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.

The products of teliospore germination in Ustilago triodiae and Sporisorium loudetiae-pedicellatae are illustrated. In U. triodiae the transversely-septate basidium is usually three-spored. The sporidia (basidiospores) either bud or form surface hyphae from which branched systems of aerial sporidia arise. A similar haplophase occurs in some other species.

In S. loudetiae-pedicellatae the basidium is unicellular and produces up to four sporidia in succession. These give rise to colonies composed largely of similar cells. No conjugation was seen in either species.

Growth of A. ochraceus at 30°C was significantly influenced by changes in both solute (NaCl, glycerol) and matric potential (PEG 8000). Optimum growth rates were at approx. −10·0 MPa with the non-ionic solute, and between −3·5 (=0·975 aw) and 14·0 MPa (=0·90 aw) with the ionic solute in a sucrose-based minimal salts medium. The optimum matric potential for growth was at −7·0 MPa (=0·95 aw). Growth rates were influenced by whether colonies were grown directly on the agar medium or on cellophane overlays. Sclerotia were profusely formed on media >−1·5 MPa (=0·99 aw), only initials at −3·5 MPa, and none under optimum conditions for growth (−7·0 to −10·0 MPa). The contents of low and high mol. wt polyols (glycerol, erythritol, arabitol and mannitol) and sugars (glucose and trehalose) were quantified in whole colonies and conidia grown under different osmotic and matric potential conditions for the first time. There was a higher total amount of polyols (μg mg−1D.W..) in conidia than in whole colonies. In glycerol-amended osmotic media maximum total polyols were present at −4·5 (=0·965 aw) and −17·0 MPa (=0·88 aw) in mycelial colonies and conidia respectively, with glycerol and mannitol being predominant in both, with small amounts of erythritol also present in conidia. By contrast, in matrically-modified media, the high mol. wt mannitol was the major component (>75% of the total) with small amounts of arabitol and the low mol. wt erythritol and glycerol, regardless of matric potential. For sugars, trehalose was predominant in conidia (>90%) from osmotically-modified media with low levels of glucose present in both conidia and mycelial colonies at all potentials tested. By contrast, in matric media trehalose was the predominant sugar in both conidia and whole colonies. This study suggests that xerophilic fungi may use different mechanisms for overcoming osmotic and matric stress.

A new species of Menisporopsis was collected from submerged wood in the Lam Tsuen River, Hong Kong. It differs from other species in having conidia with 5–6-setulae. The generic concept of Menisporopsis is briefly discussed and six species are accepted. A composite illustration of their conidial morphology is provided for comparison, and a synopsis of and a key to the genus are provided. The new species is described and illustrated with light and scanning electron micrographs.

Phylogenetic analyses of partial sequences (1002 basepairs) from the nuclear encoded small subunit ribosomal RNA (18S rRNA) were performed to determine the phylogenetic relationships of the ascostromatic fungi, Botryosphaeria, Guignardia, Leptosphaerulina, Didymella, Venturia, Sporormia, and Sporormiella. Our studies confirm the concept that the subclass Loculoascomycetidae is paraphyletic if Chaetothyriales is included, as this order and the plectomycetes constitute a monophyletic clade that is highly supported in our and some previous phylogenetic analyses. In general, taxa that lack pseudoparaphyses (Dothideales) or have reduced hamathecial tissue, such as Botryosphaeria and Guignardia (sensu Sivanesan), are a sister group to the Pleosporales, the most derived group, whose members have pseudoparaphyses. The two species of Leptosphaerulina, which also lack pseudoparaphyses, clustered within the Pleosporales, rather than within the Dothideales, with high confidence from bootstrap values. Didymella nested within the Pleosporales with a high degree of confidence, and is the sister group to Leptosphaerulina. The taxonomic placement of Venturia and Sporormiella corresponds to the Pleosporales.

The effect of carbon source on the production of a single extracellular β-glucosidase by Acremonium persicinum was investigated using fast protein liquid chromatography. The enzyme was detected in the medium during growth of the fungus on a wide range of β-glucans, particularly those containing a high proportion of (1→6)-β-linkages. While growth of A. persicinum on gentibiose, cellobiose and laminari-oligosaccharides moderately increased β-glucosidase production, growth on sophorose provoked 10-fold greater levels of this enzyme in the culture medium. Levels of the β-glucosidase were minimal in the presence of even low concentrations of glucose, sucrose, fructose or galactose. The results are consistent with the extracellular A. persicinum β-glucosidase being an inducible enzyme, subject to carbon source control by readily metabolizable sugars, with sophorose possibly directly inducing its synthesis.

