Two PCR primers (MSU1 and MSU7) were designed to amplify part of the mt SSU rDNA gene from filamentous ascomycetes. The MSU1-MSU7 and MSU1-NMS2 primer pairs were tested with 32 isolates of 23 species in 10 genera. Minimum evolution, parsimony, and likelihood methods were used for the phylogenetic analyses of mt SSU rDNA sequences from Botryosphaeria species and related taxa. These analyses indicated that: Botryosphaeria is not monophyletic; the separation of Botryosphaeria from Guignardia is supported; except for B. vaccinii, other Botryosphaeria species and related taxa form a monophyletic group with strong jackknife support; Diplodia pinea f. sp. cupressi is different from RAPD marker groups A and B of Sphaeropsis sapinea; the anamorph genera Sphaeropsis and Diplodia are not indicative of close relationships among different species; and the name B. stevensii may have been applied to more than one species. Botryosphaeria needs additional study and substantial taxonomic revision.

This issue of Mycological Research News points out that Micropeltopsis quinquinecladiopsis belongs in Lichenopeltella, and features a record number of ascomycetes found on a single tree in Papua New Guinea, and the rules of fungal melanins.

This part of Mycological Research includes 14 papers. The first reports the transformation of the endophyte Neotyphoidiumlolii with a green fluorescent protein gene. Molecular systematic studies clarify generic and species concepts in Phaeoacremonium and Phaeomoniella, reassess Neofabraea species, compare species of Hypoxylon and allied genera, clarify the identities of Oidium powdery mildews on tomato, and provide evidence for the reproductive mode in Phellinus nigrolimitatus.

The intracellular peptidases and proteinases of Serpula lacrymans have been investigated, and conidial discharge in Erynia neoaphidis examined.

Developmental studies are reported on apothecia of Pyrenopeziza brassicae and basidiomes in Mycena stylobates, and the germination of ascospores of Monosporascus cannonballus documented. The moss parasites Eocronartium and Jola have been studied in culture to elucidate their life-histories and revealed anamorphs.

Additional fungi have been found in the hypersaline Dead Sea, and seven new neotropical smut species are described.

The following new scientific names are introduced: Aurantiosporium pallidum, Kuntzeomyces ruiziana, Moreaua bulbostylidis, Oidium neolycopersici, Phaeoacremonium mortoniae, Thecaphora amaranthicola, T. smallanthi and Tilletia boliviana spp. nov.; and Lichenopeltella quinquinecladiopsis (syn. Micropeltopsis quinquinecladiopsis) comb. nov.

The development of a transformation system is a significant technical hurdle in the study of many filamentous fungi. Recently Agrobacterium tumefaciens was shown to transform several filamentous fungi, but until now only one independent confirmation of this promising finding has been published. Fusarium circinatum (teleomorph Gibberella circinata) is an important pathogen of pine that has not been transformed previously. A. tumefaciens strain AGL1(pPK2) transformed three different isolates of F. circinatum with an efficiency of 2–150 transformants 10−5 conidia. The T-DNA was integrated into the genome and was stable through mitotic and meiotic cell divisions. Most F. circinatum transformants contained a single T-DNA copy. Half of the tested transformants also contained non-T-DNA vector sequences. These findings should facilitate future analysis of F. circinatum pathogenicity and stimulate wider use of this valuable transformation method in fungal research.

Laccaria bicolor and L. laccata are ectomycorrhizal basidiomycetes which fruit abundantly over 1 or 2 months in autumn and sometimes in summer. We investigated the phenology of fruiting of the various Laccaria spp. genets in a Douglas fir plantation in the Nièvre (France), in which all genets had been identified by molecular methods. During the fruiting period of the population, the genets did not fruit synchronously: some fruited early, others later, and some over the whole period. For some genets that survived over 2–3 years (1995–97), the fruiting phenology remained similar from year to year, even when fruiting occurred in summer. We took advantage of the presence of a L. bicolor genet, which had been used to artificially inoculate Douglas firs in this plantation, to assess whether the timing of fruiting was influenced by microsite differences in the plantation (soil, host tree, etc.). In summer 1997, fruit bodies of this genet appeared synchronously everywhere at the beginning of the fruiting period. We conclude that the timing of Laccaria fruiting may be genetically determined, rather than explained by the size and position of the genet or the environmental conditions. The consequences for population studies are discussed.

