous fungi with very similar morphologies or ITS sequences, dissecting species complexes, and justifying newly described species.

In a polyphasic classification approach, stromata and cultures of Daldinia and allied Xylariaceae from around the world were studied for: (1) morphology of teleomorphs and anamorphs; (2) metabolite patterns in stromata and cultures, employing analytical HPLC-UV-visible (diode array) detection and mass spectrometric detection; (3) amplified 18S rDNA restriction patterns (ARDRA); and (4) PCR amplified minisatellite regions. Comparison of type material, herbarium specimens, cultures, and freshly collected material revealed new evidence on their geographic distribution. Several Daldinia spp. were identified in Europe and other locations for the first time. The results point towards the existence of further undescribed species. Stromata of a given species never contained the same major metabolites as corresponding cultures. Most cultures of Daldinia spp. produced naphthalene and chromane derivatives, differing from allied genera by the absence of mellein. Stromata of Daldinia spp. did not produce mitorubrin, but generally contained binaphthyls. Metabolite profiles were correlated with colours of KOH-extractable stromatal pigments. The yellow azaphilones and benzophenones found in D. childiae were lacking in species with purple stromatal pigments. Cytochalasins were found in stromata of D. eschscholzii. Genetic fingerprints helped to distinguish morphologically closely related taxa. ARDRA gave specific results for species whose 18S rDNA contained insertions, while minisatellite PCR provided specific genetic fingerprints. A combination of both PCR based techniques provided a fair resolution of genetic subtypes, reflecting the intrageneric variance in Daldinia as established from morphological data and secondary metabolite profiles.

Ephelis japonica and E. oryzae are biotrophic fungi that form systemic epiphytic associations with warm-season grasses. Ephelis has been recognized as the anamorph of Balansia and Myriogenospora, and a synanamorph of Atkinsonella; all three genera belong to Clavicipitaceae (Ascomycota). The teleomorphs have not been detected for E. japonica and E. oryzae. Balansia oryzae had been regarded as the teleomorph of Ephelis oryzae, however, this teleomorphic name is not validly published for the teleomorph alone (Art. 59·6). We analyzed the sequences of the ITS1, ITS2 and 5·8S rDNA regions of 33 Ephelis isolates from Japan, Korea, China, Nepal and India. Phylogenetic relationships of these isolates were analyzed, together with other clavicipitaceous fungi for which sequences were obtained from GenBank. All Asian Ephelis isolates formed a single cluster, which comprised two subgroups. One subgroup had strong affinity with B. andropogonis, an epibiont of grasses in tropical regions of Asia. The second subgroup had strong affinity with B. discoidea, an epibiont of grasses in the Americas. A close relationship was also shown to B. asperata, another epibiont of grasses of tropical regions of Asia. Our results do not justify the separation of Asian Ephelis epibionts into E. oryzae and E. japonica. E. japonica, established in 1904, ten years prior to the name E. oryzae, is the appropriate name for the asexual Ephelis epibionts of warm season grasses of Asia.

The distribution of the major components of the bolete mycobiota of South East Asia is considered and suggestions made as to their possible origin, mycogeography and relationships. The final number of species of boletes in South East Asia is likely to be well in excess of the number compiled in Corner’s 1972 monograph. Molecular information is incorporated into the theme where of interest.

Correct identification of fungi to species level is important because a specific epithet embodies a set of characters that enables us to predict, for example, the mycotoxin production of a species. Many small-spored Alternaria isolates have been misidentified due to inappropriate growth conditions and the use of spore size as the only identifying character. In this study 39 Alternaria isolates were grown under standardised conditions and subjected to chemical, morphological and physiological analyses. All isolates were extracted and analysed by HPLC–DAD. Analysis showed that both A. gaisen and A. longipes were able to produce altertoxin I, which has not previously been reported. The resulting metabolite profiles were subjected to cluster analysis and principal component analysis. A subset of the isolates was grown at five different temperatures. Colony colour and diameter were recorded and the diameter measurements were subjected to principal component analysis. Analysis of chemical and physiological data showed that the 39 isolates segregated into the same distinct groups that are morphologically identifiable as A. alternata, A. longipes or A. gaisen. The results showed that A. longipes, A. gaisen and A. alternata are different species that can be distinguished morphologically, physiologically and chemically. Therefore, the continued use of the name Alternaria alternata for A. longipes and A. gaisen is unwarranted and pathotypes should not be used.

