Isolates of Verticillium fungicola, collected over a period of 45 yr from various geographical sites, were analysed for their responses to four fungicides (benomyl, thiabendazole, chlorothalonil and diethofencarb), and for colony morphology, virulence and DNA polymorphisms. Three different colony morphologies were observed for this population of isolates including fluffy, dense and appressed. The majority of isolates had the appressed colony morphology. Most isolates exhibited high levels of virulence. The appearance in this population of both benomyl and thiabendazole resistance was correlated with the introduction of benomyl for use on mushrooms. Eighty-eight percent of the isolates were cross-resistant between benomyl and thiabendazole and 80% exhibited negatively correlated cross-resistance between benomyl and diethofencarb. Seventy percent of the isolates exhibited negatively correlated cross-resistance between thiabendazole and diethofencarb. The level of resistance to chlorothalonil was relatively high even prior to its introduction and overall the response to this fungicide appears to have varied little over the last 45 yr. DNA polymorphisms, as detected by randomly amplified polymorphic DNA (RAPD) analysis, divided the isolates into four groupings. Limited correlations could be drawn between RAPD groupings, fungicide response and geographical origin. All recently collected isolates (1993–5) were extremely similar in their response to the four fungicides and had appressed colony morphology, high virulence and were members of RAPD group four. The lack of variation in the recent isolates as compared to the older isolates indicates that the V. fungicola population may be becoming more homogeneous.

Mycobiont isolation experiments were performed on six species of Ramalina from Brazil: R. celastri, R. complanata, R. dendriscoides, R. gracilis, R. peruviana and R. sprengelii. This study aimed to optimize the culture conditions and nutrient requirements of the selected mycobionts. The aposymbiotic R. complanta was successfully isolated from ascospores, while aposymbiotic R. peruviana was obtained from thallus fragments. In R. peruviana the production of secondary metabolites was investigated under aposymbiotical growth conditions using HPLC. When cultivated on solid medium, this mycobiont produced the typical chemosyndrome (sekikaic acid and satellite compounds), found in the voucher lichen thallus. When cultivated in liquid medium (immersed in malt yeast medium in the absence of agar), only one, the major lichen substance, sekikaic acid, was synthesized by the fungus. In addition, atranorin was formed, but was not detected in any of the voucher specimens. Red pigments were found in solid and liquid cultures. These were separated into two compounds, but could not be fully identified. R. celastri spores germinated, but did not form mycelia. R. dendriscoides, R. gracilis and R. sprengelii were not successfully cultivated in aposymbiotic conditions, although eight different culture media were tested.

Phytophthora megakarya is a devastating oomycete pathogen that causes black pod disease in cacao. Phytophthora species produce a protein that has a similar sequence to the necrosis and ethylene inducing protein (Nep1) of Fusarium oxysporum. Multiple copies of NEP1 orthologs (PmegNEP) have been identified in P. megakarya and four other Phytophthora species (P. citrophthora, P. capsici, P. palmivora, and P. sojae). Genome database searches confirmed the existence of multiple copies of NEP1 orthologs in P. sojae and P. ramorum. In this study, nine different PmegNEP orthologs from P. megakarya strain Mk-1 were identified and analyzed. Of these nine orthologs, six were expressed in mycelium and in P. megakarya zoospore-infected cacao leaf tissue. The remaining two clones are either regulated differently, or are nonfunctional genes. Sequence analysis revealed that six PmegNEP orthologs were organized in two clusters of three orthologs each in the P. megakarya genome. Evidence is presented for the instability in the P. megakarya genome resulting from duplications, inversions, and fused genes resulting in multiple NEP1 orthologs. Traits characteristic of the Phytophthora genome, such as the clustering of NEP1 orthologs, the lack of CATT and TATA boxes, the lack of introns, and the short distance between ORFs were also observed.

The introduction of recombinant DNA technology in the field of mushroom research has resulted in the cloning and characterization of a large number of genes. In order to study the genetics of compost colonization of A. bisporus, genes encoding enzymes involved in utilization of this substrate have been isolated. In addition, a number of genes which are induced in fruit bodies during fruit body development have been cloned and they will provide more insight in the genetics of this economically important aspect of the life cycle. Other genes that were cloned encode proteins of basic biochemical routes. They provide knowledge on the importance and regulation of these routes in the life cycle of A. bisporus and add to knowledge on the general architecture of A. bisporus genes. Here we present an overview of the currently available biochemical and molecular data of A. bisporus and we discuss the importance of the available genes as genetic markers for breeding purposes.

