The symbiotic phenotype of a lichen arises through the interaction and cooperation of two or more genetically unrelated partners. Ultrastructural and molecular methods were used to investigate the changes that take place during early stages of the lichenization process. The resynthesis of prethallus stages of Baeomyces rufus was studied by co-culturing under sterile conditions the isolated, axenically grown mycobiont and its green algal photobiont Elliptochloris bilobata. The lichenization process was monitored by SEM. One day after co-culture, symbionts were bound together by a newly secreted mucilage. By day 12, photobiont induced, morphological changes in the mycobiont were visible. Aerial hyphae grew around photobiont cells, showed a high frequency of branching and formed appressoria on the algal wall surface. By day 28, many photobiont cells were completely engulfed by hyphae and soredia-like clusters were observed. These morphological developments resemble lichenized structures formed in the natural lichen. cDNA-AFLP was used to investigate gene expression profiles on day 12 of co-culture. Differential gene expression patterns revealed that few genes were induced, and many fungal and algal genes seemed to be suppressed in the early stages of lichenization.

Microsporidia are intracellular parasitic microbes, which were once thought to be among early diverging eukaryotes, mainly because they lack mitochondria. However, this evolutionary scheme has been challenged by protein-based molecular phylogenetic studies, which suggest a more recent origin of the Microsporidia, which clustered Microsporidia with or within ‘true’ fungal lineages. However, most of these studies did not include sequences from basal fungal phyla (Chytridiomycota and Zygomycota), and unrepresentative taxon sampling could have resulted in misleading relationships being suggested. To further investigate the evolutionary origin of Microsporidia relative to Fungi, we performed molecular phylogenetic analyses with newly determined RPB1 (DNA-dependent RNA polymerase II largest subunit) and EF-1α (translation elongation factor I alpha) including sequences from basal fungi. Although the phylogenetic position of Microsporidia in the EF-1α tree still might be misplaced due to the unusually high rate of sequence divergence of the microsporidian EF-1α gene, the phylogenies recovered based on these two protein sequences do not provide strong evidences for a close relationship between Microsporidia and Fungi. Moreover, we have identified within the EF-1α genes a characteristic two amino acid deletion conserved in all fungal sequences currently available, whereas this deletion is absent in microsporidian sequences. These results argue against the view of Microsporidia as highly degenerate fungi, and whether Microsporidia and Fungi are sister taxa remains unresolved.

Persistence of Aschersonia aleyrodis, a fungal pathogen of whitefly, was studied on cucumber, gerbera and poinsettia. Germination capacity and infectivity of conidia, which stayed on the plants for up to 1 month, were assessed. Average germination of conidia on the leaves was low (< 14%), whereas most of the conidia transferred from the leaf to water agar were viable, even after having been on the leaf surface for 1 month. Germination capacity was highest on cucumber, followed by poinsettia and lowest on gerbera. On cucumber leaves, conidia stayed viable and were able to infect 90% of whitefly nymphs 31 d after spore application. On gerbera, germination capacity decreased considerably from 80% (day 0) to 40% (day 31). This was reflected in nymphal mortality, which declined from 75% to 40%. Despite the high germination capacity (60%) of conidia on poinsettia after an exposure of one month, nymphal mortality decreased from 70% at the day of spore application to 10% after 3 d at leaf surface, and remained low throughout the monitoring period. Relations between germination capacity, infectivity and the host plant environment such as phyllosphere microorganisms, secondary plant metabolites and microclimate are discussed.

Two new genera, Neophaeosphaeria and Phaeosphaeriopsis, are described to accommodate species of Paraphaeosphaeria that are not congeneric based on morphological characters and results of 18S rDNA sequence analyses. Paraphaeosphaeria s. str. is restricted to species with two-septate ascospores and anamorphs that produce non-septate, smooth, pale brown conidia enteroblastically from phialides which have some periclinal thickening. Species in Neophaeosphaeria have 3–4-septate ascospores and anamorphs that produce ovoid to ellipsoid, non-septate, brown, verrucose or punctate conidia from percurrently proliferating conidiogenous cells. Paraphaeosphaeria barrii, P. conglomerata, P. filamentosa and P. quadriseptata are transferred to Neophaeosphaeria. At present all species in Neophaeosphaeria occur on Yucca (Agavaceae). Phaeosphaeriopsis is described for species that produce 4–5-septate ascospores. Known anamorphs produce cylindrical, 0–3-septate, brown, punctate conidia from percurrently proliferating conidiogenous cells or bacillar conidia from simple phialides. P. agavensis, P. glauco-punctata, P. nolinae and P. obtusispora are transferred to Phaeosphaeriopsis. P. amblyspora is described as a new species.