An in vitro conifer ectomycorrhizal model system was developed to study changes in gene expression during the establishment of symbiotic relationship. Using the model system and the mRNA differential display technique, we have identified cDNA clones from Laccaria bicolor that appear to be involved in establishing and maintaining the symbiotic relationship between L. bicolor and red pine. These differentially expressed cDNA clones were obtained from mRNA extracted from the fungus 6–24 h after initiation of interaction. BLAST analysis showed that sequences of these clones did not match with any of the previously characterized genes. The cDNAs had regions with varying degrees of similarity to genes involved in signal transduction and transcriptional activation.

Fifty-five Phytophthora sojae isolates were collected from soil samples and diseased soybean plants from Illinois, Indiana, Iowa, and Minnesota in 1994 and 1995. Races for the isolates were determined. DNA of P. sojae isolates was amplified with 16 Operon decanucleotide primers. Twenty-three of 75 amplified fragments were polymorphic. Based on the 23 RAPD markers, a dendrogram depicting the relatedness of the isolates was constructed using UPGMA. The P. sojae isolates clustered into four distinct groups. The isolates of races 3, 4 and 25 clustered into group I. The isolates of races 1, 8 and 13 clustered into group II. The isolates of race 5 clustered into group III, and the isolates of race 7 clustered into group IV. Genetic diversity was detected among isolates of races 1, 3, 4, 5, 7 and 25 but not among isolates of races 8 and 13.

The importance of pneumocandin B0 as the fermentation-derived starting material for the antifungal drug candidate, MK-991, along with the identification of our production strain as Z. arboricola (ATCC 20868) as CBS prompted a search for other strains of Z. arboricola or Zalerion species with improved titres or that might produce natural pneumocandin analogues. Analysis of morphology, secondary metabolites profiles, and DNA fingerprinting demonstrated that ATCC 20868 was not congeneric with Z. arboricola. Ribosomal DNA sequences were compared among Zalerion species and pneumocandin-producing fungi and with rDNA sequences in GenBank. No good matches with sequences in GenBank were obtained for Z. arboricola or Z. maritimum, but for Z. varium, P. carpinea and ATCC 20868, relevant similarities were observed with ITS1 sequences from fungi of Leotiales. ATCC 20868 was phylogenetically more akin to P. carpinea, another pneumocandin producer, than initially suspected. The closest relative of ATCC 20868 seemed to be Hymenoscyphus monotropae. We conclude that the genus Zalerion is artificial; its species bear no phylogenetic relation among themselves. ATCC 20868 and Z. varium were related to fungi of the Leotiales. We propose a new anamorph genus and species, Glarea lozoyensis, to accommodate ATCC 20868.

Observations on cytological and ultrastructural changes in Plasmopara viticola oospores were carried out during the overwintering period. Three types of oospores were observed. Type I, characterized by a thin inner oospore wall (IOW), large lipid globules and two nuclei, was recovered only in samples collected in October. These oospores were considered to be immature. Maturation occurred during November and involved a noticeable increase in thickness of the IOW, fusion of nuclei, formation of an ooplast and break up of large lipid globules into smaller ones (type II oospores). A few oospores (type III) showed abnormal organization with very large lipid globules and less frequently discernible nuclei. IOW solubilization, dissolution of the ooplast and lipid globules and nuclear division were the first detectable events during oospore germination. Germinating oospores produce a germ tube which was terminated by a sporangium. In its young stage, the sporangium had a thick wall and an unusual multi-layered membrane. During this phase, nuclear divisions took place in the sporangium. While sporangium development progressed, the ribosome density in the cytoplasm decreased and mitochondria, initially roundish with evident cristae, became their usual tubular profile. The plasma membrane had a typical structure and storage organelles, such as finger print vacuoles and lipid globules, became more numerous in the cytoplasm. Larger vacuoles contained the flagella of differentiating zoospores.

Two fruitbodies each of Russula messapica and Macowanites messapicoides, identified by morphological characters, were screened for polymorphism in the ITS and the IGS of their ribosomal DNA, as well as in mitochondrial rDNA by RFLP analysis of PCR-products. The two species were monomorphic for all markers with the exception of the IGS. This limited genetic differentiation indicates a close relationship between these two species. The overall correspondence of the RFLPs between R. messapica and M. messapicoides adds molecular evidence to the relatedness of Russula and Macowanites, and suggests the possibility that M. messapicoides is recently derived from R. messapica.

Protostelids, dictyostelids and myxomycetes are well known from terrestrial ecosystems in temperate regions but poorly known from the tropics. This study examined the distribution and ecology of these three groups of ‘slime moulds’ in the forest floor litter microhabitat of five different forest types (tabonuco forest, secondary tabonuco forest, palo colorado forest, palm forest, and elfin forest) within the Luquillo Experimental Forest in Puerto Rico. Protostelids associated with aerial litter (dead but still attached plant parts) also were studied. At least 13 species of protostelids, 13 species of dictyostelids and 24 species of myxomycetes were recovered. Both species richness and abundance of dictyostelids and myxomycetes were higher in the secondary tabonuco forest than in any of the other forest types. The tabonuco forest yielded the most species of protostelids, but the next highest total was recorded from the secondary tabonuco forest. Both of these forest types are located at the low end of the elevation gradient. Lowest levels of species richness and abundance occurred in the elfin forest, located at the high end of the elevation gradient. The general pattern observed was that of decreasing diversity with increasing elevation.