Seven hundred and eighty two isolates corresponding to 52 fungal taxa have been obtained from 920 segments of healthy and symptomatic twigs of Eucalyptus grandis, incubated on media with water potential −0·138 MPa and −4·19 MPa. Only thirty taxa were isolated at frequencies of 2% and greater. The composition of species isolated from bark of healthy twigs incubated on media with both water potentials was similar, with Aureobasidium pullulans being dominant. Sistrotrema brikmannii was the main species isolated from xylem of healthy twigs at −0·138 MPa whereas Pleospora sp. and Sphaeropsis eucalyptus were the dominant species isolated from this material on medium at −4·19 MPa. Fusicoccum eucalypti and Cytospora chrysosperma were most commonly isolated from bark and xylem of symptomatic twigs. Correspondence analysis showed the fungal species associated with each tissue. Summer drought followed by frequent frost in early autumn may have incited wounds allowing fungal invasion of the bark of twigs.

The white-rot basidiomycete, Inonotus hispidus, produces a characteristic pattern of decay in naturally infected London plane (Platanus × hispanica) in which zones of severely decayed wood, traversed by relatively undegraded xylem rays, alternate with relatively undegraded, gummous ‘reaction zones’. Microscopic observations of London plane wood both naturally colonized and artificially inoculated with I. hispidus indicated that the resistance of reaction zones and xylem rays to degradation is due respectively to obstruction of cell lumina by tyloses and polyphenols, and infiltration of intercellular spaces by polyphenols. Inonotus hispidus was apparently able to circumvent these impedances by forming soft-rot like tunnels through the wood cell walls.

Two distinct and unrelated groups of ophiostomatoid fungi are associated with Protea infructescences. Two species, Gondwanamyces proteae and G. capensis, have unique anamorphs that reside in Knoxdaviesia. This anamorph genus is infrequently found amongst species of Ceratocystis sensu lato. The second group includes Ophiostoma splendens and O. protearum that have Sporothrix anamorphs typical of Ophiostoma. During recent collections of Protea gaguedi infructescences from the Northern Province in South Africa, an apparently new species of Ophiostoma with a Sporothrix anamorph was discovered. It can be distinguished from O. splendens and O. protearum based on its short ostiolar hyphae and its associated plant host. This teleomorph is described as O. africanum and the anamorph as S. africanum. Five species of ophiostomatoid fungi are now known from Protea infructescences and a key is presented for them.

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.

Clonal isolates of Uncinula necator, the grapevine powdery mildew pathogen, were characterized by mating type, RFLP and PCR analyses. Five random genomic clones, consisting of three multi-copy and two low-copy sequences, were used as probes for hybridization. The oligonucleotides (CAC)5, (GACA)4, the core sequence of the M-13 minisatellite region and the consensus region from a plant intron splice junction (R1), were used as primers in PCR reactions. The DNA probes detected high levels of genetic diversity in U. necator, identifying 34 unique haplotypes among the 81 isolates analysed, with a Shannon diversity index of D′=0·85. In comparison, 15 haplotypes were resolved in PCR reactions with the four primers, resulting in a diversity index of D′=0·59. The overall diversity within the collection, by combining RFLP and PCR data, was D′=0·88, distinguishing 37 haplotypes. The RFLP analysis provided greater discrimination of individual haplotypes than did PCR. Phenetic analysis of the RFLP and PCR data separately and in combination revealed two broad phenetic groups. The average overall similarity between the two groups was 40%. Mating of isolates from the two phenetic groups resulted in viable ascospores. The combination of RFLP, PCR and mating techniques was effective in determining levels of diversity and provided insight into the genetic relatedness of U. necator isolates.

Eudarluca caricis is assumed to be a nonspecific mycoparasite of rust fungi. The evidence for its mycoparasitism has rested on constant association with uredinia. In this study, stable isotopes provided additional evidence of mycoparasitism, as E. caricis was enriched with 15N relative to its associated rust fungus, as were parasites and mycoparasites generally with respect to their hosts. Host specificity was directly tested in inoculations in the greenhouse. Isolates of E. caricis from Puccinia on two Eurasian grasses (i.e. Holcus lanatus and Phalaris arundinacaea) did not infect Melampsora on Populus that, in contrast, was successfully infected by a poplar isolate of E. caricis. An isolate from M. medusae on P. deltoides infected a significantly greater percentage of uredinia of M. medusae on P. deltoides than uredinia of M. occidentalis on P. trichocarpa. The host specificity of the three isolates was reflected in their divergence in a phylogenetic analysis based on ITS sequences. Interestingly, the analysis revealed that mycoparasites of rust and powdery mildew fungi have evolved from a common ancestor.

