Two papers have been fast-tracked for publication in this issue. The first reports the transformation of the genome of Verticillium fungicola using PEG- and Agrobacterium-mediated methods; both were successful and provide a new tool for the study of the epidemiology of this important pathogen of cultivated Agaricus bisporus (pp. 4–11). In the second, double-stranded RNA mycovirus elements have been found in Rhizoctonia solani anastomosis group three (AG 3) and its transmission and elimination investigated (pp. 12–22).

Other molecular papers in this issue describe the development of primers for the identification of Alternaria species in carrots and compare the approach with other techniques (pp. 23–33); the diversity and molecular strain characterization of Lentinula edodes in Japan (pp. 34–39); the genotypic variation within Beauveria bassiana has been examined by a variety of methods and eight phylogenetic clusters found – which were not significantly correlated with the groups of insects from which they were obtained (pp. 40–50); the Eucalyptus pathogen Coniothyrium zuluense is established as present in Thailand, the first time the fungus has been found outside South Africa (pp. 51–59); and molecular data has been sued to show that the causal agent of Rhododendron anthracnose is Colletotrichum dematium (pp. 60-69).

Verticillium fungicola, a severe mycopathogen of the cultivated mushroom Agaricus bisporus, was successfully transformed using both PEG-mediated and Agrobacterium-mediated techniques. PEG-mediated co-transformation was successful with hygromycin B resistance (hph), uidA (β-glucuronidase GUS), and green fluorescent protein (GFP) genes. Agrobacterium-mediated transformation was successful with the hph gene. Transformation frequencies of up to 102 transformants per μg DNA and 4068 transformants per 105 conidia were obtained for PEG-mediated and Agrobacterium-mediated transformation respectively. Expression of integrated genes in co-transformants was stable after 18 months of successive sub-culturing on non-selective medium, and following storage at −80 °C in glycerol. Molecular analysis of PEG-mediated transformants showed integration of the transforming genes into the target genome. Molecular analysis of Agrobacterium-mediated transformants showed integration of transforming DNA as single copies within the target genome. Co-transformants exhibited symptoms of disease in inoculation experiments and were at least as virulent as the wild-type fungus. GFP and GUS expression were observed in-vivo with the GFP-tagged strain showing great potential as a tool in epidemiological and host-pathogen interaction studies. The development of transformation systems for V. fungicola will allow in-depth molecular studies of the interaction of this organism with A. bisporus.

All isolates of Rhizoctonia solani (teleomorph Thanatephorus cucumeris) anastomosis group (AG) 3 from potato tubers in commercial fields in Scotland were found to contain more than one double-stranded RNA (dsRNA) element ranging in size from 0·3 to 15 kb. Strains of other AGs (2-1, 3, 4, 5) from culture collections also contained dsRNA elements. Isolates of AG 3 from different black scurf lesions on single tubers showed similar dsRNA profiles and, when opposed on agar plates, underwent compatible hyphal fusion where the colonies met. Isolates from different tubers in the same commercial field often showed somatic incompatibility (mean 44%) leading to cytoplasmic death when colonies fused. Isolates from different fields always showed somatic incompatibility after hyphal fusion, suggesting that the complex population structure of AG 3 could significantly limit the transmission of dsRNA between strains. The incidence of different dsRNA elements in AG 3 isolates could not be related to pathogenicity in seedling tests on tomato, carrot, lettuce, radish, cress or onion. Double-stranded RNA elements could not be eliminated from AG 3 isolates by repeated hyphal-tip subculture or extended (8 week) incubation on agar containing cycloheximide. Some isolates lost the smaller dsRNA elements (1·0 to 1·6 kb) during these procedures, and one isolate (of AG 4) lost a 6·5 kb dsRNA element, but the larger elements (up to 9·5 or 12 kb) were always retained. When assessed by low stringency hybridisation, the dsRNA elements of R. solani AG 3 and of strains from AGs 2-1, 4 and 5 showed no homology with a plasmid containing cDNA of the complete hypovirus dsRNA genome of Cryphonectria parasitica.