Cryphonectria cubensis is an important canker pathogen of plantation Eucalyptus spp. in tropical and sub-tropical areas of the world, including South Africa. It is best known on Eucalyptus spp., but it also occurs on Syzygium aromaticum (clove). In 1998, C. cubensis was found to cause cankers on the non-myrtaceous hosts Tibouchina urvilleana and T. lepidota in Colombia. In this study, we report on a similar canker disease that has recently been found in South Africa on T. granulosa, commonly grown as an ornamental tree. The identity of the pathogen was determined through morphological comparisons and phylogenetic analyses of ITS and β-tubulin gene sequences. The pathogenicity of the fungus was also tested on T. granulosa and E. grandis. Morphological as well as DNA sequence comparisons showed that the fungus on T. granulosa is the same as C. cubensis occurring on Eucalyptus spp. in South Africa. Pathogenicity tests on T. granulosa and E. grandis clones showed that the fungus from T. granulosa is able to cause cankers on both hosts.

A search for double-stranded RNA (dsRNA) was conducted among 106 isolates of the pathogenic basidiomycete Heterobasidion annosum. Of these isolates, 47 were tissue isolates from fruit bodies and 59 were isolated from decayed wood. Nucleic acids were extracted from freeze-dried mycelia and dsRNA was separated by cellulose CF-11 chromatography and confirmed by digestions with specific nucleases. dsRNA was present in 19 and 14% of the tissue and wood isolates, respectively. From five of the fruit bodies containing dsRNA basidiospores were investigated and 10–84% of the germinated basidiospores contained dsRNA. On high nutrient media, the germination frequency of basidiospores was reduced by presence of dsRNA in the fruit body (P<0.05); germination frequencies were 34 and 78% for spores from fruit bodies with and without dsRNA, respectively. The same trend was present also on low nutrient media, although not statistically significant; germination was 3 and 10% for spores from infected and dsRNA free fruit bodies, respectively. Transmission of dsRNA in H. annosum from mycelia into basidiospores together with the lowered germination frequency are likely to play a significant role in the life cycle of the fungus. The relative importance of different transmission routes for dsRNA in H. annosum is discussed.

Metarhizium anisopliae has potential as a biological control agent. Included among its hosts are certain insect pests of brassica crops. Brassica species produce isothiocyanates, some of which are known to be fungitoxic. In our study, isothiocyanates inhibited both germination and subsequent growth by M. anisopliae in vitro and its ability to infect insects. Conidia were more sensitive than the mycelium to these compounds, the most fungistatic of which were phenylethyl-, 2-chlorophenyl- and allyl-isothiocyanates. Appressorium production in vitro was suppressed by all isothiocyanates except allyl- and propyl-isothiocyanates, which appeared to stimulate appressorium formation. Phenylethyl- and 3-butenyl isothiocyanates, which are present in several of the plant hosts of Phaedon cochleariae, reduced the pathogenicity of M. anisopliae when inoculated insects were exposed to their vapours. These findings have implications for the efficacy of biocontrol of brassica pests by this fungus.

Effect of temperature and water potential on extension rate, extra-resource biomass and fractal dimension of Stropharia caerulea and Phanerochaete velutina growing in trays of soil was determined non-destructively with time, by image analysis. S. caerulea responded differently from P. velutina, mycelial extension and biomass production rates of the former being considerably affected by changes in temperature and water potential, with greater aggregation of mycelia into cords at 25 °C and at and below −0·02 MPa. These morphological changes were reflected in the fractal dimension values. Mycelial extension of S. caerulea on agar was sensitive to temperatures above 25 ° and water potentials below −1·3 MPa. This is the first reported work on abiotic effects on fractal dimension of cord-forming fungi on soil, and these results are discussed in terms of the function of mycelial cords and foraging strategies.

Substantial polymorphism was detected between isolates from five populations of Alternaria brassicicola attacking Cakile maritima along the New South Wales coast of Australia, with a maximum of two genotypes being shared between population pairs. Of ten pair-wise population comparisons, six had no pathogen genotypes in common; only one genotype occurred five times, and most (93%) were found only once. Although an UPGMA based on Nei's measure of genetic distance separated the five populations, a cluster analysis using individual isolates failed to group them according to population, indicating significant gene flow. An analysis of molecular variance indicated ca 14% of the variation occurred between populations, representing moderate population differentiation over the spatial scale of the study. Tests of the relative contribution of clonality and sexual recombination indicated low, albeit significant levels of linkage disequilibrium in all populations. The level of linkage disequilibrium, and the high genotype diversity, provides support for the contention that a hitherto unidentified sexual stage might be a significant factor in the life-cycle of A. brassicicola.