The phylogenetic relationships among fungi that cause spring dead spot disease of Bermuda grass in Australia and the United States were studied from nucleotide sequence data of the ITS region of the rDNA. High levels of sequence similarity were observed among Ophiosphaerella herpotricha, O. korrae and Leptosphaeria narmari. These species clustered into a distinct clade that was distant from other Leptosphaeria and Phaeosphaeria species. Based on sequence data and previous descriptions of the ascoma and ascospore characteristics of these species, we propose a taxonomic transfer of L. narmari to O. narmari. Oligonucleotide primers specific for O. narmari were developed from ITS1 and ITS2 regions to help identify non-sporulating isolates. These primers amplified all isolates of O. narmari, including those from the United States, but not those of O. herpotricha, O. korrae or other ectotrophic root-inhabiting fungi. This is the first report of O. narmari in North America. Two Group I introns were found in the small subunit rDNA of some isolates of O. korrae and O. narmari, but not O. herpotricha. A 425-nucleotide intron in O. narmari and O. korrae was inserted two nucleotides downstream from the ITS5 conserved primer sequence. These introns exhibited an 80% sequence identity. In addition, a second 431-nucleotide intron in O. narmari was similar in size and location to one in O. korrae.

The cultivated mushroom Agaricus bisporus was grown and fruited in wheat straw compost. RNA was extracted from samples of mycelium growing in compost during colonization and fruit body development. Laccase gene (combined lcc1 and lcc2) expression and cel3 gene (cellobiohydrolase) expression were determined by Northern blot analysis and by competitive RT-PCR. The level of laccase transcripts was highest in colonized compost (prior to the onset of fruiting), declined during the period of fruit body enlargement and increased again after harvesting and during the second flush of fruit body production. No transcripts of the cel3 gene were detectable in colonized or pre-fruiting cultures, but the level became detectable and rose to a maximum at the veil-break stage of fruiting, declined after harvesting and rose again during the second flush. Gene expression for laccase and cellobiohydrolase correlated with the known changes of laccase and cellulase enzyme activities during the fruiting life cycle.

The ITS region of the rRNA genes was amplified using DNA extracted from Terfezia pfeilii fruit-bodies. Two types of restriction fragment profiles were revealed among 11 fruit-bodies by the Hinf I restriction enzyme. An additional four truffles collected from the same field one year later were found to have a mixed profile combining both types. Germinated spores from one of these mixed-profile individuals had a single profile belonging to one or the other of the two types. Roots collected around and underneath one Terfezia fruit-body were traced to different plants. Roots of a watermelon plant (Citrullus vulgaris) showed a non-vesicular-arbuscular endomycorrhizal association. Restricted ITS amplification fragments of mycorrhized roots from C. vulgaris were shown to match T. pfeilii fruit-body ITS-RFLP fragments. The significance of this finding with regard to T. pfeilii potential host range is discussed.

Comparison of type specimens of A. gerdemannii and A. appendicula revealed the spores to be identical in organization and subcellular structure, indicating they are conspecific. The Glomus morph of A. appendicula was not assigned a name in the protologue, but 11 living cultures started from or producing spores of A. gerdemannii also generated Glomus-like spores of identical morphology to those in type specimens of Glomus leptotichum. These spores were indistinguishable from those in type specimens of G. fecundisporum, indicating they are conspecific. Both species were described in the same paper, so the name G. leptotichum is given priority because type specimens are in better condition and specimens from a reference culture have been identified by an author of both prologues. Absence of a teleomorph and confirmation of dimorphism from single and multiple-spore inoculations of host plants in culture provide the basis for designating A. gerdemannii and G. leptotichum as synanamorphs of one vesicular-arbuscular mycorrhizal fungus.

Erynia neoaphidis is the most widespread aphid pathogen in temperate regions. A few authors have identified this species as infecting some non-aphid hosts. It exhibits high variability with respect to morphology and cultural characteristics that underlines the need for assessment of the genetic variability within this species. We used size of PCR-amplified ribosomal DNA internal transcribed spacer (ITS) coupled with RAPD-PCR pattern in order to investigate genetic variability within a sample of 30 isolates identified as E. neoaphidis and one isolate of E. kondoiensis from diverse countries and hosts. Isolates fell into four discrete ITS size groups: 575 base pairs (bp) in isolates from non-aphid hosts, 1000 (including the E. kondoiensis isolate), 1100, or 1450 bp in those from Aphididae. The 1100-type seems to be the one of most widespread, since two-thirds of the isolates from diverse locations belong to this type. ITS types can be correlated with morphological and physiological characteristics. A great deal of genetic variability exists within ITS types, as shown by RAPD-PCR. RAPD groupings could be related with geographical origin in some cases. There was no apparent relationship between host and ITS type or RAPD pattern among isolates from Aphididae. PCR-based technologies provide evidence that could be used to clarify the taxonomy of E. neoaphidis and closely related species, and will help identify markers related to epidemiological characteristics.

