Two distinct groups (A and B) of the conifer shoot blight and canker pathogen Sphaeropsis sapinea have been recognized on pine hosts in the northcentral United States. Their occurrence in a collection of isolates from much more diverse hosts and locations was studied by analysis of RAPD markers and isozymes. Seventy-nine isolates were used for RAPD marker analysis, and 37 for isozyme analysis, with 33 isolates in common. Hosts included 19 species in Pinus and species of Cedrus, Larix, Picea, Pseudotsuga, located in Africa, Australasia, Europe, and North America. Relationships among these isolates were determined using cluster analyses of presence or absence data for each amplification product or isozyme. Analyses of either RAPD marker or isozyme data placed most isolates in a group that contained previously characterized RAPD marker group A isolates. There was no strong indication of either host or geographic clustering within this highly similar, widely distributed group. Analysis of RAPD marker data placed several other isolates in a separate group that contained previously characterized RAPD marker group B isolates. This is the first report of isolates from hosts other than Pinus banksiana or P. resinosa, and from outside Michigan, Minnesota, or Wisconsin, that have been characterized as members of the RAPD marker group B.

The influence exerted by the non-pathogenic binucleate Rhizoctonia (np-BNR) isolate 232-CG in stimulating plant defence reactions in young bean plants inoculated with the root rot fungus Rhizoctonia solani (AG-4) was examined using light and electron microscopy and further investigated by gold cytochemistry. Severe necrotic lesions on hypocotyles of diseased beans were observed, and the pathogen invaded the cortical tissue causing extensive damage including cell disorganization and cell wall degradation. In contrast, these host reactions were not seen in bean plants inoculated with the non-pathogenic BNR or in plants that were inoculated with BNR and subsequently challenge-inoculated with R. solani. Microscopic examination of hypocotyls inoculated with the non-pathogenic BNR, showed a different host reaction typical of plant defence reactions. In these samples, epidermal and outer cortical cells were impregnated with an electron-dense material. Histochemical assays of this material confirmed the substantial presence of phenols, pectic substances and suberin. Electron microscope observations clearly showed that in non-pathogenic BNR-inoculated plants, fungal cells were confined to the epidermal layer which was darkly stained. Gold cytochemistry confirmed the presence of pectic substances in the electron dense material. The possibility that pectic oligogalacturonides released after hydrolysis by the non- pathogenic BNR enzymes may act as elicitors of defence responses is discussed. The present ultrastructural observations corroborate that non-pathogenic BNR isolates may function as potential inducers of plant disease resistance.

Isolates of Phytophthora infestans of A1 mating type from central Mexico and Wales, when mated to a range of A2 isolates from Russian and Mexico, produced a proportion of abnormal oogonia with several oospheres. Similar oogonia were observed in self- fertile lines resulting from treatment of a Russian A1 isolate with a chemical mutagen followed by the fungicide, metalaxyl.

Changes caused by Septoria apiicola during the development of leaf spot disease in celery were quantified stereologically in susceptible cvs, a partially resistant parental wild celery line, fully resistant parsley and lovage. Thick sections, cut in vertical orientation, were stained and using a camera lucida, were superimposed on a lattice grid for stereological analysis. The volume of each component within the leaf space was calculated using the Cavalieri method.

In healthy tissue volume fractions of intercellular space, palisade layers and, to a lesser extent, collenchyma, vascular tissue and oil ducts were greater in the more resistant genotypes. This coincided with a reduction in the volume fraction of mesophyll tissue. Estimates of absolute volume fraction were similar. In both resistant and susceptible celery the principal response to infection was a substantial decrease in the volume fractions and absolute volumes of palisade and mesophyll tissue. Volume fractions of mesophyll and palisade tissues declined by 97% in susceptible lesions and by 98% in resistant lesions. The fungus was not observed in xylem or phloem cells. No appreciable changes were observed in either collenchyma or oil duct tissues in response to infection in resistant and susceptible genotypes. Infection had no effect on the epidermis or stomata and these remained intact until late in the infection cycle, when pycnidia erupted through the epidermis of the upper and lower leaf surfaces.

