The aim of the present paper is to investigate if Mycocalicium subtile as presently circumscribed is a morphologically highly variable species, or in fact represents two or more cryptic species, and further to investigate the status of M. minutellum. The morphological variation of 19 specimens of Mycocalicium subtile from five continents was investigated and the ITS1-5.8S-ITS2 region of their rDNA sequenced. Sequences from closely related taxa, 4 specimens of M. albonigrum, 2 of Mycocalicium sp., and Chaenothecopsis nana were also included. For comparison the corresponding sequence of M. victoriae and C. pusilla were also determined. We used the sequence of Monascus purpureus as the outgroup for the analysis. The sequences were used for phylogenetic inference using parsimony and distance methods. All the specimens assigned to M. subtile with the exception of two, form a well-supported monophyletic group. Those two specimens represent a morphologically cryptic, but genetically distinctive taxon. The infraspecific clades of M. subtile in the molecular phylogeny did not correspond to geographical origin. Only a weak correlation with geographical origin was found in the morphological analysis. The molecular analysis supports the suggestion that Mycocalicium minutellum is a taxonomic synonym of M. subtile.

The present work reports the development of a PCR assay to detect the ascomycete Biscogniauxia mediterranea in asymptomatic tissues of Quercus cerris. To this purpose, two specific primers (MED1 and MED2) were designed by comparison of sequences comprised between ITS1 and ITS4 generic primers of 21 isolates of B. mediterranea and related species. Specificity of the primers was tested against 19 isolates of B. mediterranea and 20 isolates of related and unrelated genera, and against Quercus cerris DNA. The reliability of the results was confirmed by Southern Blot analysis. The 2 specific primers MED1 and MED2 were able to detect B. mediterranea DNA in the host tissues at picogram quantity of target DNA. This assay would greatly enhance the possibility to study the presence and localization of B. mediterranea during its latent phase in Quercus species.

Trichoderma harzianum (CCM F-470) was found to produce large amounts of extracellular mutanase (0·33 U ml−1, 1·85 U mg protein−1) when grown aerobically on the optimized mutan medium in fermenter culture with an automatic pH control. The mutanase from this source was purified to homogeneity by a rapid procedure, using ion-exchange chromatography, hydrophobic interaction chromatography and chromatofocusing. The enzyme was recovered with a 94-fold increase in specific activity and a yield of 73%. The molecular weight of the purified enzyme proved to be 67 kDa, as estimated by SDS–PAGE, and to be 274 kDa, as determined by size-exclusion HPLC. These results indicate that the native mutanase is probably a tetramer protein. The isoelectric point was at 7·11, and the carbohydrate content in the purified enzyme was 4·42%. The pH and temperature optima were 5·5 and 40 °C, respectively. The enzyme remained stable over a pH range of 4·5–6·0 and up to 35 ° for 1 h. The values of Km and Vmax under standard assay conditions were 1·2×10−3 g ml−1 and 8·48×10−2 U mg−1, respectively.

Arbuscular mycorrhizal fungi are common in moderately saline soil (colonized by Distichlis stricta) in an inland boreal salt pan in north-central Manitoba, but are absent from adjacent soil that is extremely saline (colonized by Puccinellia nuttalliana). In order to determine if this absence was due to the lack of suitable plant hosts or to edaphic factors, mycorrhizal colonization, vegetation composition, soil salinity and water content were examined along transects between the two vegetation zones. Correlation and principal component analyses revealed that mycorrhizal colonization of P. nuttalliana was positively linked to cover of Distichlis stricta and soil water content, and negatively linked to bare ground. The area closest to the point of salt seepage was uncolonized by mycorrhizal fungi. A transplant experiment confirmed that mycorrhizal fungi are unable to sustain colonization in this area of the salt pan. In this inland salt pan, colonization by mycorrhizal fungi is limited by edaphic factors, and not by the absence of suitable host plants, suggesting that mycorrhizal fungi have a lower limit of salinity tolerance than halophytes such as P. nuttalliana.

