A study was designed to isolate and identify the endophytic mycobiota in leaves and twigs of Rubus spectabilis and R. parviflorus, the long-term goal being biological control of these plants with endophytes. Endophytic fungi were isolated from healthy-looking leaves and twigs collected from each species on Vancouver Island, British Columbia, Canada. More than 90% of the leaves of both species were colonised by endophytes. In addition, one third of the twig samples of each species were also colonised. Most isolates belonged to Phomopsis, and five species were identified and characterised according to morphology of the cultures and fructification structures. All Phomopsis species occurred on both Rubus species, but, one primarily colonised R. parviflorus, whereas another preferentially colonised R. spectabilis. The suitability of the isolated endophytes to inhibit growth of R. spectabilis and R. parviflorus, which compete with conifers in reforestation sites, is discussed.

A new species of Chaetomium, C. cuyabenoensis, is described from the Ecuador rain forest. It is similar to Chaetomium longicolleum but differs by having smaller and, in face view, strongly limonifom to quadrangular ascospores.

The nematophagous fungus Pochonia chlamydosporia is a potential biocontrol agent against root knot and cyst nematodes. Genetic transformation of the fungus to introduce visual marker genes, novel traits, or changes in expression levels of endogenous genes, would greatly enhance understanding of its behaviour on nematode-infested roots and of its interactions with other soil and rhizosphere microorganisms. A transformation system for the introduction of novel genes into P. chlamydosporia has been developed. Methods to generate protoplasts, introduce DNA and regenerate transformed viable fungal mycelium have been optimised, using plasmids carrying the green fluorescent protein marker gene gfp and the hygromycin resistance gene hph. Cultures of P. chlamydosporia were resistant to high levels of a range of fungal inhibitors, including hygromycin, that are commonly used with dominant selectable marker genes in the transformation of other fungi. However, regenerating protoplasts transformed with hph could be selected by their ability to grow through an agar overlay containing 1 mg ml−1 hygromycin. Green fluorescence was observed in protoplasts and regenerating mycelium after transformation with gfp, but the GFP phenotype was lost on subculture. Maintenance of introduced genes was not stable, and during subculture, PCR assays indicated that the transformants lost both hph and gfp. When these genes were introduced on the same plasmid, segregation of hph and gfp was observed prior to their loss. It was unclear whether the introduced plasmids were able to replicate autonomously in P. chlamydosporia, or if they integrated transiently into the fungal genome. Possible reasons for the instability of the transformants are discussed.

Unidentified isolates of Cylindrocladium with 1–3-septate conidia and clavate vesicles were compared with C. theae, C. colhounii var. colhounii, C. colhounii var. macroconidiale, C. gracile, C. pteridis and Calonectria gracilis. Ribosomal DNA (rDNA) of these isolates were digested with the restriction enzymes EcoR I, Hind III and Xho I, and Southern analysis performed with the 6·3-kb rDNA repeat unit of Neurospora crassa as DNA probe. Based on differences in general morphology, supported by their rDNA restriction fragment length polymorphisms, isolates of all species and varieties could be distinguished. Furthermore, the Cylindrocladium anamorph of Calonectria gracilis was shown to be distinct from Cylindrocladium pteridis and C. gracile. The name Cylindrocladium pseudogracile sp. nov. is, therefore, proposed for the undescribed anamorph of Calonectria gracilis.

Stilbohypoxylon is resurrected. The type species, S. moelleri, is redescribed and S. samuelsii is described as new. The third species, S. quisquiliarum, represents a new combination. A key to these taxa is provided.

A study on the mangrove leaf litter fungi of two tropical mangroves in Singapore was carried out. Five Halophytophthora species and four isolates which did not fit the descriptions of known species were isolated from leaves of Avicennia alba, A. rumphiana, Rhizophora apiculata and R. mucronata. Halophytophthora exoprolifera, H. kandeliae, H. operculata, H. spinosa var. lobata and H. vesicula are new records for Singapore; the first three also being new records for tropical mangroves. The isolate of H. exoprolifera sporulated at 28 °C and may be a tropical strain. H. operculata is reported for the first time outside of Australia. The results of this study contribute to the little known distribution of the Halophytophthora in the Indian Pacific region. Thirteen deuteromycetes were also isolated from the mangrove leaf litter and represented both phylloplane fungi as well as possible marine fungi.

The relationship between hyphal growth and branching of the grape pathogen Botrytis cinerea was determined on solid media containing either glucose, fructose, sucrose, tartaric acid or malic acid. The concentration of the carbon source had little effect on specific growth rate or the specific rate of tip formation, but growth was inhibited at high concentrations of tartaric and malic acids. Hyphal growth unit length and hyphal extension rate increased with increasing sugar concentration and were always significantly greater than values on tartaric or malic acids. The data provide an explanation for colonization patterns of grape berries. Growth will be poor during the period from setting to the onset of ripening, when organic acids are the main carbon source produced by the berry. Following the onset of ripening, the production of sugars provides more favourable carbon sources for the fungus, enabling achievement of higher specific growth rates, greater hyphal extension rates and, hence, greater colonizing potential.