No hyphae were found in leaf sections of lovage or parsley, and fungal growth was severely restricted in all resistant material bred from wild celery. In wild celery lines, S. apiicola did not produce any, or produced very few, pycnidia and the volume fraction of vegetative hyphae was lower than that in susceptible cvs, where the fungus proliferated extensively within the lesions and many pycnidia were produced. This accounted for 30–50% of the fungal material in lesions on susceptible celery plants. The amount of fungus (21%) was less in lesions on resistant celery and in this material 96% of the fungus was present as vegetative hyphae. In lesions formed on susceptible celery 26% of the fungal tissue was reproductive and of that 11% was conidia whereas in resistant celery, where pycnidia were present, these structures were morphologically abnormal and did not contain conidia. Stereological analysis demonstrated clear differences in plant and pathogen response during infection of resistant and susceptible celery by S. apiicola.

Gaeumannomyces graminis var. graminis produces 1,8-dihydroxynaphthalene (DHN) melanin in its hyphal and hyphopodial cell walls. We isolated G. graminis mutants that were affected in their melanin biosynthesis. One was unable to synthesize DHN-melanin and, because it accumulated 2-hydroxyjuglone, a DHN melanin pathway shunt product, it is most likely to be defective in the reductase that catalyzes the conversion of 1,3,8-trihydroxynaphthalene to vermelone, the penultimate reaction in DHN synthesis. Genetic crosses with our wild-type strain indicated that this trihydroxynaphthalene reductase deficiency was the result of a single mutation. Another mutant constitutively synthesized DHN melanin and genetic crosses with our wild-type strain suggested that this heavily melanized mutant had a single mutation responsible for its phenotype. This mutant produced more melanin than the wild-type strain as measured by Azure A binding to melanin. The wild type and constitutively melanized mutant hyphae were more hydrophobic and more resistant to lytic enzymes, benomyl, restrictive temperature, and uv light than the non-melanized mutant, which also autolysed more readily. The non-melanized mutant was not more sensitive to heavy metal than the melanized strains. In addition, the non-melanized mutant was unaltered in pathogenicity to rice, whereas the constitutively melanized mutant was less pathogenic. The constitutively melanized mutant produced less extracellular lytic enzymes than the wild-type and the non-melanized mutant, which may explain its reduced virulence.

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.

Aspergillus flavus and A. parasiticus are closely related species belonging to the Aspergillus section Flavi. Both species can produce aflatoxins, but not all isolates of either species do so. We examined the genetic relationship between toxigenic and non-toxigenic isolates using RAPD and Neighbour Joining analysis. Twenty-four RAPD amplifications using a combination of 17 primers discriminated between 20 isolates of A. flavus and 15 isolates of A. parasiticus. A. flavus isolates divided into two distinct groups with both toxigenic and non-toxigenic isolates occurring in each group. No association was seen between RAPD genotype and the ability to produce toxin. Five non-toxigenic isolates of A. parasiticus separated into two groups, in which the isolates were similar but not identical. These groups of non-toxigenic isolates occurred on branches in which toxigenic isolates also occurred. This study suggests that either multiple losses of toxigenicity in A. flavus and A. parasiticus have occurred, or that recombination has reassorted this phenotype into a variety of different genetic backgrounds.

The aim of this study was to compare the ectomycorrhizal community structure in spruce stands treated with different levels of dolomite lime. ITS-typing of randomly sampled mycorrhizas, without prior morphotyping, was used. Sixteen different ITS-RFLP patterns were found. By comparison with the available reference material, nine of these could be identified at least to genus level. Variation within treatments was large and statistically there were no significant differences between treatments with respect to specific taxa. A similarity assessment did, however, show less similarity between control and high dolomite (HD) than between low dolomite (LD) and either control or HD, suggesting a shift in the community structure as a result of the treatment. The fruitbody production at Hasslöv had been recorded in a different study during 1985–92 and 28 ectomycorrhizal species had been found in the treatments examined in the present study. Except for three species, all were present in the reference material used for identification of the ITS-types. Only three species, Russula ochroleuca, Lactarius necator and Boletus chrysenteron were found as both mycorrhizas and fruitbodies. There were five taxa which occurred on over 5% of the screened roots. These were Thelephora terrestris, 21.5%; Tylopilus felleus, 13%; Tylospora fibrillosa, 13%; and two unidentified taxa, 10% and 6%. Together these five taxa colonized over 60% of the mycorrhizal roots investigated, yet none of them was found among the fruitbodies recorded in the above-ground study. Despite the differences in taxa found below and above ground, similarity tests between treatments using the fruitbody data also revealed a shift in community structure.