Thirty South American species of Cortinarius belonging to the subgenera Telamonia, Dermocybe, Myxacium, Phlegmacium, and Cystogenes were studied using an integrated approach that included morphological, anatomical, and ultrastructural data, and also molecular phylogenetic analysis of nuclear rDNA sequences. The micromorphology of the basidiomes was studied by light microscopy, and the principal structures were illustrated by line drawings. Basidiospore ornamentation was studied by scanning electron microscopy (SEM). Nuclear internal transcribed spacers (ITS, including the 5.8S gene) and the DNA coding for the D1/D2 domains of the large ribosomal subunit (LSU) were sequenced and analysed using a Bayesian Markov chain Monte Carlo method to estimate phylogenetic relationships between the studied Cortinarius species. Morphology and anatomy of the pileus surface and basidiome pigmentation appeared to be the most useful characters to delimit some natural groups, whereas microcharacters related to the structure of pileus context, hymenophoral and stipe trama were of little taxonomic value. Basidiospore morphology and cheilocystidia seem to be taxonomically relevant at the species level. The following five infrageneric groups were supported by the morphological, chemical and molecular data: (1) Telamonia characterized by wide hyaline hyphae of the veil and by small basidiomes; (2) Dermocybe spp. with an epicutis as the most external layer of the pileus, and skyrin and hypericin pigments; (3) Dermocybe spp. with a thin viscid layer on the pileus, and endocrocin and dermolutein pigments; (4) Phlegmacium spp. characterized by a long and radicating stipe; and (5) Phlegmacium spp. that overlap in some macrocharacters with Telamonia species. Our analyses suggest that classification concepts based mainly on macromorphological characters are likely to lead to artificial grouping, whereas certain microscopical and chemical characters seem to be useful in constructing a more natural classification system for Cortinarius.

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.

A method to effect rapid, near-synchronous colonization of tomato root systems by a vesicular arbuscular mycorrhizal fungus was developed. This procedure was used to compare the time course of colonization in roots of tomato that were grown under varying phosphate conditions. It was found that the primary symbiotic interaction developed through a series of regularly timed stages. Furthermore, the formation of intercellular hyphae, arbuscules and vesicles was significantly reduced in plants grown with high soil phosphate. These results show that this method is ideal for the production of material for molecular biological studies of VAM symbiosis, including investigation of the regulatory role of phosphate in the symbiosis and the processes that control the fungal life cycle.

The production of laccase, an enzyme of industrial interest, was screened among species of the genus Pycnoporus, in particular P. sanguineus. Strains were isolated from various tropical Chinese environments and phylogenetically compared to ones deposited in international collections. Molecular clustering, based on ribosomal ITS1-5.8S-ITS2 genomic sequence analysis, showed that the Chinese strains of P. sanguineus formed an homogeneous phylogenetic group distinguished by its laccase-overproducing character. The dikaryotic strain P. sanguineus G05 was selected for its ability to produce up to 40 000 U l−1 laccase in the presence of 2,5-xylidine, Tween 80 and maize bran. Since fruit bodies of P. sanguineus could be formed in the laboratory, monokaryotic laccase-hyperproducing strains were isolated using classic genetical methods. Among these isolates, strain G05.10 synthesized up to 71 000 U l−1 laccase, with a productivity of 5069 U l−1 d−1. The laccase was purified and identified as a 70 kDa protein with an acidic pI, and was very stable at high temperatures.

The causal agent of tobacco anthracnose was identified as Colletotrichum destructivum based on the morphology of the fungus and a comparison of the sequence of the rDNA ITS with those of other Colletotrichum species. The infection process on tobacco (Nicotiana tabacum and N. benthamiana) was examined by light microscopy, which revealed that the pathogen acted as an intracellular hemibiotroph. Penetration occurred preferentially at the anticlinal walls of epidermal cells by an appressorium and penetration peg. An infection vesicle formed in the penetrated host cell by 48 h after inoculation, and out of this, a multi-lobed infection vesicle grew which remained limited to the initially infected cell. The interaction at this point was biotrophic, which was confirmed by plasmolysis and accumulation of a vital stain by the infected host cells. Thin secondary hyphae arose from multi-lobed infection vesicles at 60 h after inoculation, which then penetrated the host cell wall and began the necrotrophic phase of the infection. Acervuli formed on the plant surface by 96 h after inoculation, typically with a single melanized seta. In addition to tobacco, the fungus could infect alfalfa, cowpea, and Medicago truncatula, but not soybean. The process of infection of C. destructivum in tobacco was very similar to that previously reported in alfalfa and cowpea.