Oidiophores with uninucleate oidia are produced on the aerial mycelium of Coprinus cinereus and occasionally also on the submerged mycelium. Oidiophore development starts with the formation of a stem cell and proceeds with stem cell elongation, followed by the formation of oidial hyphae at the tip of the stem cell and subsequent release of matured oidia. However, analysis of more than 20 different C. cinereus strains revealed that oidiophore formation is a flexible process. Based on morphological variations, we defined four main types of oidiophores. Types 1 and 2 oidiophores produced oidia at the tip(s) of simple (type 1) or branched stems (type 2). Types 3 and 4 oidiophores were characterized by the absence of stem cell elongation (type 3) or stem cell formation (type 4). All strains examined produced types 1 and 2 oidiophores and some produced also types 3 and 4 oidiophores but the frequency of the different oidiophore types varied strongly from strain to strain.

Mycelial cord systems, of the basidiomycete Phanerochaete velutina, a common woodland saprotroph, were grown on unsterile soil in model laboratory microcosms from 4 cm3 wood inocula. Systems were supplied after 37 d with a fresh 4 cm3 beech wood ‘bait', placed behind the foraging colony margin. Systems were subject to dry shift (−0.056 MPa) or wet shift (−0.009 MPa) over an 11 d period either immediately after, or 20 d after baits were supplied. Controls were maintained at constant soil matric potential (−0.019 MPa). 57-d-old systems were supplied with NH4K2PO4 including 32P tracer within soil compartments local to inoculum or bait. Image analysis was used to quantify morphological responses to water regime and resource supply, and tracer movement monitored non-destructively with a scintillation probe for 57 d. 32P uptake was greatest when tracer was supplied local to the inoculum. Dry shift concurrent with bait supply caused system wide cord-thickening, prevented polarised growth towards the newly supplied bait, had a significant carbon (energy) cost compared to controls, significantly reduced 32P acquisition, and significantly increased 32P relocation to the bait. Wet shift concurrent with bait supply caused considerable loss of extra-resource mycelium in the unbaited region, resulting in highly polarised development along the bait-inoculum line, but did not affect 32P uptake and partitioning. Delayed wet shift caused swifter polarisation towards the bait, quantified in terms of fractal dimension, did not result in system wide regression of extra-resource mycelium, and resulted in correspondingly increased rates of 32P acquisition. Delayed dry shift prevented polarised growth towards the bait and had only transient effects on 32P uptake and partitioning. Results suggest that resource capture took priority over coordination of C reserves and reallocation of mycelial effort, and that mycelium colonising the new resource was more dependent on P translocate during desiccation stress.

The patterns of conidial discharge of Erynia neoaphidis were measured from three species of aphids: nettle aphid (Microlophium carnosum), grain aphid (Sitobion avenae) and pea aphid (Acyrthosiphon pisum). The effects of release height, morph of aphid and temperature on the horizontal and vertical discharge of conidia were studied. Numbers of conidia deposited in the dorsal and lateral directions were distributed with distance in truncated bell shaped patterns. Discharge distances ranged from 2 to 11 mm and half the conidia caught travelled further than 5 mm. There was little difference in the horizontal dispersal patterns of conidia when ejected from cadavers of different morphs of the same species, or from different species. The weight of infected aphids showed little correlation with discharge patterns. Values of maximum discharge distance, Dm, were generally between 6 and 9 mm. Temperature had a significant effect on conidia discharged from the dorsal surface of apterous A. pisum cadavers. Dm was greater at 18 °C than at 10 or 25 °. Vertical discharge distances for conidia released from A. pisum cadavers ranged between about 2 and 8 mm. The maximum height to which conidia were projected vertically was not affected by temperature. However, the average height tended to be greater at 18 °. Half of the conidia reached heights of about 3.5 mm while the maximum heights reached were about 8 mm. The initial speeds of conidia were estimated from measured discharge distances. The results suggest that conidia of E. neoaphidis may be ejected at speeds of about 8 ms−1.