Experimental systems were established for the molecular analysis of different developmental stages of Glomus mosseae. RNA and DNA were simultaneously extracted from ectocarpic spores, chlamydospores, dormant and germinating sporocarps and extraradical hyphae. A β-tubulin gene fragment was obtained by PCR from spore genomic DNA and further analysed. β-tubulin RNA expression could be monitored during the fungal life cycle with a gene-specific primer by RT–PCR using an internal cRNA standard.

Partial rDNA sequences were obtained from 26 isolates representing species of Cercospora, Passalora, Paracercospora, Pseudocercospora, Ramulispora, Pseudocercosporella and Mycocentrospora. The combined internal transcribed spacers (ITS) including the 5·8S rRNA gene and 5′ end of the 25S gene (primer pairs F63/R635) on rDNA were amplified using PCR and sequenced directly. The ITS regions including the 5·8S varied in length from 502 to 595 bp. The F63/R635 region varied from 508 to 519 bp among isolates sequenced. Reconstructed phylogenies inferred from both regions had highly similar topologies for the taxa examined. Phylogenetic analysis of the sequences resulted in four well-supported clades corresponding to Cercospora, Paracercospora/Pseudocercospora, Passalora and Ramulispora, with bootstrap values greater than 92% for each clade. Based on the results of the analysis, a new combination for Pseudocercosporella aestiva is proposed in Ramulispora, and Paracercospora is reduced to synonymy with Pseudocercospora.

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.

A new staining method for the examination of active regions of hyphae is presented. Using a simple protocol, application of the fluorescent stain Mag fura (tetra-potassium salt) in low pH buffer to two filamentous fungi resulted in bright responses to active hyphal regions. Not only were active apices delineated, but more distal compartments were also found to respond to the stain. Some apices did not stain, presumably because they were inactive. It is believed that all stained regions retained cell membrane integrity and are thought to have had high membrane ATPase activity. All showed high nuclear and mitochondrial complements. The stain was apparently held within the cell wall structure, close to the cell membrane. It is hypothesized that it was responding to a localized flux of divalent cations from the cell membrane. With non-active regions, only a low level of response to the stain remained, at the exterior of the cell wall. This low emission is thought to be a response to contaminating ions in the buffer. It could be clearly distinguished from the bright response associated with the active regions. Counter-staining with other commonly used fluorescent probes showed these regions of low response (unless they were completely degenerated) still contained nuclear material and mitochondria.

The fluorescent signal from active regions of fungal hyphae was sufficiently bright and persistent for visualization by a conventional CCD camera. This will allow the development of image analysis protocols to measure the effects of environmental conditions on fungal physiology, using commonly available equipment.

The correct identity of the ergot strains Claviceps fusiformis PRL 1980 (ATCC 26245), C. fusiformis 129 and 59, and Pepty 695/ch-I, formerly described as Claviceps purpurea, was proven by means of RAPD, genomic DNA restriction patterns as well as by a literature search about the strain origin.

Pseudocercospora bolkanii sp. nov., and P. luzardii sp. nov., were collected in the Brazilian cerrado on leaves of Strychnos pseudoquina, and Hancornia speciosa, respectively. Prathigada backmanii sp. nov., is described on leaf spots of Bowdichia virgilioides. Pseudocercospora curatellae on Curatella americana is illustrated in detail.

Using leaf disc techniques, nine isolates of the mycoparasite Sphaerellopsis filum were examined for pathogenicity to Melampsora epitea, the most important species of rust in renewable energy willow plantations. Five isolates were derived from Melampsora spp. on willows and poplar, one from Puccinia coronata on couch grass, two from Phragmidium violaceum on blackberry and one from Triphragmiopsis laricinum on larch. Two inoculation experiments were carried out. In the first, S. filum and rust were applied simultaneously on to leaf discs of Salix burjatica cv. Korso. In the second, leaf discs were inoculated with rust initially, then the resulting rust uredinial pustules were inoculated with S. filum. In both experiments, all S. filum isolates from Melampsora spp., and that from P. coronata, developed pycnidia on willow rust. No pycnidia were produced from the other three isolates. Among isolates which produced pycnidia, the frequency of rust pustules colonized by S. filum and suppression of rust spore production differed markedly. In the first experiment, 55–99% pustules were colonized and rust spore production was suppressed by 64–98%. In the second, the proportions were 33–97% and 53–73%, respectively. Suppression of rust sporulation was closely correlated with the frequency of rust pustules bearing S. filum pycnidia. The results suggest that S. filum is composed of pathogenically specialized populations differing widely in their virulence.