Alternaria alternata, A. radicina and A. dauci are important seed-borne fungi on carrot, with the first two species having a high toxigenic potential, for which a specific and sensitive detection method is required. Because both the traditional deep-freeze-blotter method and plating on selective medium are time consuming and laborious, a PCR-based assay was developed. Sequences of the internal transcribed spacer regions of the ribosomal gene repeat (rDNA) from 45 different Alternaria isolates were determined, a restriction fragment length polymorphism analysis was performed and a phylogenetic tree was constructed. Based on the sequences, specific primers for detection and identification of the three Alternaria species on carrot seeds and roots were designed. The primers were highly sensitive and were shown to be able to differentiate between the three Alternaria species. A. alternata and A. radicina could be detected in DNA isolated from carrot material applying the specific primers, even at low infection levels. The PCR-assay was compared to the deep-freeze-blotter method (DFBM) and plating on Alternaria radicina Selective Agar (ARSA, for A. radicina) by testing naturally infected seed samples and root material. Results of the PCR-assay were similar to those of the blotter method and plating on ARSA for the detection of A. alternata and A. radicina. A positive correlation was found between the percentage of seed infection established by the blotter method and the intensity of the amplified, specific product. The PCR-assay based on the specific primers developed seems to be a good alternative for the deep-freeze-blotter method and plating on ARSA, especially when time is an important issue.

Amplified fragment length polymorphism (AFLP) analysis is based on selective PCR (polymerase chain reaction) amplification of genomic DNA restriction fragments. The present study was performed to: (1) assess the extent of AFLP variation among major cultivated strains of Lentinula edodes (the shii-take mushroom) in Japan; (2) evaluate the usefulness of AFLP as genetic marker for typing the cultivated strains; and (3) infer the genetic relatedness among them. Six AFLP primer pairs detected a total of 304 DNA fragments in a sample of 13 cultivated strains for wood log cultivation and 2 strains for sawdust cultivation, of which 179 DNA fragments (58·9% of detected fragments) were polymorphic between two or more strains. These polymorphic DNA fragments could differentiate all of the cultivated strains. Cluster analysis and principle coordinate analysis based on AFLP data showed two distinct groups; one group was composed of the strains in which fruiting occurred during the middle to low temperature period from autumn to next spring under outdoor wood log cultivation, and the other strains in which fruiting could be induced in the summer under outdoor cultivation and strains for sawdust cultivation under indoor air-controlled condition.

DNA sequence alignment of the nuclear 5·8S ribosomal RNA (rRNA) gene and internal transcribed spacers (ITS) from Beauveria bassiana demonstrated that 6·62% sequence variation existed between nine isolates. A higher level of mutation was observed within the ITS regions, where 8·39% divergence occurred. Polymerase chain reaction restriction fragment length polymorphism, PCR–RFLP, and DNA sequence alignment of the ITS1 and ITS2 regions identified seven polymorphic restriction endonuclease sites, Alu I, Hha I, Hinf I, Sin I, Tru 9AI and two Tha I restriction sites. The allelic frequency of each genetic marker was determined from 96 isolates. PCR–RFLP defined 24 B. bassiana genotypes within the sample set, from which eight phylogenetic clusters were predicted to exist. AMOVA and Fst (θ) indicated that no significant correlation existed between B. bassiana haplotype and insect host range as defined by insect order from which each isolate was derived.

Coniothyrium canker, caused by Coniothyrium zuluense, is a serious stem canker disease of Eucalyptus in subtropical parts of South Africa. A Coniothyrium associated with similar symptoms to those in South Africa was observed on E. camaldulensis in 1996 in Thailand. It was previously thought that C. zuluense was restricted to South Africa. This study compares South African isolates of C. zuluense with isolates of the Coniothyrium from Thailand. Results of morphological comparisons indicate that the South African and Thailand isolates are the same. This was further confirmed when all Coniothyrium isolates associated with stem cankers on Eucalyptus spp. grouped together in a single major clade for both rDNA sequence data and AFLP analysis. This clade was distant from isolates of other Coniothyrium spp. included for comparative purposes. Although the Coniothyrium isolates from South Africa and Thailand resided in two separate clades, these were closely related and, we believe that the isolates from Thailand represent C. zuluense. This is the first record of the important Eucalyptus stem canker pathogen outside South Africa.