The electrophoretic karyotype (EK) of 24 strains of Botryotinia fuckeliana was resolved to determine the extent of chromosomal polymorphism among them. Based on EK variation, nine different profiles with a number of bands ranging from 5 to 8 were found. Those profiles were not related to the origins of the strains, hosts, or year of isolation. Inheritance of chromosomal bands was studied by analyzing the EK of the progeny from crosses between sexually compatible B. fuckeliana strains that had different karyotype. The EKs of 44 monoascospore strains showed 12 new EK profiles, suggesting that chromosomal rearrangements generated after meiotic recombination provides EK variability in this fungus. Also, we report here that the degree of EK variability indicates that sexual reproduction under natural conditions might be quite important in the life cycle of B. fuckeliana. Hybridization analysis showed that, in most strains, the rDNA is located in both a chromosomal band of high molecular weight and different size depending on the strain and a band of low molecular weight, revealing evidence for chromosome rearrangements in this plant pathogen.

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

The occurrence of an undescribed ergot species, Claviceps citrina, on the halophytic chloridoid grass Distichlis spicata, in the Texcoco region of central Mexico, is reported. RAPD and ITS1 sequences of this fungus were compared with other Claviceps species and the morphological uniqueness of the fungus was reinforced. Its sclerotia do not contain any alkaloids of the ergoline type. Germination of sclerotia occurred immediately after placing them on humid sand. After removal of the capitula, the remaining stipe was able to regenerate the capitulum.

As a later homonym of Mycosphaerella aggregata (Schweinitz) Stevenson the name Mycosphaerella aggregata Carnegie & Keane is illegitimate. The name Mycosphaerella gregaria is proposed as a replacement with similar etymology. The original description is revised following further collections and includes new hosts and cultural characteristics. Originally thought to be a saprophyte, this species may be a primary pathogen of leaves of Eucalyptus.

A xylanase gene, XYL2, was identified and characterised in Cochliobolus sativus (anamorph Bipolaris sorokiniana), a necrotrophic cereal pathogen that attacks both shoots and roots. The fungus was grown on a xylanase inducing medium containing mineral salts, oat spelt xylan, cellulose, and peptone, RNA was isolated, and a complementary DNA (cDNA) library constructed. The library was screened with a xylanase (XYL1) cDNA clone from the maize pathogen Cochliobolus carbonum. Xylanase cDNA clones, all representing a single gene, were identified. Corresponding genomic DNA was amplified by PCR. Sequencing of the cDNA and the PCR products gave a nucleotide sequence of 2211 bp containing two introns in an open reading frame of 693 bp that codes for a xylanase from glycosyl hydrolase family 11. The most similar sequences to this gene in nucleotide sequence databases are the XYL2 gene of C. carbonum and a xylanase (XYL1) cDNA from a saprophytic fungus, Humicola insolens. Northern blot analysis and reverse transcription PCR (RT-PCR) showed expression of the gene when the fungus was grown on xylan or cellulose, but not when peptone or sucrose was the only carbon source. Expression of XYL2 in inoculated barley seedlings was detected by RT-PCR.

A monoclonal antibody (MAb), UB31, is described that binds to the extracellular matrix (ECM) surrounding germ-tubes and appressoria, but not conidia, of the bean anthracnose fungus, Colletotrichum lindemuthianum. Comparative localisation studies with MAb UB26, which has the same cell type specificity, suggest that the ECM is heterogeneous in composition. Immunofluorescence showed that UB31 labelled appressoria more intensely than germ-tubes, whereas UB26 labelled these structures to a similar extent. Immunofluorescence and TEM-immunogold labelling showed that UB31 antigens were located close to the appressorial wall, while UB26 antigens extended further away from the wall. MAb UB31 bound to the ECMs of all six Colletotrichum species tested. Western blotting and ELISA indicated that the antibody recognises a high Mr glycoprotein (>200000) that may be linked to melanin. The glycoprotein recognised by UB31 was not removed from substrata by ultrasonication, suggesting it may contribute to germling adhesion.

Mycoëmilia scoparia gen. sp. nov. is described as a new member of Kickxellales. It is characterized by lageniform sporocladia produced acrogenously in mass and bears wet and fusiform spores on the sporocladia. Ramicandelaber brevisporus sp. nov. is distinguished from the type species of the genus, R. longisporus, by producing much shorter asexual spores, (3–)8(–13) long fertile branches arising from a globose body, and lateral branches.