In vitro, antibiotic effects of the culture filtrates of 29 species of aquatic hyphomycetes against Gram-negative and Gram-positive bacteria, yeasts and hyphomycetes were studied. The culture filtrates of 38% of isolates were active in at least one of our tests for biological activity. Fifteen species inhibited growth of bacteria and four demonstrated antifungal activity. Filtrates of 35% of the isolates were antibacterial. About 50% of the isolates, those with the largest in vitro inhibitory activities (Dimorphosphora foliicola, Flagellospora sp. and Mycocentrospora sp.) were obtained from wood. Filtrates of Articulospora tetracladia were not only active against Gram −ve bacteria, but were also antiviral against bacteriophage T4 and influenza virus.

Five ophiostomatoid taxa have been found associated with the infructescences of Protea species, an ancient group of flowering plants endemic to South Africa. Two of these fungi are characterized by their unusual Knoxdavesia anamorphs and have been placed in Gondwanamyces. The three remaining species have Sporothrix anamorphs and have accordingly been accommodated in Ophiostoma. The phylogenetic relationships between the fungi associated with Protea spp. and other ophiostomatoid fungi in Ceratocystis and Ophiostoma are unknown. Large subunit ribosomal RNA sequence data was obtained for the fungi associated with Protea infructescences as well as for the type species of Ceratocystis and Ophiostoma. Both groups of ophiostomatoid fungi were phylogenetically distinct from either Ceratocystis or Ophiostoma, despite sharing morphological and physiological characters with these genera. The species of Ophiostoma associated with Protea infructescences group within the Ophiostomatales while species of Gondwanamyces group within the Microascales. Furthermore, the Ophiostoma spp. from Protea should reside in a separate genus and are a fascinating example of convergent evolution towards insect dispersal.

The electrophoretic karyotype of the Chinese straw mushroom, Volvariella volvacea, was determined. The haploid strain V34 of V. volvacea has 15 chromosomes ranging in size from 1.4 to 5.1 Mb. No chromosomal polymorphism in terms of size and number was seen in either of two growth stages: vegetative mycelia or fruit-body gill tissues. DNA fingerprints were prepared by the arbitrarily- primed polymerase chain reaction. Those obtained from different stages during fruit-body morphogenesis were identical. However, variation in DNA fingerprints was evident in protoplast regenerants derived from the same vegetative mycelium. Thus the haploid mycelium of strain V34 is heterokaryotic but the bulk genotype is stable during fruit-body development. F1 and F2 progenies germinated from the haploid, uninucleate basidiospores from self-fertilized fruit bodies also regularly segregate a range of mycelial morphological variants as well as phenotypic variation revealed by DNA fingerprints. We overview the known mechanisms to generate genetic variation and propose a novel mechanism that could account for the 1:1 segregation ratio of self-fertile to self-sterile progeny regularly obtained from selfed Volvariella volvacea fruit bodies.

Conidia of Metarhizium anisopliae need water activity (Aw)>0·98 for germination irrespective of whether they are formulated in an aqueous or oil carrier. The presence of nutrients in the carriers or culture medium accelerated germination but did not increase the range of Aw (or humidities) over which conidia could germinate. Insect extract stimulated more conidia to germinate than yeast extract. Oils appeared to extract fungistatic and stimulatory compounds from the insect cuticle. The ratio of these compounds depends on both the insect species and the carrier. Conidia of M. anisopliae formulated in oils were observed with SEM to flow over the surface of insect and plant cuticles. The conidia were probably deposited at sites which are conducive for germination and infection and consequently increased the overall mortality of insects. In contrast, aqueous formulations of conidia remain as drops on the leaf surface immediately after application with a pipetteman or sprayer.

Strains of Heterobasidion annosum belonging to the S and P intersterility groups (IGs) were compared in respect of laccase activity on various substrates. Biomass production was significantly lower in P cultures than in S cultures. By contrast, laccase activity, measured in relation to growth rate, was significantly higher for the P strains tested, compared with a similar number of S strains, particularly in substrates, rich in carbohydrates as well as in inorganic and organic nitrogen. Using the Bradford assay method, P cultures were shown to contain significantly higher amounts of protein than S. The pH optimum of laccase for both IGs was 4·5 with guaiacol as substrate and 5·3 with syringaldazine. Isozyme patterns varied greatly, depending on strain, substrate and incubation time. In P cultures 4–5 bands were obtained, whereas S strain laccase was mostly separated into 2–3 isozyme bands. Only one was significantly different in position between the IGs. The results may help to explain why P strains are more aggressive than S strains as pathogens and wood decayers.