Sclerotinia sclerotiorum differentiates by forming terminal-type sclerotia. We found that the fungus produces β-carotene in low and high levels during its undifferentiated and differentiated stages, respectively, and at low (in dark) and high (in light) oxidative growth conditions, respectively. In contrast, a sclerotium non-producing strain of S. sclerotiorum produces very low amounts of β-carotene in constant levels throughout its entire growth period, irrespective of oxidative stress degree of growth conditions. When β-carotene was added to the growth medium of the sclerotium producing strain, it caused a concentration-dependent inhibition of sclerotia formation. We propose that β-carotene inhibits sclerotial differentiation in S. sclerotiorum by lowering oxidative stress via its antioxidant scavenging properties.

Samples of standing senescent culms of Panicum maximum, Pennisetum purpureum, Phragmites australis, Miscanthus floridulus, Saccharum arundinaceum and Thysanolaena maxima (Gramineae), and Schoenoplectus litoralis (Cyperaceae) were collected in Hong Kong between 1997 to 1999. A total of 205 fungal taxa were identified on these samples, including 61 ascomycetes, and 144 mitosporic taxa. Common fungal genera included Diaporthe, Leptosphaeria, Massarina, Ophiobolus and Ophioceras (ascomycetes), and Monodictys, Phaeoisaria, Periconia, Phoma, Phomopsis, Rhinocladiella, Septoria and Sporidesmium (mitosporic taxa). Different grass species were host to different fungal communities and diversities of taxa. Diversity indices for fungi on the hosts varied from 3·3 to 8·7, the highest index being from Pennisetum purpureum, and were overall higher from species offering more durable, strongly sclerenchymatic substrates. No single saprobic fungus collected in this study is thought to be specific to any one grass, however, certain fungi tended to reoccur on single grass species, but not on adjacent grasses. A ‘core fungal group’ was commonly associated with the decaying grasses and this ‘core’ was thought to be important in nutrient cycling in the grasses. A comparison of the fungi occurring on grasses with those on other monocotyledonous hosts is made. The numbers of fungi known to occur on Juncus roemerianus and Phragmites australis are briefly summarised.

The arbuscular mycorrhizal (AM) fungus Glomus intraradices was grown in vitro with RIT-DNA transformed Daucus carota roots on plates divided into two compartments. One side of the plates contained only fungal hyphae and spores while the other contained mycorrhizal roots. Using this technique, the limitations of insufficient biological material available for molecular analysis of this obligate symbiont were overcome. Fungal material from the first compartment was used as a pure source of G. intraradices for genomic DNA extraction. PCR amplification as well as southern hybridization were conducted using this DNA. Sufficient pure genomic DNA and mRNA was obtained to carry out Southern analysis, achieve optimum PCR results with 25–35 cycles and conduct RT-PCR. Differential expression of chitin synthase class I and II was detected in G. intraradices. Using degenerate primers specific to fungal chitin synthase sequences, a single amplification product obtained after 25 cycles of PCR was cloned. Sequencing of this fragment revealed similarity to other fungal chitin synthase genes. PCR with these primers and additional primers allowed the amplification of chitin synthase fragments from spores of different isolates of G. intraradices as well as from G. mosseae, Gigaspora margarita, Acaulaspora scrobiculata, Scutellospora calospora and Entrophosphora colombiana. A total of 21 chitin synthase sequences from different species and isolates of various AM fungi were successfully amplified. Sequencing of these fragments permitted their classification into class I and II of the chitin synthase groups.

To determine the feasibility of using internal transcribed spacers (ITS1 and ITS2) and 5.8S ribosomal DNA sequences as genetic markers in molecular systematic and phylogenetic studies of Ophiostoma, the presence of multiple types of ITS in the two sibling sapstain fungi, Ophiostoma piceae and O. quercus were examined in 22 isolates from diverse geographic regions. PCR and sequence analysis revealed that two different versions of ITS sequences were present in the PCR-products amplified with the ITS primers, ITS1-ITS4 or ITS5-ITS4 or ITS1-F-ITS4, in every isolate of the two Ophiostoma species tested. Surprisingly, the two ITS sequences were shared by both species. One ITS sequence which was major in O. piceae was designated as the OPC type, and the other ITS sequence which was minor in O. piceae was designated as the OPH type. In O. quercus the OPH type was major and the OPC type was minor. The major ITS comprised more than 99% of the amplicons in both species. The results suggest that common ITS gene pools have been maintained at different levels within these sibling species.