Anthracnose caused by Colletotrichum is a severe foliar disease in rhododendron plantations in Sweden and Latvia. Isolates of this pathogen were collected and characterised based on morphological and molecular criteria. Out of 37 isolates examined, two with falcate spores were identified as C. dematium. The remainder of the isolates had straight cylindrical conidia, which were too variable to be reliably assigned to C. gloeosporioides (syn. C. azaleae) the reported causal agent of anthracnose of Rhododendron spp. These isolates were compared with the reference strains of C. gloeosporioides, with holo- and paratypes and with reference isolates of C. acutatum. The light and scanning electron microscopy examinations revealed that conidial shape was not a reliable criterion for separation of these two taxa and for the identification of our isolates. The two internal transcribed spacers (ITS1 and ITS2) and the 5·8S rRNA gene were PCR amplified and sequenced from all the rhododendron isolates and the reference strains, including the holo- and paratype materials. In addition, a segment of mitochondrial small subunit ribosomal DNA (mtSSU rDNA) and a fragment of a β-tubulin gene from several representatives of isolates of rhododendron as well as the reference strains were amplified and sequenced. The sizes of the amplified fragments among the isolates studied were heterogeneous only for mtSSU rDNA, which allowed separation of C. acutatum from C. gloeosporioides. Parsimony analysis of the individual and combined nucleotide sequence data sets were concordant and indicated that all isolates originating from rhododendrons belonged to C. acutatum. The grouping of the isolates was further supported by bootstrap analysis. Thus, C. acutatum was identified as the causal agent of anthracnose of Rhododendron in Sweden and Latvia. To our knowledge, this is the first report of C. acutatum and C. dematium on Rhododendron. This study is also the first to determine the genetic status of the holotype of C. acutatum and also one of the paratypes on the basis of ITS sequences in comparison with world-wide living isolates of this taxon.

The ergocalciferol (vitamin D2) content of individual fruit bodies of the edible mushroom Cantharellus cibarius (chanterelle) was measured with HPLC 2–6 years after drying. The concentrations of ergocalciferol in different fruit bodies varied between 0·12 and 6·30 μg g DW−1, with a mean of 1·43 μg g DW−1. This result indicates that dried and stored chanterelles contain significant amounts of ergocalciferol. The huge range in concentration challenges the validity of food tables based on means from pooled samples. The range may reflect differences in exposure to sun light, as fruit bodies within the same cluster showed little variation. Our study indicates that mycelial cultivation for ergocalciferol production is an interesting prospect if strains and incubation conditions are selected carefully. No significant difference in ergocalciferol concentration between pigmented and albino forms was found.

Eight species of ectomycorrhizal (ECM) fungi in the genera Amanita, Gymnoboletus, Lactarius, and Russula were isolated from subtropical plant communities in eastern Australia. Two species were isolated from each of rainforest, Nothofagus forest, Eucalyptus forest, and Eucalyptus dominated wallum (heath) forest. These communities differ strongly in their soluble soil nitrogen (N) composition. The ability of the fungi to use inorganic (nitrate, ammonium) and organic (amide, peptide, protein) nitrogen sources was determined. As the fungi did not grow in liquid culture, a ‘floating culture’ technique was devised that allows hyphal growth on a screen floating on liquid medium. With some exceptions, fungal biomass production in floating culture closely reflected fungal growth on solid media assessed by total colony glucosamine content. Most isolates grown in floating culture had similar glucosamine concentrations on all N sources, with isolate specific concentrations ranging from 6 to 12 μg glucosamine g−1DW. However, Russula spp. had up to 1·7-fold higher glucosamine concentrations when growing with glutamine or ammonium compared to nitrate, glutathione or protein. Floating cultures supplied with 0·5, 1·5, 4·5, or 10 mM N mostly produced greatest biomass with 4·5 mM N. In vitro nitrate reductase activity (NRA) ranged from very low (0·03 μmol NO2 g−1 fw h−1) in Russula sp. (wallum) to high (2·16 μmol NO2 g−1 fw h−1) in Gymnoboletus sp. (rainforest) and mirrored the fungi's ability to use nitrate as a N source. All Russula spp. (wallum, Nothofagus and Eucalyptus forests), Lactarius sp. (rainforest) and Amanita sp. (wallum) utilized ammonium and glutamine but had little ability to use other N sources. In contrast, Amanita species (Nothofagus and Eucalyptus forests) grew on all N sources but produced most biomass with ammonium and glutamine. Only Gymnoboletus sp. (rainforest) showed similar growth with nitrate and ammonium as N sources. Fungal N source use was not associated with taxonomic groups, but is discussed in the context of soil N sources in the different habitats.