Fungi developing on dung samples, from a wide range of locations and incubated in moist chambers, were recorded. Highly significant differences were found among the mycobiota of different dung types, from different latitudinal ranges, and collected at different seasons. Sheep, cattle, deer, rabbit, hare and grouse provided 86 % of the 425 samples. Highly significant differences in the community composition of the mycobiota of these six dung types were observed. Coprinus stercoreus was the commonest basidiomycete, and most frequent on sheep, cattle, deer and rabbit; C. miser was the only common species, of any taxonomic group, which showed no significant difference in its frequency on different dungs. Apothecial fungi comprised about a quarter of the records, with Ascobolus, Saccobolus and Thelebolus spp., Iodophanus carneus and Lasiobolus cuniculi the commonest. A. carletonii occurred uniquely, but frequently, on grouse. Perithecial species, especially of Schizothecium, Podospora, Coniochaeta and Sporormiella, accounted for ca 50% of all records. The grouse dung mycobiota was markedly different, with a lower species richness (mean 3.2 per sample), from that of the mammalian herbivores, which had means of 9–12 spp. per sample. In temperate latitudes more species were recorded on samples in winter. Species richness was greater in samples from lower latitudes. Significant differences in the composition of different dung types are discussed in relation to their fungal communities. The usefulness of studying the fungi of the dung microcosm as a means of rapid biodiversity assessment is considered, by comparing the results with those of a similar, but smaller, study 30 years earlier, and by examining the diversity of a sample from a subjectively poor habitat in Morocco. There was no indication of any major change in the coprophilous mycobiota that might be associated, e.g. with climatic change or changed farming practice over the last three decades. The Moroccan samples had a much lower diversity (80 spp./50 samples) than would be expected of samples from that latitude (146 spp./50 samples) based on the worldwide data set.

Video microscopy was used to examine a genetically tagged mutant (T30) of Glomerella graminicola whose conidia have a propensity to burst during the germination process. Before the germ tube is produced, the cell wall ruptures and the cytoplasm is extruded. The bursting takes place in less than 0.1 second. Bursting can be prevented by adding osmotica to the germination medium. By phase-contrast microscopy of alkali-washed conidia, we found that each ruptured conidium had a single gaping hole that was oriented parallel to the long axis of the spore. The holes were more frequent in the middle region of the conidia than at the apices. A mathematical model for stress based on conidial shape indicated that conidia ruptured where the stress on the wall was greatest. The microscopic observations on the orientation and distribution of the rupture sites are consistent with the hypothesis that T30 conidia have weaker walls than the wild-type. Comparative tests of resistance to mechanical breakage support this hypothesis.

Since 1993, a hitherto unidentified Phytophthora species has been found associated with twig blight disease in Rhododendron and, sporadically, Viburnum. The morphology and growth characteristics of fourteen isolates from Germany and the Netherlands were investigated, together with their breeding system, the internal transcribed spacer (ITS) regions of the ribosomal DNA, amplified fragment length polymorphism (AFLP) fingerprints, and isozyme profiles, which were compared to those of a number of outgroup species. Morphologically the isolates are characterized by abundant production of chlamydospores and elongate, ellipsoid, deciduous sporangia with a short pedicel, in which they resemble P. palmivora. However, sporangia were semi-papillate, chlamydospores were much larger and cardinal temperatures much lower than those of P. palmivora. Oogonia with amphigynous antheridia and plerotic oospores were produced in dual cultures with an A2 mating type strain of P. cryptogea. ITS1 and ITS2 sequences of the unidentified species were closest to those of P. lateralis, but differed in three and eight nucleotides respectively from the latter species. AFLP fingerprints and isozyme patterns of malate dehydrogenase (MDH-2) and malic enzyme (MDHP) showed that the isolates formed a homogeneous group, distinct from all examined outgroup species, including P. lateralis. It was concluded that they represent a new Phytophthora species, described here as P. ramorum sp. nov. In pathogenicity tests all isolates of P. ramorum were pathogenic to Rhododendron.