Neotyphodium endophytes have not been reported within vascular tissue of grass hosts even though hyphae were observed in close proximity to vascular bundles. However, examination by light microscopy and transmission electron microscopy of a range of natural and artificially introduced associations involving Neotyphodium spp. has now shown that hyphae of these endophytes can colonize vascular tissue. In natural associations, hyphae were seldom present within vascular bundles and if present the number was low. Small vascular bundles lacking a bundle sheath were more likely to contain hyphae than large vascular bundles that were surrounded by a bundle sheath. Examination of a range of Lolium×hybridium (hybrid rye-grass) and L. multiflorum (annual rye-grass) plants, as well as F. arundinacea (tall fescue) and F. pratensis (meadow fescue) plants, that were inoculated as seedlings with either a strain of N. lolii or a strain of an interspecific hybrid Neotyphodium sp., revealed extensively colonised vascular bundles in four Lolium plants. Each of these plants was infected with the N. lolii strain and each had strong L. multiflorum characteristics. The most extensively colonised large vascular bundles were those first formed during leaf growth. Growth within these vascular bundles was continuous along their length. Hyphal extension and branching of hyphae within these vascular bundles ceased when leaf growth ceased. Grasses with extensively colonised vascular bundles were not noticeably stunted and formed inflorescences without external stromata.

Sclerotinia sclerotiorum produced erythroascorbate at levels dependent on oxidative growth conditions, developmental stage and strain differentiating capacity. The transition of S. sclerotiorum from the undifferentiated to differentiated state under high oxidative stress growth conditions, was accompanied by a shift in the ratio of reduced/oxidized erythroascorbate towards its oxidized form. At low oxidative stress, the reduced form of erythroascorbate predominated. In a non-differentiating strain of S. sclerotiorum, reduced erythroascorbate predominated over the oxidized form during growth. The reduced/oxidized erythroascorbate ratio in this strain was 5-fold higher than the corresponding ratio in the differentiating strain. The level of oxidative stress (lipid peroxidation) in colonies of the differentiating strain (one day before they differentiated) was 3-fold higher than that formed in colonies of the non-differentiating strain. Exogenous ascorbate exhibited its antioxidant activity in the differentiating strain by causing a dose-dependent decrease in lipid peroxidation, and proportional inhibition of sclerotial development. It is suggested that during its initial growth stages, S. sclerotiorum cannot decrease lipid peroxidation to levels found in the non-differentiating strain, and then forms sclerotia. We propose that oxidative stress triggers differentiation in sclerotium-producing filamentous phytopathogenic fungi.

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.

Colletotrichum acutatum is the major causative agent of secondary leaf fall disease of rubber. All isolates of the fungus examined secreted polygalacturonase (PG), pectin lyase (PL) and the cellulolytic enzymes, β-glucosidase, and cellobiase in culture. Molecular weight determinations suggest that PG as well as the PL produced by all isolates are similar. PG activity was absent in infected rubber leaves. However, PL activity was detected in infected tissues associated with the enlargement of lesions. β-glucosidase was present in both healthy and infected leaves while cellobiase was detected only in infected leaf tissue.

Spatial distributions and species diversity of ectomycorrhizal fungi were studied. Epigeous fungal fruit bodies and mycorrhizas were investigated in a reforested stand of Pinus densiflora aged 45 yr. Forty species of fruit bodies, including the dominant genera, Russula and Inocybe, were recorded throughout the three-year investigation. A species–area curve of the fruit bodies indicated that mean species number in 1.5 m2 was ca 1.0. Pine ectomycorrhiza, which were randomly sampled throughout the same investigation period, were assigned to 51 morphotypes microscopically. Although the dominant morphotypes were not manifested due to the sampling method used, a species–area curve of the mycorrhizas indicated the mean species number in 1.5 m2 was ca 25. Thus, spatial distributions among fruit bodies and mycorrhizas were quite different, and the mycorrhizas showed more complex distributions than fruit bodies. Several species abundance and diversity coefficients changed in relation to both the calculation method and fungal material used, which suggested the necessity for a uniform method of characterizing ectomycorrhizal fungal community structure.