The aim of this study was to test the usefulness of direct PCR-amplification in analysing fungal diversity in stumps. The analysis was conducted on stumps treated against Heterobasidion spp. using a commercial formulation of Phlebiopsis gigantea (Rotstop), and carried out using denaturing gradient gel electrophoresis (DGGE) of small subunit (SSU) ribosomal DNA (rDNA) fragments PCR-amplified directly from wood DNA samples using two separate fungus-specific primer pairs. On average, two (range 0–9) different amplification products were observed by DGGE in single wood samples of approximately 500 mm3. The PCR products were classified into operational taxonomical unit (OTU) groups based on their DGGE mobility. Six OTUs could be affiliated with a known species based on a reference fungal collection of 37 species: Heterobasidion annosum, H. parviporum. Hypholoma capnoides, P. gigantea, Resinicium bicolor and Stereum sanguinolentum. Sequence analyses did not give further identifications. OTU profiles from old (6 yr-old) and fresh (1-year-old) Scots pine and Norway spruce stumps from treated and untreated forest plots were compared statistically, and some significant differences were observed in the species composition between the treated and untreated plots. However, the frequency of most of the OTUs did not seem to be affected, and the treatment did not seem to have reduced the overall level of fungal diversity. Based on these results, direct PCR-amplification seems to be useful in analyses of fungal communities in decaying conifer stumps.

Two species of Haptoglossa isolated from nematodes from cow manure from N.E. England are described. H. erumpens sp. nov., infecting Bunonema nematodes, is aplanosporic, the cysts being released after rupture of the nematode cuticle in the absence of an evacuation tube. H. dickii sp. nov. is zoosporic, infecting several species of rhabditid nematodes. Aplanospores and zoospore cysts germinated to produce secondary spores (gun cells) with which to infect further nematode hosts. Some key ultrastructural features in both species have been included.

The name Agaricus subfloccosus (J. E. Lange) Pilát, based originally on Danish specimens, has been applied to mushrooms growing in coastal northwestern Europe as well as to mushrooms associated with Picea (and Abies) at higher elevations in western Europe. Corresponding populations are also found in these two habitat zones in western North America. Material of both lowland and highland forms from Europe and North America was studied using morphological, cultural, and genotypic approaches. Homothallism was consistently observed in those isolates studied. Individual and population level genetic data are also consistent with homothallic reproduction. Dissimilarity analysis of multilocus nuclear and mitochondrial genotypes based on allozyme and DNA RFLP markers provided strong evidence that, within either ecologically distinct group, the amphiatlantic populations were genotypically very similar. Conversely, there were large genotypic differences between the two ecologically distinct groups, whether within or between continents. Supported by cultural and morphological evidence, these data indicate that two ancient, phylogenetically distinct entities exist within current concepts of A. subfloccosus. A formal taxonomic resolution is complicated by the lack of a holotype for Lange's species.

Few studies have examined faeces as a substratum for nematophagous fungi, and no information is available about colonisation of faeces in tropical regions. Sheep faeces were deposited on a Brachiaria decumbens pasture in the Mata Region of Minas Gerais, Brazil. Up to 2 wk after deposition, samples were collected and cultivated on water agar (2 %) with free-living nematodes as bait. From 150 samples, 130 isolates were recovered after 3 wk. More than 70% were predators and Arthrobotrys and Monacrosporium spp. predominated. Endoparasitic species of Drechmeria, Harposporium, Myzocytium, Nematoctonus and Verticillium were also isolated.

A 909 bp region containing a genomic clone encoding for hydrophobin (ssgA) from the entomopathogenic fungus Metarhizium anisopliae has been sequenced and the regulatory motifs analysed against those recognized in other fungi. The genomic clone was also compared with the open reading frame of the hydrophobin ssgA (starvation stress gene) cDNA sequence. The genomic clone contained a 291 bp coding sequence with one intron of 64 nucleotides. From this sequence primers were established that could be used to amplify the hydrophobin. Restriction fragment polymorphism analysis of hydrophobin amplified by the polymerase chain reaction from 80 isolates of M. anisopliae showed no variability. Analysis of the potential regulatory elements 313 bp upstream from the transcriptional start site revealed typical TATAA and CCAAT boxes. CT or GC motifs were not found. Upstream regulatory elements were also found with sequence homologies to the AREA, CREA, CRE (cAMP response element) and BRLA regions of Aspergillus nidulans as well as the CYS3 and AmyB regions of Aspergillus oryzae. The promoter regions of other fungal hydrophobins were also assessed for the presence of regulatory elements. Upstream regulatory elements are also present for the gene encoding a cuticle-degrading protease (Pr1) from M. anisopliae. We suggest that nutrient levels and cAMP mediation of thigmotropic signals in the entomopathogenic fungus, M. anisopliae, co-ordinate the regulation of the gene products required for morphological development and secretion of ‘penetration’ enzymes.