Based on 43 taxa, the relationships of the European poroid Hymenochaetales were examined by molecular sequence data of a region of the large subunit of the nuclear-encoded ribosomal DNA. The molecular data were discussed referring to morphological and anatomical features of the fruit bodies, nuclear behaviour of the vegetative mycelium, and the pattern of reproduction. As a result, the genera Phellinus s. lat. and Inonotus s. lat. are subdivided into five subunits each. The following genera are accepted: Phellinus s. str. (P. igniarius group), Porodaedalea (P. pini group), Fomitiporia (P. robustus group), Fuscoporia (P. ferruginosus and relatives, including P. torulosus and P. wahlbergii), Phellinidium (P. ferrugineofuscus and relatives), Phylloporia (P. ribis) and Inonotus s. str. (I. hispidus and relatives), Inocutis (I. rheades group), Mensularia (I. radiatus and relatives), Pseudoinonotus gen. nov. (I. dryadeus and relatives), and Inonotopsis (I. subiculosus). Their relationships to non-European members of these groups are discussed. Onnia and Coltricia were confirmed as separate genera. Phaeolus schweinitzii was placed outside of the Hymenochaetales.

Species of Calonectria represent an important group of plant pathogenic fungi that cause serious losses to crops in tropical and subtropical climates. Because these fungi are difficult to identify, selected species were subjected to DNA analysis with the aim of producing a more objective identification technique. This was done by amplifying the 5′ end of the β-tubulin gene from isolates representing more than 30 species of Calonectria. A neighbour joining analysis was performed on a total data set of 86 isolates, while a representative subset of isolates was analysed by means of maximum parsimony. Both analyses yielded dendrograms with concordant topology. Several clades were strongly supported by bootstrap, confirming the morphological and biological species concepts presently employed. At least two large groupings were evident and both contained small-spored, 1-septate species. The only morphological character that correlated with DNA phylogeny was vesicle shape. These data support Calonectria as a monophyletic genus with Cylindrocladium anamorphs. Furthermore, this study also emphasises the importance of vesicle morphology in identifying Cylindrocladium anamorphs, and hence species of Calonectria.

The phylogeny of the Agyriales was investigated using nucleotide sequences of ITS1, 5.8S, and ITS2 rDNA. Sequences of these regions from 15 new sequences of 13 agyrialean fungi were aligned to those of four representatives of Ostropales used as outgroup. Six different alignments were analysed cladistically using maximum parsimony. The Agyriales were supported as a monophyletic group in the strict consensus tree and the synonymy of the Rimulariaceae and Saccomorphaceae was confirmed. The Schaereriaceae is also shown to belong to the Agyriaceae. The distribution of selected characters in the Agyriales is investigated and it is shown that the ascus type is variable within the group, while the ascoma ontogeny is invariant within the Agyriaceae. Trapelia as usually circumscribed is polyphyletic. The T. geochroa group is not closely related to Trapelia s. str. and the new genus Ainoa is described to accommodate T. geochroa and T. mooreana.

The green fluorescent protein gene was inserted into isolates of the endophyte Neotyphodium lolii from perennial ryegrass Lolium perenne using the method with polyethylene glycol treatment of protoplasts. The transformation rate was 3·2×10−6 of the original number of protoplasts 5 weeks after the transformation. Freshly isolated mycelium from the host plant was used in the transformation experiments, since difficulties of infecting the host plant with older isolates were observed. Isolates were identified using the universal primed (UP) PCR fingerprinting method.