Gymnomyces dominguezii, a gasteroid fungus belonging to the Russulales, is described and illustrated as a new species. Its main taxonomic characters are discussed and compared with other species in the genus and those in Martellia and Zelleromyces.

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.

Colletotrichum dematium is the cause of a new fungal stem disease on cowpea in South Africa. The pre-penetration and infection process of this fungus on cowpea stems was studied by light microscopy and SEM. Conidia began germinating at 6 h post inoculation (hpi), forming appressoria directly or at the ends of germ-tubes. By 14 hpi appressoria had melanized and direct penetration of host tissue had begun. At 20 hpi infection vesicles formed in epidermal cells. Thick, knotted primary hyphae formed from these vesicles and entered adjacent cells. At ±40 hpi C. dematium produced secondary hyphae which were highly branched, and grew extensively inter- and intracellularly. After approximately 48 hpi light brown lesions appeared on the stem, associated with the invasion of secondary hyphae into cells and cell necrosis. Acervuli with one or two melanized setae were visible on lesions by 70 hpi.

The ability of Peridermium pini and Cronartium flaccidum aeciospores and mycelium to infect known (Vincetoxicum spp., Pedicularis spp., Paeonia spp.) and potential (Melampyrum spp., Pyrola sp., Dactylorhiza sp., Solidago sp., Salix sp., Geranium sp. and Maianthemum sp.) alternate hosts was tested. None of the mycelial cultures and only 10% of the aeciospore samples produced uredinia or telia on the tested species suggesting that most aeciospores in Finland belong to the autoecious P. pini. Aeciospores from three locations in northern Finland, however, produced uredinia or telia on Vincetoxicum mongolicum, V. nigrum, V. fuscatum, Paeonia anomala, three P. officinalis cultivars, Melampyrum sylvaticum and Pedicularis palustris either in vitro or in vivo, indicating that these aeciospores belong to the heteroecious C. flaccidum, which occurs sporadically in Finland. Interestingly, the host-specificity of C. flaccidum encountered in Finland was very low (e.g. one sample produced uredinia or telia on eight species). This, added to the wide distribution of Melampyrum spp. over northern Fennoscandia, suggests that the main alternate hosts for C. flaccidum in Finland may be in Melampyrum rather than Pedicularis.

Esteya vermicola gen. et sp. nov. isolated from infected pinewood nematodes, Bursaphelenchus xylophilus, is described and illustrated. The new species is characterized by the production of two types of conidiophores, conidiogenous cells, and conidia. The first type of conidiophore is subhyaline to greyish green, flask-shaped, apex tapering at the apex into a thin neck, sometimes crooked; the conidiogenous cells are integrated, phialidic, rarely percurrent; the conidia are hyaline, one-celled, lunate, concave, containing an endospore-like apparatus, adhesive. The second type of conidiophore is subhyaline to greyish green, cylindrical, subulate, septate, the base somewhat swollen; conidiogenous cells integrated, phialidic; conidia hyaline, one-celled, bacilloid, often forming an aggregate droplet at the apex, non-adhesive. Esteya vermicola exhibits high infectivity towards the pinewood nematode, and the potential for its development as a biocontrol agent against the pinewood nematode is briefly discussed.

The adhesion of conidia of Drechmeria coniospora to the nematode Panagrellus redivivus was reduced after treatment of the conidia with Pronase E, or the detergents sodium dodecyl sulphate (SDS) and dodecyltrimethylammonium bromide (DTAB) suggesting involvement of proteinaceous compounds in the adhesion process. In the TEM the thick extracellular pad covering the adhesive bud of the conidia was completely removed after treatment with Pronase E. After treatment with SDS or DTAB the proteinaceous compounds appeared to be dissolved leaving mainly carbohydrates in the pad as observed on OsO4 without and OsO4 with Ruthenium red-stained material, respectively. The detergent extracts after SDS and DTAB treatments contained nine and seven peptides, respectively, with molecular masses in the range from 6 to 80 kDa on SDS-PAGE gels, and five biotinylated peptides were found in each extract, after blotting to nitrocellulose membranes, indicating that these were surface proteins. None of the detergent extracts was able to reduce adhesion of the conidia after treatment of the nematodes. The detergent extracts contained protease- and phosphatase activity. The protease inhibitor, chymostatin, inhibited infection of nematodes and growth of the conidia, suggesting the involvement of chymotrypsin-like proteases in the infection process. On gelatin-containing substrate gel electrophoresis two proteases were clearly chymostatin sensitive.