Selected isolates of Phytophthora infestans from around England and Wales were fingerprinted using both RG57, a multi-locus RFLP probe, and Amplified Fragment Length Polymorphisms (AFLPs). The larger number of polymorphisms detectable with the AFLP method allowed resolution of several similar AFLP genotypes among isolates with identical RG57 fingerprints. However, some isolates with the same RG57 genotype had remarkably dissimilar AFLP genotypes, suggesting that there has been convergent evolution of some RG57 fingerprints. Also, some isolates with dissimilar RG57 fingerprints had similar or identical AFLP fingerprints. Both techniques distinguished isolates of mitochondrial DNA haplotype Ia from those of haplotype IIa. However, with AFLPs only, most of the isolates of A2 mating type were very similar and were distinguished from those of A1 mating type, suggesting that gene flow between A1 and A2 genotypes is limited and that sexual recombination is rare.

Suitability and reproducibility of ISSR–PCR to find strain-specific and taxon specific markers for strains of the tree-root endophytes Phialocephala fortinii and ‘Type 1’, a non-sporulating dematiaceous mycelium, were examined. The results were compared with data previously generated by isozyme analyses. P. fortinii and ‘Type 1’ are two DSE taxa and are abundant colonisers of coniferous forest-tree roots in the North Temperate zone. ‘Type 1’ was never observed to sporulate in pure culture but is well defined by its cultural characteristics. DNA of 14 strains per taxon was amplified with three short, 17–18-nucleotide-long, tandemly-repeated primers (CCA, CGA, ACA) with two (CCA) or three degenerated bases at their 5′-ends. The resulting DNA products were separated by agarose gel electrophoresis. The bands were scored for absence/presence, respectively, and the binary matrix subjected to multiple correspondence analysis (MCA). ISSR–PCR was found to be highly reproducible since amplification of DNA from several single-hyphal-tip cultures of the same strain resulted in identical banding patterns. Eighty-five (92.4%) of the 92 DNA fragments were polymorphic. The fragments ranged from 320 to 4100 bp. ISSR–PCR was found to be a powerful tool to find strain-specific and taxon-specific markers. Each strain showed a unique banding pattern and diagnostic bands for the two taxa could be identified. ISSR–PCR data correlated neither with the geographical nor the host origin of the strains. The strains grouped into similar clusters independently of whether MCA was performed with ISSR–PCR or isozyme data. However, ISSR–PCR allowed the differentiation of strains with the same allozyme phenotype.

A microsatellite-based DNA profiling system was developed that can be used to distinguish genetically diverse isolates of a forest pathogen, Dothistroma pini. All isolates of this pathogen identified in New Zealand so far appear to be isogenic and the disease is kept under control by aerial applications of copper fungicide. Although New Zealand has strict importation controls in place, the possibility of genetically diverse isolates of D. pini being introduced from other countries poses a severe biosecurity threat to forest health. Therefore a DNA-based monitoring system was developed. Two informative microsatellite loci were found serendipitously in D. pini sequence data available in our laboratory. Further microsatellite loci were obtained using a rapid 5′-anchored PCR amplification technique. For each informative locus identified, specific primers were designed to flank the repeated sequence and subsequently used to generate DNA profiles for the D. pini strains. The profiles obtained from five microsatellite loci were sufficient to distinguish most isolates tested. The anchored primer technique provides an efficient tool for the identification of polymorphic loci that can be used to screen for genetic differences between fungi.

Evidence that the biometric characters of the oogonial unit of Pythium are of significant taxonomic value, allowing for a good species separation, has been presented by other workers, based on 40 species, each represented by fairly few isolates. Since broad intraspecific variation and interspecific overlapping of biometric parameters was observed in isolates of four Pythium species from Denmark, it was suspected that a such wide variability would weaken the conclusions obtained by previous biometric approaches. Species to be studied should be represented by higher numbers of isolates to reflect intraspecific variation. In the present biometric study 49 isolates were used to test the robustness of the biometric approach and to examine the taxonomic value of biometric characters from a practical perspective.

Good species separation was obtained despite intraspecific variation. The biometric parameter set for the oogonium, consisting of oogonial, oospore and ooplast diameters, supplemented with the thickness of the oospore wall or the protoplast diameter and with derived indices of volume or linear ratios of the measured parameters were all of taxonomic value for the separation of species, as shown from canonical variate plots after linear discriminant analysis. More detailed information on species separation, isolate-species or isolate–isolate associations could be obtained by separation based on the Mahalanobis distance function in the linear discriminant analysis. Both the derived volume and linear indices contributed significantly to species separation and appeared to be equally satisfactory.