The transformed mycelium readily expressed the green fluorescent protein gene. Fluorescence was observed in the cytoplasm of the mycelium cells in pure culture, both before infection and after re-isolation from the host plant. The fluorescent hyphae were easily detected in the host plant. No disturbing autofluorescence from the host plant leaf sheaths or from the wild type isolate was observed. The importance of mitotic stability is discussed, based on the transformed phenotype and southern hybridization pattern, since the Neotyphodium lolii transformants lost 1–2 copies of the integrated gene after re-isolation from the host plant without losing their GFP phenotype. This is the first report on transformation of a Neotyphodium sp. with a gfp gene. In the future, the green fluorescent protein may serve as a tool for studying gene expression and protein localization in the endophyte/host plant interaction using heterologous fusion proteins.

In order to estimate phylogenetic relationships in the Auriculariales sensu Bandoni (1984) and allied groups we have analysed a representative sample of species by comparison of nuclear coded ribosomal DNA sequences, applying models of neighbour joining, maximum parsimony, conditional clustering, and maximum likelihood.

Analyses of the 5′ terminal domain of the gene coding for the 28 S ribosomal large subunit supported the monophyly of the Dacrymycetales and Tremellales, while the monophyly of the Auriculariales was not supported. The Sebacinaceae, including the genera Sebacina, Efibulobasidium, Tremelloscypha, and Craterocolla, was confirmed as a monophyletic group, which appeared distant from other taxa ascribed to the Auriculariales. Within the latter the following subgroups were significantly supported: (1) a group of closely related species containing members of the genera Auricularia, Exidia, Exidiopsis, Heterochaete, and Eichleriella; (2) a group comprising species of Bourdotia and Ductifera; (3) a group of globose-spored species of the genus Basidiodendron; (4) a group that includes the members of the genus Myxarium and Hyaloria pilacre; (5) a group consisting of species of the genera Protomerulius, Tremellodendropsis, Heterochaetella, and Protodontia. Additional analyses of the internal transcribed spacer (ITS) region of the species contained in group (1) resulted in a separation of these fungi due to their basidial types.

Two non-papillate species of Phytophthora are the principal cause of pistachio gummosis in Iran. Their previous description as P. megasperma and P. drechsleri was re-examined in the light of RFLPs and sequence comparisons of internal transcribed spacer (ITS) regions of rDNA. Both taxa were more closely related to a clade comprising P. sojae, P. cajani, P. vignae, P. melonis and P. sinensis than to the unrelated P. megasperma s. str. or P. drechsleri. The P. megasperma-like isolates from pistachio differed from all the above taxa in morphology, ITS sequence and AFLP patterns, and are described as a new species, P. pistaciae. The P. drechsleri-like isolates from pistachio had identical ITS sequences to those of P. melonis, P. sinensis and P. drechsleri-like isolates from cucurbits in Iran and the AFLP profiles of P. melonis, P. sinensis and the P. drechsleri-like isolates from pistachio were virtually identical. This and other published isozyme and mtDNA evidence suggests these taxa should be considered conspecific and all subsumed within P. melonis with a revised host range and geographical distribution. The relationships of P. melonis and P. pistaciae with other members belonging to the same ITS clade are considered.

Microbotryum violaceum (anther-smut) from Silene caroliniana and S. virginica produced promycelia that frequently appeared to contain more than three cells according to the number of septations. This is in contrast to samples from S. latifolia and to previous descriptions of this species. Apparent four-celled promycelia contained three nucleate cells and one anucleate zone. Anucleate zones were usually positioned between the first and third cells of the promycelium. Their presence was negatively correlated with the occurrence of intra-promycelial conjugation. Also, unlike isolates from Silene latifolia, isolates from S. caroliniana and S. virginica produced hyphae in the absence of an inducing agent.