The phylogenetic position of Uncinula forestalis within the Erysiphales has been inferred from 5.8S, 18S, and 28S rDNA sequences. Although the appendages of the ascomata are Uncinula-like, i.e. unbranched with curved-coiled apices, U. forestalis is situated at the very base of the large Erysiphales cluster, far from the ‘Pseudoidium’ clade (Erysiphe, including Microsphaera and Uncinula) and separate from the recently introduced basal genus Parauncinula. U. forestalis differs morphologically from the species of Erysiphe sect. Uncinula in having terminal, fasciculate (as in Podosphaera tridactyla), septate ascoma appendages and a Euoidium-like anamorph (conidia catenate). In Parauncinula, the appendages are also terminal but not fasciculate, the ascospores are curved, and the anamorph is lacking. Uncinula forestalis is a basal, tree-inhabiting powdery mildew with some additional ancestral characteristics, including Uncinula-like appendages and 6–8-spored asci. The new genus Caespitotheca gen. nov. is described with C. forestalis comb. nov. (syn. Uncinula forestalis) as the type species. We calculated the timing of the divergence of U. forestalis and P. septata using a molecular clock of the Erysiphales (6.5×10−10 changes per site per year in domains D1 and D2 of 28S rDNA) and a 28S rDNA data set. The results suggest that the divergence of U. forestalis and P. septata from other Erysiphales occurred between 90 and 80 Myr ago; i.e. the divergence of the two ancestral species may have occurred in the late Cretaceous.

The content of phenolic substances in freshly collected fruit bodies of several basidiomycete genera varied in the range 2·35–9·05 mg g−1, following closely the trend of a normal function.

It was necessary to use a polyphenoloxidase-inhibiting substance to obtain reliable analysis. The content of phenolics decreased, during 3 d at room temperature and following freezing at −20°C.

Cryphonectria canker caused by Cryphonectria cubensis is one of the most destructive diseases of Eucalyptus plantations in South Africa. To implement a meaningful management of plantation diseases, it is important to have an understanding of the population diversity of the pathogen. In this study, trees were surveyed to determine whether C. cubensis reproduces sexually in South Africa. The diversity of the South African C. cubensis population was assessed based on vegetative compatibility tests. Field inoculations were used to determine whether VC groups correlated with virulence. Only pycnidia were found on cankered trees, indicating that sexual reproduction does not occur. Only 23 VC groups were found amongst 100 isolates each collected from single diseased trees. A low degree of genetic diversity also indicated that sexual reproduction is absent or rare in the South African C. cubensis population. Inoculation studies revealed that isolates belonging to different VC groups differ significantly in their ability to cause lesions. The low level of genetic diversity enhances opportunities to capitalise on hypovirulence to reduce the impact of the pathogen in the future. It also supports the view that the fungus was recently introduced into South Africa.

In the mushrooms Pleurotus ostreatus and Agrocybe aegerita, expression of the hemolytic proteins ostreolysin and aegerolysin, which belong to the aegerolysin family, has been shown to be initiated specifically during formation of primordia and fruiting bodies. We used rabbit anti-ostreolysin and fluorescent rhodamine-labelled secondary goat antibodies for monitoring ostreolysin and aegerolysin in situ during the mushrooms' development. In parallel, the protein level in developing tissues was monitored with SDS-PAGE and hemolytic assay. Immunolocalization of ostreolysin, visualized by epifluorescence, together with biochemical tests, confirmed specific expression of ostreolysin and aegerolysin in the primordia and fruiting bodies but not in the vegetative mycelia. In the primordia, the proteins were disposed diffusely. In growing and mature fruit bodies they persisted in the lower part of the pileus, in particular in basidia and basidiospores, while in other parts only a few focal remains were observed. Confocal microscopy of immunolabelled sections showed that intracellular ostreolysin was located specifically along the inner edges of hyphae. Since both proteins were found preferentially in the rapidly growing primordia, and in the basidia and basidiospores of maturing fruit bodies, it is suggested that they might play a role in the processes of fructification and/or sporulation.