The ontogeny of the fruit bodies of Mycena stylobates was studied by light microscopy and scanning electron microscopy (SEM). The ontogeny was divided into two phases: first the primordium with all the structures of the mature fruit body was established, then the primordial stipe elongated rapidly and the exposed hymenium started producing spores immediately. The first detected stage of fruit body formation was an irregularly arranged hyphal structure within the substrate colonized. After the rupture of the surface of the substrate, the primordium established on the surface. Soon a layer of wrapping hyphae was differentiated, which covered the complete primordium. The structures of the stipe and the cap developed synchronously. The developing stipe, cap and basal disc together formed a secondary ring-like cavity, in which the development of the hymenophore took place. The lower side of the cap was covered by a layer of degenerated hyphae. The development of the hymenophore started with a number of small alveolae on the lower side of the cap, which were covered with a hymenophoral palisade. The margins of these alveolae formed the primary lamellae, which in the first stage of their development were covered by a layer of degenerated hyphae. The hymenophoral palisade spread from the developing alveolae to the lamellar edge; the edge of the primary lamellae was forked in the early stages. Secondary lamellae were formed by the down folding of ridges from the lower side of the cap. In contrast to the primary lamellae, they were covered with hymenophoral palisade from the beginning. Spore production started immediately after the elongation of the stipe. These results were compared with other known modes of ontogeny within the Agaricales and some comments on the terminology used for the description of basidiome morphogenesis are made.

Vesicular–arbuscular (VA) mycorrhizas are described from anatomically preserved roots of the Middle Eocene taxodiaceous conifer Metasequoia milleri, and compared to those of the living species M. glyptostroboides. Virtually identical VA mycorrhizal structures occur in the root cortex of both species, where they conform to the Paris-type. Coiled hyphae are most common within cells of the inner cortical region, and these produce numerous, highly branched arbuscules. Close similarity of fungal position and structure within roots of the living and fossil Metasequoia species demonstrates that modern Paris-type VA mycorrhizal associations characterized taxodiaceous conifers by the early Tertiary.

Competing ectomycorrhizal fungi can cause problems during the commercial production of edible Tuber species infected plants in greenhouses and later when infected plants are transplanted to the field. We investigated the effect of selective fungicides on Tuber borchii, in the presence of Hebeloma sinapizans, both in vitro and during the production of T. borchii infected Quercus pubescens. Oxycarboxin completely suppressed the growth of H. sinapizans but had only a weak fungitoxic effect on the growth of T. borchii both in vitro and on Q. pubescens roots. In contrast, other fungicides either suppressed both fungi or T. borchii more than H. sinapizans.

Five fungal strains that are able to grow on toluene were isolated from enrichment cultures. Three different techniques were used: solid state-like batches, air biofilters and liquid cultures. Fungal growth in the latter systems was favoured by combining low pH and low water activity. Soil and groundwater samples from gasoline-polluted environments were used as inocula. The isolates were identified as deuteromycetes belonging to the genera Cladophialophora, Exophiala and Leptodontium and the ascomycete Pseudeurotium zonatum. The previously isolated toluene-degrading fungus Cladosporium sphaerospermum was included in the present study. Results showed that fungi grew on toluene with doubling times of about 2 to 3 days. Some of the strains also grew on ethylbenzene and styrene. The apparent half-saturation constant (Km) for toluene oxidation ranged from 5 to 22 μM. Degradation activity was inhibited by 50% at toluene concentrations ranging from 2.4 to 4.7 mM. These kinetic parameters are comparable to analogous data reported for toluene-degrading bacteria. The ability of fungi to grow at low water activities and low pH suggest that they may be used for the purification of gas streams containing aromatic hydrocarbons in air biofilters.