Epicoccum purpurascens was evaluated as a biocontrol agent of Sclerotinia head rot of sunflower. The potential of antifungal compounds produced by E. purpurascens as fungicides was also assayed. Treatments consisted of application of E. purpurascens conidia and of partially-purified antifungal compounds produced in broth culture before inoculation with ascospores of S. sclerotiorum. Application of conidia resulted in reduced head rot incidence on greenhouse grown plants, but the application of antifungal compounds had no effect on head rot development. Capitula colonization by E. purpurascens had no negative effects on capitula development. After application of E. purpurascens to field grown plants, capitula were much less colonized than under greenhouse conditions. As a consequence, this treatment had no effect on head rot development in the field when plants were artificially inoculated with S. sclerotiorum. The search for new isolates of E. purpurascens well adapted to the fluctuating conditions typical of natural environments could contribute to achieving an acceptable level of efficacy of this organism as a biological control agent of head rot.

This study investigates the potential of emissions from a zinc smelter to induce oxidative damage to DNA and lipids in Boletus edulis, the king bolete. Concentrations of cadmium, zinc, copper, and mercury were determined in 16 fruit bodies collected near the smelter (exposed group), as well as in 15 reference samples. Frequency of apurinic/apyrimidinic (AP) sites in DNA (a pre-mutagenic DNA base modification) and concentration of lipid hydroperoxides were chosen as damage parameters. Concentrations of the four metals, as well as oxidative damage to DNA and lipids were significantly elevated in the exposed group (Mann–Whitney, P<0.001). Both damage parameters correlated positively with concentrations of cadmium, zinc or copper in fruiting bodies (Spearman's P<0.01). Frequency of AP sites correlated significantly with mercury in the fruit bodies (P<0.05), whereas the association between lipid hydroperoxides and mercury was insignificant. Frequency of AP sites correlated positively with concentration of lipid hydroperoxides (P<0.001). Negative trends for the associations between concentrations of metals and AP sites or lipid hydroperoxides in the reference group (significant only for mercury and lipid hydroperoxides; P<0.05) suggest that in B. edulis low concentrations of mercury, possibly also of other of the metals determined in the present study, may induce dose-response relationships of a hormetic (‘J-shaped’) nature.

The ability of Hymenoscyphus ericae to produce 12 carbohydrolytic activities has been investigated during growth under solid-state fermentation conditions. The fungus produced a complete cellulase enzyme complex (cellulase, cellobiohydrolase and β-D-glucosidase) along with components of the hemicellulolytic (endoxylanase, β-D-xylosidase, α-L- and β-L-arabinosidase and glucoronidase) and mannanolytic (mannanase, β-D-mannosidase, α-D- and β-D-galactosidase) complexes. Specific activities were greatest for endo-acting enzymes and lower for exo-acting and side-branch removing activities in each case. The data indicate that H. ericae possesses a considerable ability to degrade plant polymeric material and are discussed in relation to establishment of the symbiosis and interaction of mycelium with moribund plant organic matter in soil.

Mycosphaerella ambiphylla sp. nov. (anamorph: Phaeophleospora) and Mycosphaerella aurantia sp. nov., are described from diseased Eucalyptus globulus leaves. In addition, a new fungal record in Australia, M. mexicana, and two new records for Western Australia, M. gregaria and M. parva, are discussed. A key is provided to Mycosphaerella species on E. globulus in Western Australia.

The ITS II and the first part of the LSU rDNA were amplified from 26 isolates within the genus Entomophthora. The specificity of the primers allowed the use of both in vivo and in vitro material. Size polymorphism and long amplification of the ITS II regions, ranging from 1200 to 2000 bp, were observed. The PCR-products were cut with eight different restriction endonucleases and analysed by UPGMA, one analysis from each of the two regions. Conidial morphology was of predictive value for the overall taxonomy of the genus Entomophthora, as the genus clustered together in the analysis of the LSU rDNA. In both analyses the E. muscae complex clustered into three different clades, which support the validity of E. schizophorae and E. syrphi as separate species. Considerable variation was detected in the E. muscae clade, but it could not be grouped by host, geographic origin or conidial morphology, though the E. muscae s. str. isolates in both analyses grouped together. One isolate with E. muscae-like conidia found on Hymenoptera clustered out within the E. muscae clade, widening the host range for E. muscae significantly.