The ecologically and economically important arbuscular mycorrhizal (AM) fungi, crucial in the ecology and physiology of land plants, and the endocytobiotic fungus, Geosiphon pyriformis, are phylogenetically analysed by their small subunit (SSU) rRNA gene sequences. They can, from molecular, morphological and ecological characteristics, unequivocally be separated from all other major fungal groups in a monophyletic clade. Consequently they are removed from the polyphyletic Zygomycota, and placed into a new monophyletic phylum, the Glomeromycota.

The recognition of this monophyletic group, which probably diverged from the same common ancestor as the Ascomycota and Basidiomycota, gives these fungi their proper status, and provides a basis for a new and natural systematics of these fascinating, yet largely hidden organisms, with three new orders (Archaeosporales, Paraglomerales, Diversisporales) described herein. Additionally, several clades resolve at family level; their formal description is in progress.

Techniques to rapidly identify the basidiomycete fungal partner of ectomycorrhizal associations would be a major advantage for ecological, fungal population dynamics and life history studies of epigeous and hypogeous forms in plantations, forests, wild lands and other native or natural vegetation. PCR–RFLP (Polymerase Chain Reaction–Restriction Fragment Length Polymorphism) identification of DNA regions is an available technique; however, primers which have a high probability of amplifying only the basidiomycete DNA are needed. Here we have assessed the specificity, sensitivity and discrimination of six different primer pairs, three targeting nuclear and three mitochondrial regions, for use in identification of Australian basidiomycete fungi from Eucalyptus forests by matching PCR–RFLP patterns to morphologically defined species. Two sets of primers, one newly designed and targeting the nuclear ribosomal DNA internal transcribed spacers (ITS) and the other amplifying a fragment of mitochondrial large subunit ribosomal DNA met the requirements of high specificity and sensitivity, amplifying DNA from a broad range of larger basidiomycetes, with no amplification of plant, bacterial or ascomycete DNA. The specificity of the ITS primer pair was compared with that of ITS1-F/ITS4-B. PCR–RFLP of the two regions discriminated fungi to species level for 91 fungal species from 28 families. Hence these two DNA regions and the specific primers are a potential practical PCR–RFLP tool for identifying basidiomycetes associated with plants from field samples.

Optimization of protoplast release (202×104 ml−1) from a strain of Rhizoctonia solani anastomosis group three was achieved by treating 3 d old mycelia from broth cultures with Novozyme 234 for 3 h at 30 °C. The only other treatment, of many tested, that yielded any protoplasts (15·6×104 ml−1) was commercial lysing enzymes from Trichoderma harzianum. Various combinations of commercial enzyme preparations, variations of osmotic stabilisers, and mycelial age, were ineffective in raising the protoplast yield. Regeneration of colonies from protoplasts was maximal (83–87%) when protoplasts were incorporated in cooled agar containing 5% glucose, malt extract and yeast extract, supplemented with 0·8 or 1·0 M mannitol as osmotic stabiliser. Liquid culture broth and other potential osmotic protectants at various concentrations yielded fewer regenerating colonies (5% in broth and 13–75% in agar). The Rhizoctonia strain was highly sensitive to hygromycin B, so transformation of protoplasts was achieved with a polyethylene glycol-based procedure, using plasmid pES200 containing the bacterial hygromycin B phosphotransferase gene driven by a trpC promoter from Aspergillus nidulans. Transformation was achieved using the DNase inhibitor aurintricarboxylic acid and incubation temperatures below 10–12 °. It was confirmed by high stringency hybridization of DNA extracted from the transformants, using pES200 as a radiolabelled probe. The transformants grew slowly (25–35 mm diam after 14 d) then ceased growth, and never produced fast-growing sectors indicative of integrated plasmid DNA. Transformation was also achieved with pAXHY2 from Cryphonectria parasitica, and colonies reached 55–65 mm diam before they stopped growing. Southern blots confirmed the presence of non-integrated pAXHY2, but no fast-growing sectors were produced.