Production of lignin-modifying enzymes by 10 white-rot fungi, as measured by decolorization of Poly R 478 dye, varied in response to different carbon and nitrogen regimes. Fastest decolorization rates were achieved with wood polysaccharide monomers (glucose, xylose) as carbon source, although cellulose was the only carbon source to facilitate decolorization by all test fungi. Enzyme production by most isolates was strongly dependant on nitrogen levels, with high nitrogen conditions generally suppressing enzyme production. One unidentified isolate (HKUCC 4062) displayed apparent nitrogen deregulation of enzyme production. A cellulose-low nitrogen growth medium is recommended for agar-based screening procedures of lignin-modifying enzyme production by white-rot fungi.

Epichloë spp. (Clavicipitaceae) and their close asexual relatives, Neotyphodium spp., form systemic endophytic associations with Pooideae grasses. Interactions between Lolium and Festuca host grasses and fungal endophytes were examined in studies focusing on leaves of natural associations and also of plants into which the endophyte was introduced by seedling inoculation. Light microscopy as well as fungal isolation was used to locate the position of hyphae, while transmission electron microscopy was used to examine host/fungus interactions at the cellular level. These studies provide support for synchronised plant and endophyte growth. This characteristic pattern of growth was maintained when both Epichloë and Neotyphodium spp. were introduced by seedling inoculation into new host species. Hyphae, with few exceptions, grew rapidly as leaves grew and ceased when leaf growth ceased. This pattern of growth offers an explanation for the characteristic appearance of hyphae in leaf sheaths of host grasses; seldom branched and for all species other than N. occultans, aligned parallel to the leaf axis. Hyphal growth of a second group of endophytic fungi, referred to as p-endophytes, was not regulated in the same way, with growth continuing as leaves aged. This pattern of growth gives rise to high concentrations of branched ramifying hyphae in old leaf sheaths. Although host genotype did not effect the basic pattern of hyphal growth of Epichloë/Neotyphodium endophytes, it strongly influenced the concentration and distribution of hyphae throughout leaves. Examination by TEM revealed no evidence that penetration of hyphae into dense tissue was aided by the secretion of pectic enzymes that loosen the middle lamella connecting host cells. Instead it appeared that penetration occurred by hyphae physically pushing between cells. The absence of enzymatic loosening of the middle lamella during penetration could explain why hyphae of these endophytes apparently do not elicit host defence reactions in natural associations. However, subtle interactions between the hyphae of Epichloë/Neotyphodium and host cells in natural associations were observed.

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

A new pyrenomycete, Monosporascus ibericus sp. nov., isolated as an endophyte from roots and stems of three plant species growing on sand flats and salt marshes in the Ebro Delta (Spain), is described. The main characteristic of this fungus is the presence of a higher number of ascospores per ascus (up to six), compared to the other species of the genus: M. cannonballus, M. eutypoides, and M. monosporus. M. ibericus produces a cleistothecial ascomata with a tomentose peridium and lacks an anamorph. In addition to the morphological data, the comparative analysis of the ITS-region sequences of M. ibericus and the other Monosporascus spp., has supported the recognition of the new species. A phylogenetic study based on the sequences of the 18S rDNA did not allow us to assess clearly the taxonomic position of the genus Monosporascus, although the results indicated the genus might have affinities to the Xylariales rather than to the Sordariales.