The substrates used for oogonium production may influence the biometric parameters to an extent which may seriously affect the results of the biometric approach to taxonomy. These results stress the importance of standardizing substrates in morphological taxonomy and identification work.

The aim of this study was to clarify the reproductive mode in the wood-rotting fungus Phellinus nigrolimitatus, using molecular markers and pairing experiments of cultural isolates. A 511 bp segment of the translation elongation factor 1a (EF1a) gene and the internal and intergenic transcribed spacer sequences (ITS and IGS) of the nuclear ribosomal DNA (nrDNA), were amplified by PCR from somatic cultures derived directly from basidiomes and single spore cultures of the fungus. Sequence analyses of the partial EF1a sequence (511 bp) and ITS (732 bp) of single spore cultures gave five and fourteen variable sites, respectively. Five somatic isolates yielded double ITS sequences, which suggested the existence of different ITS types within the same isolate. Restriction digestion of the partial EF1a and the complete ITS and IGS (3·4±0·2 kbp) sequences with HinfI and HaeIII gave restriction fragment length polymorphisms (RFLPs) among the isolates. The sum of fragments exceeded the lengths of the initial PCR product in several somatic isolates, which indicated the existence of EF1a and nrDNA heterotypes within isolates. Segregation of ITS and IGS RFLPs in single spore isolates suggested heterokaryosis in somatic isolates of P. nigrolimitatus. The polymorphic EF1a and ITS restriction sites were used as codominant markers in a test for Hardy–Weinberg expectations. The molecular data suggests a predominant outcrossing reproductive mode in P. nigrolimitatus. Results from cultural pairing experiments with single spore isolates corresponded poorly with the molecular data, suggesting that laboratory pairing experiments do not necessarily provide reliable information of the mode of reproduction in natural populations of fungi.

We have identified ten microsatellite markers in the entomopathogenic fungus Beauveria brongniartii from three genomic libraries enriched for (AAG)n-, (TG)n-, or (TC)n-repeats. The levels of polymorphism of the identified microsatellite loci were assessed in a collection of Beauveria strains originating from different countries, areas, and host insects. Two geographically separated Swiss populations of B. brongniartii originating from European cockchafer (Melolontha melolontha) were also analysed. Microsatellites containing (AAG)n-repeats generally displayed high levels of polymorphism, whereas microsatellites containing either (TG)n- or (TC)n-repeats displayed lower levels of polymorphism. Cluster analysis revealed that strains isolated from M. melolontha larvae, and two strains isolated from Melolontha hippocastani or Amphimallon solstitiale larvae, formed one cluster which was separated from strains isolated from adult M. melolontha and other adult insects. A high degree of biodiversity was detected among B. brongniartii strains of the two separated Swiss populations. Distinct haplotypes were identified in 29 of 35 B. brongniartii strains from population A and in 12 of 28 B. brongniartii strains from population B. The high discrimination power of the identified microsatellites makes them a valuable tool, suited for the characterization and identification of B. brongniartii strains used as biocontrol agents. In addition, they may be applied to monitor biological control strains of B. brongniartii in the field and possibly to investigate their interactions with indigenous B. brongniartii isolates.

Rhynchosporium alismatis, a pathogen of several Alismataceae species, is being studied in Australia as a potential weed biocontrol agent for Alisma lanceolatum, A. plantago-aquatica and Damasonium minus. Chlamydospores of R. alismatis are described for the first time. Large numbers (5.34×105 cm−2) were produced on potato dextrose agar within 8–15 d and range in size from 4.7 to 14 μm. There was variability between isolates in mean diameter but size was not related to the host of origin. Within 24 h at 30 °C 60% of chlamydospores, 8 d to 3 months old, germinated. Each germinated chlamydospore produced up to four germ-tubes. Chlamydospores were detected in leaf lesions on naturally-infected A. plantago-aquatica. One-month-old chlamydospores were pathogenic to leaf discs cut from mature leaves of A. lanceolatum and Damasonium minus. The relative robustness of chlamydospores is an advantage over conidia for the future production of a biological control agent if they can be produced and harvested in broth culture.